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1 kbp GeneRuler:

100 bp GeneRuler:

PageRuler Plus:

Labjournal

Week 1

Monday, April 22nd

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of Phytochrome B for protein fusion.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Investigator: Jeff, Rosario

Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Sequencing of RFP-Generator (RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)

Tuesday, April 23rd

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Procedure:

pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

4 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).

Procedure:

Batch for analytical digestion for P4 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P4
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P5 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P5
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	P4	P5
	Mutation successful	Mutation successful!

Parts are compliant and do not contain RFC25 forbidden restriction sites.

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different vectors we sequenced received the following barcodes:

- ADH in pTUM100: FR01002265

- TEF1 in pTUM100: FR01002266

- TEF2 in pTUM100: FR01002266

- GAL in pTUM100: FR01002268

Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.

Wednesday, April 24th

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Florian

Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Investigator: Jeff, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.

Procedure:

Batch for analytical digestion for P7 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P7
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P8 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P8
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P9 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P9
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P10 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P10
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	P7	P8	P9	P10
	Part is correct	Part is correct	Part is correct	Part is correct

Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with EreA and EreB (pDEST14).

Procedure:

Plasmid DNA was received dried in paper from McMaster University.

DNA was resuspended in ddH2O

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Week 2

Monday, April 29th

Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Investigator: Rosario

Aim of the experiment: Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Concentrations were determined by measuring the absorption:

Plasmid	c [ng/µl]
pRK792	214,5
pRK793	154,3
pRIL	6,1
ereA	183,4
ereB	98,3

The procedure will have to be repeated for pRIL

Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Investigator: Andreas, Florian

Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Procedure:

Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

Tuesday, April 30th

Preparative digestion and gelelectrophoresis of mINF1 and Leptin with HindIII and EheI

Investigator: Louise, Johanna

Aim of the experiment: Testing the enzymes, since the earlier digestion with probably older enzymes did not work (no bands on gel) .

Procedure:

Batch for preparative digestion for mINF1 with HindIII and EheI

volume	reagent
10 µl	Plasmid DNA mINF11
5 µl	Tango Buffer (10x)
2 µl	HindIII (10 U/µl)
3 µl	EheI (10 U/µl)
30 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion for Leptin with HindIII and EheI

volume	reagent
20 µl	Plasmid DNA Leptin
5 µl	Tango Buffer (10x)
2 µl	HindIII (10 U/µl)
3 µl	EheI (10 U/µl)
20 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

Transformation of E. coli XL1 blue with PSH21 (P17)

Investigator: Johanna, Louise

Aim of the experiment: Transformation of E. coli XL1 blue with PSH21.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.

5 µl of DNA was added to 250 µl of competent cells and gently mixed.

60 min incubation on ice

5 min. heat shock at 37 °C

Adding the DNA to 2 ml LB-medium.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Thursday, May 2nd

Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system

Investigator: Katrin

Aim of the experiment: Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The concentration was measured (900 ng/µl)

Friday, May 3rd

Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry

Investigator: Andreas

Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.

5 µl of DNA was added to 250 µl of competent cells and gently mixed.

60 min incubation on ice

5 min. heat shock at 37 °C

Adding the DNA to 2 ml LB-medium.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Plating of received E. coli containing biobricks

Investigator: Jeff, Andreas

Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.

Operational sequence:

Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.

The biobricks were:

Name	Function
[[http://partsregistry.org/Part:BBa_K157004 BBa_K157004]]	Fluoresceine A -binding derivative of a Lipocalin
[[http://partsregistry.org/Part:BBa_K863000 BBa_K863000]]	bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
[[http://partsregistry.org/Part:BBa_K909009 BBa_K909009]]	cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
[[http://partsregistry.org/Part:BBa_K337013 BBa_K337013]]	constitutive promoter SV40
[[http://partsregistry.org/Part:BBa_K747096 BBa_K747096]]	constitutive promoter CMV
[[http://partsregistry.org/Part:BBa_K414002 BBa_K414002]]	constitutive promoter CaMV35S
[[http://partsregistry.org/Part:BBa_K147002 BBa_K147002]]	xylE gene coding for catechol-1,2-dioxygenase
[[http://partsregistry.org/Part:BBa_E2020 BBa_E2020]]	Cerulean
[[http://partsregistry.org/Part:BBa_K404319 BBa_K404319]]	mCFP
[[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]]	GFPmut3b
[[http://partsregistry.org/Part:BBa_K592010 BBa_K592010]]	amilGFP
[[http://partsregistry.org/Part:BBa_E2050 BBa_E2050]]	mOrange
[[http://partsregistry.org/Part:BBa_K399001 BBa_K399001]]	mStrawberry
[[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]]	mRFP1
[[http://partsregistry.org/Part:BBa_E2060 BBa_E2060]]	mCherry
[[http://partsregistry.org/Part:BBa_K592012 BBa_K592012]]	eforRed
[[http://partsregistry.org/Part:BBa_K592011 BBa_K592011]]	cjBlue
[[http://partsregistry.org/Part:BBa_K864401 BBa_K864401]]	aeBlue
[[http://partsregistry.org/Part:BBa_K592009 BBa_K592009]]	amilCP, blue chromoprotein
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414000 BBa_K414000]]	RD29A Promoter + Strong Plant Kozak (RBS) + RFP
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414006 BBa_K414006]]	35S Promoter + (Strong Plant RBS + GFP) From K414001
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414007 BBa_K414007]]	DREB1C promoter
[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K678001 BBa_K678001]]	alcA promoter
[[http://partsregistry.org/Part:BBa_K243004 BBa_K243004]]	Short linker
[[http://partsregistry.org/Part:BBa_K243005 BBa_K243005]]	Middle linker
[[http://partsregistry.org/Part:BBa_K243006 BBa_K243006]]	Long Linker
[[http://partsregistry.org/Part:BBa_K243029 BBa_K243029]]	GSAT Linker
[[http://partsregistry.org/Part:BBa_K243030 BBa_K243030]]	SEG Linker

Sunday, May 5th

Picking of the plated E. coli containing biobricks and the parts received from LMU

Investigator: Florian

Aim of the experiment: The colonies from the plates were transferred into liquid LB medium, so that minipreps can be performed the next day.

Operational sequence:

Tubes with 5 ml LB medium and the corresponding antibiotic were prepared.

A single colony from each plate was transferred into a tube with a pipet tip.

The biobricks were:

Label	Name	Function
p19		mCherry in pSB1C3
p20	[[http://partsregistry.org/Part:BBa_K823039 BBa_K823039]]	GFP in pSB1C3
p21	[[http://partsregistry.org/Part:BBa_K823029 BBa_K823029]]	mKate2 in pSB1C3
p22	[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414006 BBa_K414006]]	35S Promoter + (Strong Plant RBS + GFP) From K414001
p23	[[http://partsregistry.org/Part:BBa_K414002 BBa_K414002]]	constitutive promoter CaMV35S
p24	[[http://partsregistry.org/Part:BBa_K909009 BBa_K909009]]	cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
p25	[[http://partsregistry.org/Part:BBa_K399001 BBa_K399001]]	mStrawberry
p26	[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414007 BBa_K414007]]	DREB1C promoter
p27	[[http://partsregistry.org/Part:BBa_K863000 BBa_K863000]]	bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
p28	[[http://partsregistry.org/Part:BBa_K337013 BBa_K337013]]	constitutive promoter SV40
p29	[[http://partsregistry.org/Part:BBa_K592011 BBa_K592011]]	cjBlue
p30	[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K678001 BBa_K678001]]	alcA promoter
p31	[[http://partsregistry.org/Part:BBa_K592012 BBa_K592012]]	eforRed
p32	[[http://partsregistry.org/Part:BBa_K592010 BBa_K592010]]	amilGFP
p33	[[http://partsregistry.org/Part:BBa_K864401 BBa_K864401]]	aeBlue
p34	[[http://partsregistry.org/Part:BBa_K404319 BBa_K404319]]	mCFP
p35	[[http://partsregistry.org/wiki/index.php?title=Part:BBa_K414000 BBa_K414000]]	RD29A Promoter + Strong Plant Kozak (RBS) + RFP
p36	[[http://partsregistry.org/Part:BBa_K592009 BBa_K592009]]	amilCP, blue chromoprotein
p37	[[http://partsregistry.org/Part:BBa_K747096 BBa_K747096]]	constitutive promoter CMV
p38	[[http://partsregistry.org/Part:BBa_K147002 BBa_K147002]]	XylE dioxygenase (catechol dioxygenase)
p39	[[http://partsregistry.org/Part:BBa_K243005 BBa_K243005]]	Middle linker
p40	[[http://partsregistry.org/Part:BBa_K243029 BBa_K243029]]	GSAT Linker
p41	[[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]]	GFPmut3b
p42	[[http://partsregistry.org/Part:BBa_K243006 BBa_K243006]]	Long Linker
p43	[[http://partsregistry.org/Part:BBa_K243030 BBa_K243030]]	SEG Linker
p44	[[http://partsregistry.org/Part:BBa_K243004 BBa_K243004]]	Short linker
p45	[[http://partsregistry.org/Part:BBa_K157004 BBa_K157004]]	Fluoresceine A -binding derivative of a Lipocalin
p46	[[http://partsregistry.org/Part:BBa_E2060 BBa_E2060]]	mCherry
p47	[[http://partsregistry.org/Part:BBa_E2020 BBa_E2020]]	Cerulean
p48	[[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]]	mRFP1
p49	[[http://partsregistry.org/Part:BBa_E2050 BBa_E2050]]	mOrange

There were no colonies on the plate with K864401 (p33), therefore the LB medium could not be infected

The tubes were placed into the incubator at 37 °C

Week 3

Monday, May 6th

Miniprep of the prepared E. coli containing biobricks and the parts from LMU

Investigators: Florian, Johanna, Jefferey

Aim of the experiment: A miniprep of the overnight culture of our E. coli containig our biobricks and LMU parts was performed using the Qiagen miniprep kit.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Resulting concentrations were determined by measuring the absorption:

Label	Name	Concentration [ng/µl]
p19	mCherry in pSB1C3	40,2
p20	GFP in pSB1C3	58,5
p21	mKate2 in pSB1C3	131,3
p22	35S Promoter + (Strong Plant RBS + GFP) From K414001	42,8
p23	constitutive promoter CaMV35S	39,3
p24	cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana	30,0
p25	mStrawberry	46,6
p26	DREB1C promoter	34,8
p27	bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag	52,5
p28	constitutive promoter SV40	58,7
p29	cjBlue	44,4
p30	alcA promoter	26,9
p31	eforRed	64,8
p32	amilGFP	59,3
p34	mCFP	78,4
p35	RD29A Promoter + Strong Plant Kozak (RBS) + RFP	61,2
p36	amilCP, blue chromoprotein	89,2
p37	constitutive promoter CMV	83,6
p38	XylE dioxygenase (catechol dioxygenase)	57,4
p39	Middle linker	64,3
p40	GSAT Linker	109,6
p41	GFPmut3b	118,1
p42	Long Linker	133,0
p43	SEG Linker	107,5
p44	Short linker	60,2
p45	Fluoresceine A -binding derivative of a Lipocalin	82,7
p46	mCherry	230,2
p47	Cerulean	25,7
p48	mRFP1	299,3
p49	mOrange	181,2

Sequencing of FluA, SV40, CaMV35S, xylE, LMU mKate2, LMU GFP, LMU mCherry, DREB1C, RD29A

Investigators: Andreas, Florian, Johanna, Jefferey, Rosario

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode
p45	FluA	FR01002355
p28	SV40	FR01002356
p23	CaMV35S	FR01002357
p38	xylE	FR01002358
p21	LMU mKate2	FR01002359
p20	LMU GFP	FR01002360
p19	LMU mCherry	FR01002345
p46	mCherry	FR01002346
p26	DREB1C	FR01002347
p35	RD29A	FR01002348

Preparation of ordered primers

Investigators: Andreas, Jefferey, Rosario

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label	Oligonr.
6	EreA_for
7	EreA_rev
8	EreA_SDM1_for
9	EreA_SDM1_rev
10	EreA_SDM2_for
11	EreA_SDM2_rev
12	EreA_SDM3_for
13	EreA_SDM3_rev
14	EreB_for
15	EreB_rev
16	EreB_SDM1_for
17	EreB_SDM1_rev
18	EreB_SDM2_for
19	EreB_SDM2_rev
20	EreB_SDM3_for
21	EreB_SDM3_rev

Tuesday, May 7th

PCR, purification and analytical geleletrophoresis of EreA (P15) and EreB (P16)

Investigator: Jeff, Louise, Florian, Rosario, Andi, Johanna

Aim of the experiment: PCR of EreA (P15) and EreB (P16).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P15: O46 (EreA_for); P16: O31 (EreB_for))
1 µl	10 µM Reverse Primer (P15: O47 (EreA_rev); P16: O32 (EreB_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P15; P16)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The gradient PCR program was performed after following scheme with following conditions (T_m=54 °C; ΔG=2 °C; P15 in row 11(=52 °C); P16 in row 1 (=54.0 °C)):

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	T_m=54 °C; ΔG=2 °C	60 s
	68 °C	80 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

9 µl of the purified PCR products were mixed with 1 µl DNA loading buffer (10x) for analytical gelelectrophoresis

Analytical gelelectrophoresis was performed at 90 V for 60 min in 1% agarose gel.

1 kbp ladder	F1	F2
	Fragment-size like expected	Fragment-size like expected

Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV

Investigator: Jeff, Andi, Florian, Rosario, Louise, Johanna

Aim of the experiment: Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV .

Procedure:

Batch for preparative digestion of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI

volume	reagent
20 µl	Plasmid DNA P9/P50
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	PstI-HF
13.6 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of 20aalinker-PIF3 (P51) with EcoRI and NgoMIV

volume	reagent
20 µl	Plasmid DNA P51
4 µl	NEBuffer 4
1 µl	EcoRI-HF
1 µl	NgoMIV
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 3 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the reaction batches after digestion and were loaded on an 1% low-melting agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 70 V for 90 min.

P9 digestion = F3	1 kbp ladder	P50 digestion = F4	P51 digestion = F5
Fragment-size like expected; band was cut out		Fragment-size like expected; band was cut out	Fragment-size like expected; band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Wednesday, May 8th

Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.

Procedure:

Batch for preparative digestion of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.

volume	reagent
25 µl	PCR product F1/F2
5 µl	NEBuffer 4
0.5 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
17.5 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P52 for preperation of pSB1C3 vector:

volume	reagent
20 µl	Plasmid DNA P52
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
13.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the reaction batches for F1 & F2 and 4 µl of DNA loading buffer (10x) were added to the reaction batches for Pxx after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 60 min.

F1 digestion = F6	F2 digestion = F7	1 kbp ladder	P52 digestion = F8
Length was like expected	Length was like expected		Length was like expected; the lower band was extracted

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3)

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3). Quickchanges have still to be performed.

Procedure:

Ligation batch for F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3):

Ligation batch for F8+F6

volume	reagent
1.35 µl	F8 (74.3 ng/µl, 2086 bp)
6.76 µl	F6 (25.9 ng/µl, 1218 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
8.89 µl	ddH2O
=20 µl	TOTAL

Ligation batch for F8+F7

volume	reagent
1.35 µl	F8 (74.3 ng/µl, 2086 bp)
8.78 µl	F7 (20.6 ng/µl, 1257 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
6.87 µl	ddH2O
=20 µl	TOTAL

Negative control was als prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batches.

Cycled ligation has been performed after following protocol in a thermocycler:

100 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Midiprep of cloning vector RFC25 RFP generating device

Investigators: Andreas, Rosario

Aim of the experiment: Midiprep of our cloning vector RFC25 RFP generating device.

Procedure: Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

The yield of our vector was 1854 ng.

Thursday, May 9th

Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB)

Investigator: Florian, Jeff, Louise, Katrin, Ingmar

Aim of the experiment: Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB). Quickchanges has now to be performed to remove forbidden restriction sites.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of ligation products F8+F6, F8+F7, F8 negative control were added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Friday, May 10th

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Andi, Jeff, Rosario

Aim of the experiment: Deletion of found mutations.

Operational sequence

1. PCR general setup

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	DNA template
1 µl	1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	DNA template
1 µl	1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	12	95 °C	30 s
		55 °C	1 min
		68 °C	6 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.

After the PCR was finished 1 µl of the DpnI restriction enzyme (10 U/µl)was added

Now the reaction was mixed gently and thoroughly, spinned down in a microcentrifuge for 1 minute, and immediately incubated at 37 °C for 1 hour to digest the parental supercoiled dsDNA

2. Used constructs and primers

Quickchange number	Plasmid number	Geneconstruct	Oligo number
QC 1	P27	BPul_Laccase+ pSB1C3	O26 & O27

Picking of transformed Plasmids F8 + F6

Investigator: Andi

Aim of the study: Picking from the overnight transformation of the ligated F8+F6, to see whether ligation is successful or not.

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

PCR of TEV (P14, pRK793)

Investigator: Katrin, Jeff

Aim of the experiment: PCR, purification and analytical geleletrophoresis of TEV (P14, pRK793) to produce TEV with RFC25 pre- and suffx (still contains forbidden SpeI-site).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (O34 (TEV_fw))
1 µl	10 µM Reverse Primer (O35 (TEV_rv))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P14)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme with following conditions:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	55 °C	60 s
	68 °C	150 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Resulting product was labeled as F12.

Digestion of P61 with S1 Nuclease

Investigator: Katrin

Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)

Investigator: Andi

Aim of the experiment: Oligohybridization of MinMCS_for (O22) and MinMCS_rev (O23

Procedure:

25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev

Heating up to 95 °C for 5 min

Cooling at RT in a styropor box overnight

Preparation of ordered primers

Investigators: Andreas, Rosario, Katrin, Ingmar

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label	Oligonr.
24	BPU_for
25	BPU_rev
26	BPU_SDM1_for
27	BPU_SDM1_rev
28	BPU_SDM2_for
29	BPU_SDM2_rev
30	BPU_SDM3_for
31	BPU_SDM3_rev
32	BPU_SDM4_for
33	BPU_SDM4_rev
34	TEV_for
35	TEV_rev
36	TEV_t387c_for
37	TEV_t387c_rev
38	N_Split_TEV_for
39	N_Split_TEV_rev
40	C_Split_TEV_for
41	C_Split_TEV_rev
42	PIF6_2-100_for
43	PIF6_2-100_rev
44	p104AgeI a71g fw
45	p104AgeI a71g rev
46	xylE_for
47	xylE_rev
48	xylE_c312a_for
49	xylE_c312a_rev
50	xylE_g483a_for
51	xylE_g483a_rev
52	xylE_a834g_for
53	xylE_a834g_rev

Saturday, May 11th

Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI

Investigator: Louise

Aim of the experiment: Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI. Quickchange has still to be performed (forbidden SpeI restriction site).

Procedure:

Batch for preparative digestion of PCR product S219V pRK793 (F12) with XbaI & AgeI.

volume	reagent
20 µl	PCR product F12
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
13.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 3 h at 37 °C

Digested products were purified to remove nucleotides and enzymes with QIAquick PCR Purification Kit, QIAGEN.

The digested fragment was labelled F15

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA)

Investigator: Louise

Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA) of 2 clones picked the day before

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The plasmids were labelled as P63 and P64

Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Investigator: Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Procedure:

Batch for analytical digestion of P63 and P64 with AgeI-HF and XbaI

volume	reagent
2.5 µl	Plasmid DNA P63/P64
2 µl	NEBuffer 4 (10x)
0.2 µl	BSA (100x)
0.25 µl	XbaI(10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
14.8 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Analytical digestion of P63 with AgeI-HF and XbaI	1 kbp ladder	Analytical digestion of P64 with AgeI-HF and XbaI
Insertion successful		Insertion successful

Preparative digestion of PCR product of EreB (F2) with XbaI & AgeI

Investigator: Louise, Johanna

Aim of the experiment: Preparative digestion of PCR product EreB (F2) with XbaI & AgeI (second try since there did not grow any transformed bacteria in the first time).

Procedure:

Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

volume	reagent
20 µl	PCR product F2
5 µl	NEBuffer 4
0.5 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
22.5 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C
The digested fragment was labelled F14

Purification and ligation of digested PCR product EreB (F14)

Investigator: Louise, Rosario

Aim of the experiment: PCR Purification and ligation of digested PCR product EreB (F14).

Procedure: PCR Purification of EreB PCR (F14)

To purify the digested Fragment(F14) the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be 5 ng/µl via Nano drop.

Ligation of F14 + F8(pSB1C3)

Ligation batch for F8+F14 (positive control)

volume	reagent
1.35 µl	F8 (74.3 ng/µl, 2086 bp)
15.65 µl	F14 (5 ng/µl, 1257 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Transformation of ligation products of F10 + F11 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the ligation products of F10 + F11 and the negative control in pTUM104 in XL1 Blue.

Operation Sequence

melting of 2x 200 µl Ca-competent E.coli XL1-Blue cells on ice
Preparing 4 Tubes with 100 µl of Ca-competent E.coli XL1-Blue cells
addition of 5 µl of the ligation products
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Transformation of Quickchange Product P62 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the Quickchange Product P62 and the negative control in XL1 Blue.

Operation Sequence

melting of 1x 200 µl Ca-competent E.coli XL1-Blue cells on ice
Preparing 4 Tubes with 50 µl of Ca-competent E.coli XL1-Blue cells
addition of 1 µl of the Quickchange Product
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Quickchange mutagenesis of pTUM104

Investigator: Ingmar

Aim of the experiment: Sequencing results showed a forbiden AgeI restriction site in this vector. The sdm will delete this restricition site

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P6 template
0.5 µl	1:10 dilution of O44 (10 pmol/µL)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P6 template
0.5 µl	1:10 dilution of O45 (10 pmol/µL)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	10	95°C	30 sec
		55°C	1 min
		67°C	6 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C. The resulting product was named fragmet 14.

Transformation into E.coli Xl1-Blue Operation Sequence

melting of 100 µl Ca-competent E.coli XL1-Blue cells on ice
addition of 1 µl of the PCR product
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Sunday, May 12th

Analytical gelelectrophoresis of F15 (digested TEV PCR product)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F15 (digested TEV PCR product) to check whether PCR has worked.

Procedure:

5 µl of F15 was mixed with 0.55 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

PCR product has expected length

Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25). Further quickchanges still have to be done.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with ligation products F8+F14 (EreB in pSB1C3, RFC25)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F14 (EreB in pSB1C3, RFC25). Further quickchanges still have to be done.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P23

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P23 for insertion of miniMCS.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of transformed Plasmid F10+F11 (IRES from polio virus, blunt ligated with pSB1C3 for sequencing)

Investigator: Andi

Aim of the study: Picking of transformed Plasmid F10+F11 (AlcR blunt ligated with pSB1C3 for sequencing).

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

10 colonies were picked.

Week 4

Monday, May 13th

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F10+F11

Investigator: Rosario, Jeffery, Johanna, Flo

Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue F10+F11 of 10 clones picked the day before

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analyzing sequencing results

Investigator: Johanna

Aim of the experiment: Comparison of the sequencing results with the Biobrick (BB) sequences.

Procedure: Using Geneious to align the sequencing results with the BB sequences.

LMU GFP in RFC 25(p20)

The provided sequence in the parts registry is totally wrong.
The comparison of the amino acid seqeunces reveals several point mutations.

FluA in RFC 25 (p45)

Did not yield sequencing results (does the primer bind ?)
No presence of RCF 25 standard restriction enzymes

mCherry in RFC 25 (p46)

Did not yield sequencing results (does the primer bind ?)
No presence of RCF 25 standard restriction enzymes

CaMV35S in RFC 25 (p23)

high identity
high match with HQ699544 (Blast)

LMU mKate2in RFC 25 (p21)

no continous ORF
Did not yield sequencing results

DREB1C promoter in RFC 25 (p26)

Present of fluorecent protein gene

xylE in RCF 25 (p38)

Did not yield sequencing results

SV40 in RCF 25 (p28)

sequencing result agrees with the laccases
the sequencing result can't be the one of SV40

Quickchange mutagenesis SDMI of p27

Investigator: Rosario, Jeff, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 65.

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid P27 template
0.5 µl	1:10 dilution of O26 (10 pmol/µL)
20. 5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid P27 template
0.5 µl	1:10 dilution of O27 (10 pmol/µL)
20.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	10	95°C	30 sec
		55°C	1 min
		67°C	4 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles was increased to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of p38

Investigator: Jeff, Flo, Andi, Rosario

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 67.

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0,5 µl	Plasmid P38 template
0.5 µl	1:10 dilution of O48 (10 pmol/µL)
20.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid P38 template
0.5 µl	1:10 dilution of O49 (10 pmol/µL)
20.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	10	95°C	30 sec
		55°C	1 min
		67°C	4 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of P63

Investigator: Jeff, Rosario, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled P66.

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0.25 µl	Plasmid P63 template; 1:2 dilution
0.5 µl	1:10 dilution of O8 (10 pmol/µL)
20.75 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
0.25 µl	Plasmid P63 template; 1:2 dilution
0.5 µl	1:10 dilution of O9 (10 pmol/µL)
20.75 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	10	95°C	30 sec
		55°C	1 min
		67°C	6 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, the amount of cycles was raised to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Transformation of E. coli XL1 blue with P65

Investigator: Jeff, Rosario, Johanna, Andi, Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P65 .

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P66

Investigator: Jeff, Rosario, Flo, Johanna, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with P66.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P67

Investigator: Jeff, Rosario, Johanna, Andi, Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P67.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Tuesday, May 14th

Analytical digestion of P15 and P19 to P52

Investigator: Florian, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P15 and P19 to P52 to check the sequencing results.

Procedure:

Batch for analytical digestion of P15 and P19 to P52

volume	reagent
5 µl	Plasmid DNA
5 µl	Mastermix
=10 µl	TOTAL

Batch of Mastermix

volume	reagent
40 µl	NEBuffer 4 (10x)
4 µl	BSA (100x)
0.2 µl	NotI(10 U/µl)
148 µl	ddH2O
=200 µl	TOTAL

Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.

Discussion:
Gel 1, Row 1:

lane	part	plasmid	band 1	band 2	result
2	EreA	pDEST14	3,25 kb		no cut, no restriction sites in pDEST14
3	mCherry(LMU)	pSB1C3	2,0 kb	750 bp	probably right
4	GFP(LMU)(845 bp)	pSB1C3	2,0 kb	750 bp	maybe switched with mKate2(LMU)
5	mKate2(LMU)(702 bp)	pSB1C3	2,0 kb	900 bp	maybe switched with GFP(LMU)
6	35S + Strong Plant + GFP(1775 bp)	pSB1C3	2,0 kb	750 bp	?
7	UVR8(1332 bp)	pSB1C3	2,0 kb	1,1 kb	?
8	mStrawberry(708 bp)	pSB1C3	2,0 kb	1,4 kb	?
9	DREB1C promoter(463 bp)	pSB1C3	2,5 kb		probably only one cut -> one NotI site mutated
10	Laccase(1587 bp)	pSB1C3	2,0 kb	1,3 kb	?
11	SV40(419 bp)	pSB1C3	2,0 kb	1,6 kb	this could be the laccase
12	cjBlue(696 bp)	pSB1C3	2,0 kb	0,5 kb	?

Gel 1, Row 2:

lane	part	plasmid	band 1	band 2	result
2	alcA (843 bp)	pSB1C3	2,0 kb	0,7 kb	?
3	eforRed (681 bp)	pSB1C3	plasmid		plasmid bands (coiled, supercoiled, etc.)
4	amilGFP (699 bp)	pSB1C3	2,0 kb	0,7 kb	right part
5	mCFP (714 bp)	pSB1C3	2,0 kb	0,7 kb	right part
6	RD29A promoter (1535 bp)	pSB1C3	2,0 kb	1,5 kb	right part
7	amilCP (669 bp)	pSB1C3	2,0 kb	0,7 kb	right part
8	CMV promoter (580 bp)	pSB1C3	2,0 kb	0,6 kb	right part
9	middle linker (24 bp)	pMA-BBFR	2,4 kb		short insert went through the gel
10	GSAT (108 bp)	pMA-BBFR	2,4 kb	0,1 kb	right part
11	GFP(720 bp)	pSB1A2			?
12	long linker (36 bp)	pMA-BBFR	2,4 kb		short insert went through the gel

Gel 2:

lane	part	plasmid	band 1	band 2	result
2	SEG linker (108 bp)	pMA-BBFR	2,4 kb		short insert went through the gel
3	short linker (12 bp)	pMA-BBFR	2,4 kb		short insert went through the gel
4	FluA (522 bp)	pMA-BBFR	2,4 kb	0,5 kb	right part
5	mCherry (769 bp)	pSB2K3	4,4 kb	0,75 kb	right part
6	Cerulean (778 bp)	pSB2K3			?
7	mRFP1 (706 bp)	pSB2K3	4,4 kb	0,7 kb	right part
8	mOrange (769 bp)	pSB2K3	4,4 kb	0,75 kb	right part
9	PIF3-20aa (366 bp)	pSB1C3	2,0 kb	0,35 kb	right part
10	20aa-PIF3 (366 bp)	pSB1C3	2,0 kb	0,35 kb	two plasmids and two insert can be seen
11	LexA (ca. 6 kb)	pSB1C3	> 8 kb		only one cut, huge plasmid

Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV)

Investigator: Jeff, Florian

Aim of the study: Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV) for Miniprep.

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

1 colony of P23 were picked.

7 colonies of F8 + F15 were picked.

Sequencing of P63 & P64

Investigators: Louise

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
p63	EreA	FR01002349	FR01002350
p64	EreA	FR01002351	FR01002352

Wednesday, May 15th

Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI

Investigator: Louise,Johanna

Aim of the experiment:Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI to recover EreA and prepare two new vectors for cloning.

Procedure:

Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

volume	reagent
20 µl	Plasmid (P8,P60,P64)
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
13.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C
The digested fragments were labelled F17(P8), F18(P60), F19(P64)

PCR, purification and analytical geleletrophoresis of and EreB (P16)

Investigator: Jeff, Louise

Aim of the experiment: PCR of EreB (P16).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (O31 (EreB_for))
1 µl	10 µM Reverse Primer (O32 (EreB_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P15; P16)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program TMKS was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	52 °C	60 s
	68 °C	80 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

4 µl of the purified PCR products were mixed with 0.4 µl DNA loading buffer (10x) for analytical gelelectrophoresis

Analytical gelelectrophoresis was performed at 90 V for 30 min in 1% agarose gel.

1 kbp ladder	F16
	Fragment-size like expected

Purification and ligation of digested products F17, F18, F19

Investigator: Jeff, Louise

Aim of the experiment: Purification and ligation of digested products F17, F18, F19.

Procedure:

To purify the digested Fragments the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be XX ng/µl via Nano drop.

Ligation of F19 + FXX(pSB1C3)

Ligation batch for F19+FXX (positive control)

volume	reagent
1.35 µl	F8 (74.3 ng/µl, 2086 bp)
X µl	F14 (5 ng/µl, 1257 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI)

Investigator: Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI).

Procedure:

Mastermix for analytical digestion of P90-P96 with XbaI+AgeI-HF:

volume	reagent
16 µl	NEBuffer 4 (10x)
1.6 µl	BSA (100x)
2 µl	XbaI (20 U/µl)
2 µl	AgeI-HF (20 U/µl)
118.4 µl	ddH2O
=140 µl	TOTAL

Batch for digestion of P90-P96 with XbaI+AgeI-HF:

volume	reagent
2.5 µl	Plasmid DNA
17.5 µl	Mastermix
=20 µl	TOTAL

Batch for digestion of P90-P96 with XbaI+AgeI-HF:

volume	reagent
2.5 µl	Plasmid DNA
2 µl	NEBuffer 4 (10x)
0.2 µl	BSA (100x)
0.25 µl	XbaI (20 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
14.8 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	Digestion of P90	Digestion of P91	Digestion of P92	Digestion of P93	Digestion of P94	Digestion of P95	Digestion of P96	Digestion of P97
	Insert has expected length	Insert has expected length	Insert has expected length	Insert has expected length	Corrupt	Insert has expected length	Insert has expected length	Corrupt

The inserts for TEV semm to be right in the most cases, but the backbone has mutated and it should be cloned into a new pSB1C3 with new same restriction enzymes (XbaI+AgeI)!

The forward sequencing of CaMV p35S is right but the analytical digestion is wrong. Maybe further parts are downstream of the promoter?! Reverse sequencing should be performed for further analysis.

Thursday, May 16th

Preparative digestion of pSH21 (P61), psB1A2(P41) and P96 (TEV S19V)

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion of pSH21 (P61)and psB1A2(P41) with EcoRI and P96 (TEV S19V) with XbaI & AgeI for subsequent ligation.

Procedure:

Batch for preparative digestion of pSH21 with EcoRI

volume	reagent
20 µl	Plasmid DNA P61
4 µl	Buffer 4(10x)
1 µl	EcoRI (20 U/µl)
15 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of psB1A2 with EcoRI

volume	reagent
20 µl	Plasmid DNA P41
4 µl	Buffer 4(10x)
1 µl	EcoRI (20 U/µl)
15 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P96 (TEV S219V) with XbaI & AgeI.

volume	reagent
20 µl	Plasmid DNA P96
4 µl	Buffer 4(10x)
1 µl	XbaI (20 U/µl)
1 µl	AgeI-HF (20 U/µl)
13.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 3 h at 37 °C.

The resulting fragments were named: F23 (P41) and F22 (P61)

4 µl of DNA loading buffer (10x) were added to the reaction batch containing pSH21 (P61) after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 70 V for 90 min.

1 kbp ladder	P61 digestion = F23
	Fragment-size like expected; band was cut out

The band had the expected length. They were cut out and purified using the QIAquick Gel Extraction Kit, QIAGEN

Preparative digestion of ereB (P64) with XbaI & AgeI.

Investigator: Jeff, Louise, Katrin

Aim of the experiment:Preparative digestion of EreB (P64) with XbaI & AgeI.

Procedure:

Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

volume	reagent
20 µl	Plasmid
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
13.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C
The digested fragments were labelled

Dephosphorylation of F23

Investigator: Jeff, Katrin

Aim of the experiment: Dephosphorylation of cut vector psB1A2 to prevent re-ligation

Procedure:

the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer

Batch for the dephosphorylation of F23

volume	reagent
30 µl	Plasmid DNA F23 (2 µg)
4 µl	10X Fast AP buffer
2 µl	FastAP Thermosensitive Alkaline Phosphatase
4 µl	ddH₂O
=40 µl	TOTAL

the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
the reaction was stopped by heating to 75 °C for 5 minutes

Ligation of pSH21 (F22) with psB1A2(F23), TEV (P96), EreA (P64), pSB1A2(P41)

Investigator: Jeff, Katrin

Aim of the experiment: ligation of pSH21 (F22) with pSB1A2(F23), TEV (F20) with pSB1C3 (F17), EreA (F19)with pSB1C3 (F17) and EreB (F21) with pSB1C3 (F17)

Procedure:

Ligation batch for EreB (F21) with pSB1C3 (F17)

volume	reagent
1.6 µl	F17 (61,4 ng/µl)
15,4 µl	F21 (8,3 ng/µl)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for TEV (F20) with pSB1C3 (F17)

volume	reagent
1.6 µl	F17 (61,4 ng/µl)
4,1 µl	F20 (24,7 ng/µl)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
11,3 µl	ddH20
=20 µl	TOTAL

Ligation batch for pSH21 (F22) with pSB1A2(F23)

volume	reagent
0,85 µl	F23 (118,1 ng/µl)
2,3 µl	F22 (119,0 ng/µl)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
13,85 µl	ddH20
=20 µl	TOTAL

Ligation batch for EreA (F19) with pSB1C3 (F17)

volume	reagent
1.6 µl	F17 (61,4 ng/µl)
6,4 µl	F19 (8,3 ng/µl)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
9 µl	ddH20
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
the ligation was performed at 16 °C overnight

Friday, May 17th

Transformation of E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23, F17 (negative control), F23 (negative control)

Investigator: Jeff, Katrin

Aim of the experiment: Transformation of ligation products F17+F21 (pSB1C3, EreB), F17+F20 (pSB1C3, TEV), F17+F19 (pSB1C3, EreA), F23+F22 (pSB1A2, pSH21) and negative controls

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on chloramphenicol plates (F17+F19, F17+F20, F17+F21, F17 negative) or ampicillin plates (F23+F22, F23 negative control)

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (F17+F19, F17+F20, F17+F21) or ampicillin plates (F23+F22)

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Jeff

Aim of the experiment: removal of forbidden restiction sites from laccase

Operational sequence

PCR general setup

volume	reagent
5 µl	10x Pfu Ultra II buffer
1 µl	DNA template (p28)
0,5 µl each	1:10 dilution of appropriate Oligos (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33 )
42 µl	ddH2O
1 µl	dNTP mix
1 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	12	95 °C	30 s
		55 °C	1 min
		68 °C	4 min
3	1	4 °C	hold

Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on chloramphenicol plates 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33)

Saturday, May 18th

Picking of transformed Plasmid E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23

Investigator: Andi, Jeff

Aim of the study: Picking of transformed Plasmid F17+F19, F17+F20, F17+F21, F22+F23 .

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR/AmpR LB-medium)

2 colonies were picked for every Transformation product.

Sunday, May 19th

Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23

Investigator: Jeff, Andi

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23 of 2 clones picked the day before

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and analytical gelelectrophoresis of F17+F19, F17+F20, F17+F21 and F22+F23

Investigator: Jeff, Andi

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of ligation products F17+F19, F17+F20, F17+F21, F22+F23.

Procedure:

Mastermix for analytical digestion of F17+F19, F17+F20, F17+F21 with XbaI+PstI-HF:

volume	reagent
14 µl	NEBuffer 4 (10x)
1.4 µl	BSA (100x)
1.75 µl	XbaI (20 U/µl)
1.75 µl	AgeI-HF (20 U/µl)
103.6 µl	ddH2O
=122.5 µl	TOTAL

Batch for digestion of F22+F23 with EcoRI-HF:

volume	reagent
2.5 µl	Plasmid DNA
2 µl	NEBuffer 4 (10x)
15.25 µl	ddH2O
0.25 µl	EcoRI-HF (20 U/µl)
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	Digestion of F17+F19	Digestion of F17+F19	Digestion of F17+F20	Digestion of F17+F20	Digestion of F17+F21	Digestion of F17+F21	Digestion of F22+F23	Digestion of F22+F23
	Insert has expected length	Insert has expected length, the first line represents the rest of the uncut Plasmid	Insert has expected length	Insert has expected length, the first line represents the rest of the uncut Plasmid	Insert has expected length	Insert has expected length	Insert has expected length, insert and cut Plasmid overlay each other, because of the same length	Corrupt

Quick Change mutagenesis to remove forbidden restriction sites of P28 (Laccase), P100 (TEV S219V), P102 (EreB)

Investigator: Jeff, Andi

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).

Operational sequence

PCR general setup

Procedure:

PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid template (P28/P100/P102)
1 µl	1:10 dilution of O26 (for P28, 10 µM)/O16 (for P102, 10 µM)/O36 (for P100, 10 µM)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P38 template
1 µl	1:10 dilution of O27 (for P28, 10 µM)/O17 (for P102, 10 µM)/O37 (for P100, 10 µM)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	10	95 °C	30 s
		55 °C	1 min
		68 °C	4 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator:

Aim of the experiment:

Procedure:

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Week 5

Tuesday, May 21st

Quick Change mutagenesis to remove forbidden restriction sites - SDMI - of P28 (Laccase), P100 (TEV S219V), P102 (EreB)

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).

Operational sequence

PCR general setup

Procedure:

PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid template (P28/P100/P102)
1 µl	1:10 dilution of O26 (for P28, 10 µM)/O16 (for P102, 10 µM)/O36 (for P100, 10 µM)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P38 template
1 µl	1:10 dilution of O27 (for P28, 10 µM)/O17 (for P102, 10 µM)/O37 (for P100, 10 µM)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	10	95 °C	30 s
		55 °C	1 min
		68 °C	4 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator:

Aim of the experiment:

Procedure:

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P105, P28, P100, P102 & P98

Investigators: Jeff, Andi,Johanna

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
p105	IRES	FR01002288	FR01002269
p102	EreB	FR01002270	FR01002275
p100	TEV S219V	FR01002278	FR01002279
p98	EreA	FR01002276	FR01002277
p28	Laccase		FR01002280

Preparation of Knop-Medium for Moss

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Preparation of Knop-Medium for Moss

Procedure:

All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
Volume of prepared Medium: 1L
10 ml of each stock were converted into a 1,8L Erlenmeyer flask and filled up to 1L with destilled water (ELGA); 12,5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH
The prepared Volume of 1L was shared equally to 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium
All flasks have been autoclaved

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)

Investigator: Andi, Jeff, Johanna, Rosario

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17) from Sunday, May 19th.

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

3 colonies for P100QC (TEV, QC with O36/O37) and 5 colonies from P102QC (EreB, QC with O16/O17) were picked from the plates.

Wednesday, May 22nd

Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)

Investigator: Jeff

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27)

Investigator: Jeff

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27).

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

8 colonies for P28QC (Laccase, QC with O26/O27) were picked from the plates.

Analytical digestion and analytical gelelectrophoresis of P106, P107, P108, P109, P110, P111, P112, P113

Investigator: Jeff, Rosario

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of Quickchanges to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P100 (TEV, QC with O36/O37: P111, P112, P113) and P102 (EreB, QC with O16/O17: P106, P107, P108, P109, P110) were successful

Procedure:

Mastermix for analytical digestion of P106, P107, P108, P109, P110, P111, P112, P113 with AgeI-HF and XbaI:

volume	reagent
20 µl	NEBuffer 4 (10x)
2 µl	BSA (100x)
2.5 µl	AgeI-HF (20 U/µl)
2.5 µl	XbaI
148 µl	ddH2O
= 175 µl	TOTAL

17,5 µl Mastermix with 2,5 µl Plasmid

Mastermix for analytical digestion of P111, P112, P113 with XbaI and SpeI and P106, P107, P108, P109, P110 with XbaI and PstI-HF:

volume	reagent
20 µl	NEBuffer 4 (10x)
2 µl	BSA (100x)
2.5 µl	XbaI
148 µl	ddH2O
= 172,5 µl	TOTAL

17,25 µl Mastermix with 2,5 µl Plasmid and 0,25 µl SpeI or PstI respectively

Mastermix for analytical digestion of with P106, P107, P108, P109, P110 EcoRI and SpeI:

volume	reagent
12 µl	NEBuffer 4 (10x)
1,2 µl	BSA (100x)
1,5 µl	EcoRI-HF
1,5 µl	SpeI-HF
88.8 µl	ddH2O
= 105 µl	TOTAL

17,5 µl Mastermix with 2,5 µl Plasmid

Mastermix for analytical digestion of with P106, P107, P108, P109, P110 XbaI and SpeI:

volume	reagent
12 µl	NEBuffer 4 (10x)
1,2 µl	BSA (100x)
1,5 µl	XbaI
1,5 µl	SpeI-HF
88.8 µl	ddH2O
= 105 µl	TOTAL

17,5 µl Mastermix with 2,5 µl Plasmid

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	P111 XbaI + AgeI	P112 XbaI + AgeI	P113 XbaI + AgeI	P111 XbaI + SpeI	P112 XbaI + SpeI	P113 XbaI + SpeI
	Ctrl	Ctrl	Ctrl	QC succesful	QC failed	QC succesful

1 kbp ladder DNA ladder	P106 XbaI + AgeI	P107 XbaI + AgeI	P108 XbaI + AgeI	P109 XbaI + AgeI	P110 XbaI + AgeI	P106 XbaI + PstI	P107 XbaI + PstI	P108 XbaI + PstI	P109 XbaI + PstI	P110 XbaI + PstI
	Ctrl	Ctrl	Ctrl	Ctrl	Ctrl	PstI inside as should be; useless experiment	PstI inside as should be; useless experiment	PstI inside as should be; useless experiment	PstI inside as should be; useless experiment	PstI inside as should be; useless experiment

1 kbp ladder DNA ladder	P106 XbaI + SpeI	P107 XbaI + SpeI	P108 XbaI + SpeI	P109 XbaI + SpeI	P110 XbaI + SpeI	P106 EcoRI + SpeI	P107 EcoRI + SpeI	P108 EcoRI + SpeI	P109 EcoRI + SpeI	P110 EcoRI + SpeI
	Ctrl	Ctrl	Ctrl	Ctrl	Ctrl	QC succesful	QC succesful	QC succesful	QC succesful	QC failed

Thursday, May 23rd

Sequencing of P111, P113 and P109 (?)

Investigator: Jeff

Aim of the experiment: Sequencing of P111, P113 and P109 to check whether the QuickChange brought further unwanted mutations.

Procedure:

PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV

Investigator: Jeff

Aim of the experiment: PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV. To generate N-TEV, the primer O38 and O39 were used. To generate C-TEV, the primer O40 and O41 were used.

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (O38 (N_Split_TEV_fw) for generating N-TEV; O40 (C_Split_TEV_fw) for generating C-TEV)
1 µl	10 µM Reverse Primer (O39 (N_Split_TEV_rv) for generating N-TEV; O41 (C_Split_TEV_rv) for generating C-TEV)
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P111)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme with following conditions:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	51 °C	60 s
	68 °C	30 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Resulting product was labeled as F24 (N-TEV) and F25 (C-TEV).

Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV) to check whether PCR has worked.

Procedure:

4 µl of F24 and F25 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

PCR products has expected length, but there are unspecific products. So we have to be cautious and have to check if we ligate the right products.

Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI

Investigator: Jeff, Rosario

Aim of the experiment: Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI to clone it into pSB1C3.

Procedure:

Batch for preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI.

volume	reagent
25 µl	PCR product F24/F25
5 µl	NEBuffer 4
0.5 µl	BSA (100x)
1 µl	AgeI-HF
1 µl	XbaI
17.5 µl	ddH₂O
=50 µl	TOTAL

Incubation for 150 min at 37 °C.

Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.

The digested fragments were labelled F26 (digestion of F24 with XbaI&AgeI) and F27 (digestion of F25 with XbaI&AgeI)

Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3).

Procedure:

Ligation batch for F17+F26:

volume	reagent
1.63 µl	F17 (61.4 ng/µl, 2086 bp)
3.20 µl	F26 (15.9 ng/µl, 354 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
12.17 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F17+F27 (positive control)

volume	reagent
1.63 µl	F17 (61.4 ng/µl, 2086 bp)
2.41 µl	F27 (21.1 ng/µl, 354 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
12.96 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange (SDMI) Product of P28

Investigator: Jeff, Andi, Rosario, Johanna

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange Product of P28. 8 clones were picked the day before.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and analytical gelelectrophoresis of P28, P117, P118, P119, P120, P121, P122, P123

Investigator: Rosario, Andi, Johanna, Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P28 (promoter SV40) and quickchange products P117, P118, P119, P120, P121, P122, P123 (all QC - SDMI - of Bpul Laccase to remove forbidden AgeI).

Procedure:

Mastermix for analytical digestion of P28, P117, P118, P119, P120, P121, P122, P123 AgeI-HF:

volume	reagent
20 µl	NEBuffer 4 (10x)
2 µl	BSA (100x)
2.5 µl	AgeI-HF (20 U/µl)
150.5 µl	ddH2O
=175 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	Digestion of P28	Digestion of P116	Digestion of P117	Digestion of P118	Digestion of P119	Digestion of P120	Digestion of P121	Digestion of P122	Digestion of P123
	as expected	QC successful	QC successful	QC successful	QC successful	QC successful	QC successful	QC successful	QC successful

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P28 (Laccase), P106 (EreB)

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P106 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P106

volume	reagent	Additional information
5 µl	10x Pfu Ultra II buffer
2 µl	Plasmid template (P106 - 1:10 dilution)	need to get to 50 ng/µl
1.25 µl	1:10 dilution of O18 (for P106, 10 µM)
1.25 µl	1:10 dilution of O19 (for P106, 10 µM)
38.5 µl	ddH2O
1 µl	dNTP mix
1 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P106

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	18	95 °C	30 s
		52 °C	1 min
		68 °C	4 min

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Reaction batch - P116 (Laccase)

volume	reagent	Additional information
5 µl	10x Pfu Ultra II buffer
2 µl	Plasmid template - 1:5 dilution	need to get to 50 ng/µl
1.25 µl	1:10 dilution of O28 for P116
1.25 µl	1:10 dilution of O29 for P116
38.5 µl	ddH2O
1 µl	dNTP mix
1 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P116

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	18	95 °C	30 s
		52 °C	1 min
		68 °C	4 min

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P106 and P116.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Friday, May 24th

Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Procedure:

CaCl₂ competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions was plated again on a new chlorampenicol plates.

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P116 (Laccase), P109 (EreB)

Investigator: Volker, Louise, Katrin, Flo

Aim of the experiment: Removal of forbidden restiction sites from P116 (Laccase), P109 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P109

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P109 - 1:10 dilution)	need to get to 50 ng/µl
1 µl	1:10 dilution of O18 (for P109, 10 µM)
1 µl	1:10 dilution of O19 (for P109, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P109

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	21	95 °C	30 s
		52 °C	1 min
		68 °C	4 min 50 s

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Reaction batch - P116 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template - 1:5 dilution	need to get to 50 ng/µl
1 µl	1:10 dilution of O28 for P116
1 µl	1:10 dilution of O29 for P116
18.0 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P116

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	21	95 °C	30 s
		52 °C	1 min
		68 °C	4 min 50 s

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products into E. coli XL1 blue

Investigator: Volker

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P109 and P116.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Saturday, May 25th

Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator: Volker, Louise, Katrin, Flo

Aim of the experiment: Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Procedure:

5 µl of the PCR Quickchange products were either mixed with with 0.5 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Volker

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

5 colonies for P116QC (Laccase, QC with O28/O29) and 6 colonies for P109QC (EreB, QC with O18/O19) were picked from the plates.

Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)

Investigator: Volker

Aim of the study: Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Procedure:

Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

5 colonies were picked from plates for E. colis XL1 blue transformed with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).

Sunday, May 26th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Katrin, Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19) were successful

Investigator: Katrin, Louise

Aim of the experiment:Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Procedure:

analytical digestion of Quickchange products of P109 (EreB, QC with O18/O19)

volume	reagent
2 µl	NEB CutSmart Buffer (10x)
0,25 µl	PstI-HF (20 U/µl)
2,5 µl	DNA from Miniprep
15,25 µl	ddH2O
=20 µl	TOTAL

in order to check if the QC was successful, P109 (before QC) was also digested

analytical digestion of Quickchange products of P116 (Laccase, QC with O28/O29)

volume	reagent
2 µl	NEB CutSmart Buffer (10x)
0,25 µl	EcoRI-HF
0,25 µl	NgoMIV
2,5 µl	DNA from Miniprep
15 µl	ddH2O
=20 µl	TOTAL

in order to check if the QC was successful, P116 (before QC) was also digested

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder#	P116 (Laccase before QCII) digested with EcoRI-HF, NgoMIV	Laccase after QCII (P128) digested with EcoRI-HF, NgoMIV	Laccase after QCII (P129) digested with EcoRI-HF, NgoMIV	Laccase after QCII (P130) digested with EcoRI-HF, NgoMIV	Laccase after QCII (P131) digested with EcoRI-HF, NgoMIV	Laccase after QCII (P124) digested with EcoRI-HF, NgoMIV	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment
	as expected	as expected	as expected	as expected	as expected	as expected

Laccase P124 was chosen for sequencing

1 kbp ladder DNA ladder	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	EreB after QCII (P125) digested with PstI	EreB after QCII (P132) digested with PstI	EreB after QCII (P133) digested with PstI	EreB after QCII (P134) digested with PstI	EreB after QCII (P135) digested with PstI	EreB after QCII (P136) digested with PstI	EreB before QCII (P109) digested with PstI
					as expected	as expected	as expected	as expected	as expected	as expected	as expected

EreB P125 was chosen for sequencing

Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3

Investigator: Katrin, Louise

Aim of the experiment: Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of ligation products F17 + F26, F17 + F27

Investigator: Katrin, Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis to check whether the ligation of N-TEV (F17+F26) and C-TEV (F17+F25) in pSB1C3 were successful

Procedure:

analytical digestion of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25)in pSB1C3

Mastermix:

volume	reagent
22 µl	NEB Buffer 4 (10x)
2,2 µl	BSA (100X)
2,75 µl	XbaI
2,75 µl	AgeI-HF
162,8 µl	ddH2O
=192,5 µl	TOTAL

2,5 µl of miniprep products were added to 17,5 µl Mastermix

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder#	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	N-TEV ligated into pSB1C3 (F17 + F26) (P126) digested with XbaI, AgeI-HF	N-TEV ligated into pSB1C3 (F17 + F26) (P137) digested with XbaI, AgeI-HF	N-TEV ligated into pSB1C3 (F17 + F26) (P138) digested with XbaI, AgeI-HF	N-TEV ligated into pSB1C3 (F17 + F26) (P139) digested with XbaI, AgeI-HF
							as expected	as expected	as expected	as expected

N-TEV ligated into pSB1C3 (F17 + F26) (P126) was chosen for sequencing

1 kbp ladder DNA ladder	C-TEV ligated into pSB1C3 (F17 + F27) (P127) digested with XbaI, AgeI-HF	C-TEV ligated into pSB1C3 (F17 + F27) (P140) digested with XbaI, AgeI-HF	C-TEV ligated into pSB1C3 (F17 + F27) (P141) digested with XbaI, AgeI-HF	C-TEV ligated into pSB1C3 (F17 + F27) (P142) digested with XbaI, AgeI-HF	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment	belongs to other experiment
	as expected	as expected	as expected	as expected

C-TEV ligated into pSB1C3 (F17 + F27) (P127) was chosen for sequencing

Sequencing of P124-P127

Investigators: Louise, Katrin

Aim of the experiment: Sequencing of QCII products (EreB, Laccase) and ligation product of split-TEV

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
p124	Miniprep of transformation of QC product of P116 (Laccase, QC with O28/O29)(QC - SDMII - of Bpul Laccase to remove forbidden NgoMIV)	FR01002337 (VR)	FR01002338 (VF2)
p125	Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)	FR01002339 (VR)	FR01002340 (VF2)
p126	Miniprep of ligation product F17 + F26 (N-TEV)	FR01002341 (VR)	FR01002342 (VF2)
p127	Miniprep of ligation product F17 + F27 (C-TEV)	FR01002343 (VR)	FR01002344 (VF2)

Week 6

Monday, May 27th

Oligohybridization of MiniMCS (O56,O57) and Spytag (O54,O55)

Investigator: Johanna

Aim of the experiment: Oligohybridization of MiniMCS (MiniMCSII_fw (O56) and MiniMCSII_rv (O57) and of Spytag (SpyTag_fw (O54) and SpyTag_rv (O55))

Operational sequence

25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev in one Eppi. 25 µL of 100 pmol/µl of Spytag fw and 25 µL of 100 pmol/µl Spytag rev in a second Eppi

Heating up to 95 °C for 5 min

Cooling at RT in a styropor box overnight

PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114)

Investigator: Johanna

Aim of the experiment: PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction GC Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P114:O62 (pActin_for); P114:O60 (t35S_for); P115:O58 (npt-Kasette_for); P51:O66 (PIF3_for))
1 µl	10 µM Reverse Primer (P114:O63 (pActin_rev); P114:O61 (t35S_rev); P115:O59 (npt-Kasette_rev); P51:O67 (PIF3_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P114; P115; P51)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	49 °C	60 s
	68 °C	100 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Results:

PCR did not work properly because primer concentration was wrong.

Preparative digestion and gelelectrophoresis of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI

Investigator: Andi

Aim of the experiment:Preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI to clone fragment of P45 into F17 (pSB1C3) and to fusion Middle linker of P39 with GFP of P20.

Procedure:

Batch for preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeIwith XbaI & AgeI.

volume	reagent
20 µl	P45
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
2 µl	AgeI-HF
2 µl	XbaI
11.6 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.

volume	reagent
20 µl	P39
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
2 µl	AgeI-HF
2 µl	PstI
11.6 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.

volume	reagent
20 µl	P45
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
2 µl	NgoMIV
2 µl	PstI
11.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 120 min at 37 °C.

Preparative gelelectrophoresis was performed at 90 V for 1.5 h.

1 kbp ladder	P20 digested with NgoMIV&PstI (=F30)	P20 digested with NgoMIV&PstI (=F30)	P39 digested with AgeI&PstI (=F29)	P39 digested with AgeI&PstI (=F29)	P45 digested with XbaI&AgeI (=F28)	P45 digested with XbaI&AgeI (=F28)
	lower band was cut out	lower band was cut out	band was cut out	band was cut out	lower band was cut out	lower band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124(Laccase), P125 (EreB)

Investigator: Andi

Aim of the experiment: Removal of forbidden restiction sites from P124 (Laccase), P125 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P124

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P124 - 1:6 dilution)	need to get to 50 ng/µl
1 µl	1:10 dilution of O30 (for P125, 10 µM)
1 µl	1:10 dilution of O31 (for P125, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch - P125

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P125 - 1:6 dilution)	need to get to 50 ng/µl
1 µl	1:10 dilution of O20 (for P125, 10 µM)
1 µl	1:10 dilution of O21 (for P125, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters for both batches

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	21	95 °C	30 s
		52 °C	1 min
		68 °C	4 min 50 s

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Preparation of ordered primers

Investigators: Andi

Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl

Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.

Primers were labeled as follows:

Label	Oligonr.
O54	Spytag_fw
O55	Spytag_rev
O56	MiniMCSII_fw
O57	MiniMCSII_rev
O58	Npt_for
O59	Npt_rev
O60	t35S_for
O61	t35S_rev
O62	pACT_for
O63	pACT_rev
O64	NucA_for
O65	NucA_rev
O66	Pif3_for
O67	Pif3_rev

Transformation of P45, P51, P100 and P102 into E. coli XL1 blue

Investigator: Andi, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P45, P51, P102 and P100.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of transformed Plasmid E. coli XL1 blue with P114, P115

Investigator: Andi

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P114, P115.

Procedure:

Picking and overnight culture after standard laboratory's protocol. (AmpR LB-medium)

5 colonies of P114 and P115 were picked from the plates.

Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3)

Investigator: Jeff

Aim of the experiment: Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3).

Procedure:

Ligation batch for F29+F30:

volume	reagent
4.22 µl	F29 (23.7 ng/µl, 2110 bp)
10.48 µl	F30 (9.5 ng/µl, ~700 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
2.3 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F17+F28:

volume	reagent
1.63 µl	F17 (61.4 ng/µl, 2086 bp)
7.8 µl	F28 (9.6 ng/µl, 522 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
7.57 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Tuesday, May 28th

Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67)

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67).

Procedure:

4 µl of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67) were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.

1 kbp ladder	PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67)	PCR products of P114 (O60&O61)	PCR products of P115 (O58&O59)	PCR products of P51 (O66&O67)
	PCR did work, but strong primer dimer signal	PCR did work, but strong primer dimer signal	PCR did not work	PCR did work, but strong primer dimer signal

Wrong buffer (GC buffer, instead of standard reaction buffer, was used and 10x too much primers were also used)

Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 into E. coli XL1 blue

Investigator: Andi, Johanna

Aim of the experiment: Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 + ligation product of F17+NK + ligation product of F29+NK into E. coli XL1 blue.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P133 and P134

Investigators: Louise, Johanna

Aim of the experiment: Sequencing of two more QCII products (EreB) Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
p133	Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)	FR01002287 (fw)	FR01002296 (rev)
p134	Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)	FR01002295 (fw)	FR01002294 (rev)

Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115

Investigator: Andi

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102

Investigator: Jeff, Rosario

Aim of the experiment: Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102.

Procedure:

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (P51, P100, P102)/4 µL Ampicillin (1000x) (P45).

1 colony for each plasmid were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143)

Investigator: Rosario, Jeff

Aim of the experiment: PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P143:O62 (pActin_for); P143:O60 (t35S_for); P144:O58 (npt-Kasette_for); P51:O66 (PIF3_for))
1 µl	10 µM Reverse Primer (P143:O63 (pActin_rev); P143:O61 (t35S_rev); P144:O59 (npt-Kasette_rev); P51:O67 (PIF3_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P114; P115; P51)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	47 °C	60 s
	68 °C	120 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Analytical gelelectrophoresis of F33, F34, F35, F36

Investigator: Jeff, Rosario

Aim of the experiment: Analytical gelelectrophoresis of F33, F34, F35, F36.

Procedure:

4 µl of F33, F34, F35 and F36 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.

1 kbp ladder	F33	F33	F33	F33
	PCR did not work, maybe high GC content?	PCR products has expected length, some byproducts	PCR products has expected length, some byproducts	PCR products has expected length

Wednesday, May 29th

Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18

Investigator: Andi

Aim of the experiment: Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45 .

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Picking of of E. coli XL1 blue transformed with ligation product of F17+F28

Investigator: Andi

Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product of F17+F28.

Procedure:

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (

3 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P133 (EreB)

Investigator: Louise

Aim of the experiment: Removal of forbidden restiction site from P133 (EreB).

Procedure: Quickchange - PCR

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P133 - 1:10 dilution)	need to get to 50 ng/µl
1 µl	1:10 dilution of O20 (for P133, 10 µM)
1 µl	1:10 dilution of O21 (for P133, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P133

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	3 min 30 s

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange product into E. coli XL1 blue

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM III of P133.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI

Investigator: Louise

Aim of the experiment: Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI.

Procedure:

Batch for preparative digestion of t35s(F34) with XbaI and PstI

volume	reagent
25 µl	Plasmid DNA P61
5 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
1 µl	PstI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of npt(F35) with EcoRI and SpeI

volume	reagent
25 µl	Plasmid DNA P61
5 µl	CutSmart Buffer
1 µl	EcoRI (20 U/µl)
1 µl	SpeI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of PIF3(F36) with XbaI and AgeI-HF

volume	reagent
25 µl	Plasmid DNA P61
5 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
1 µl	AgeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P10 with XbaI & PstI for backbone isolation.

volume	reagent
20 µl	Plasmid DNA P96
4 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
1 µl	PstI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P10 with EcoRI and SpeI for backbone isolation.

volume	reagent
20 µl	Plasmid DNA P96
4 µl	CutSmart Buffer
1 µl	EcoRI (20 U/µl)
1 µl	SpeI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

The resulting fragments were named: ?????

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 90 min.

1 kbp ladder DNA ladder	Digestion of PCR product t35s (F34) with XbaI & PstI = F38	Digestion of PCR product npt casette (F25) with EcoRI & SpeI = F39	Digestion of PCR product PIF3 (F36) with XbaI & AgeI-HF = F40
	as expected, lower band was cut out	as expected, bright band was cut out	as expected, bright band was cut out

1 kbp ladder DNA ladder	Digestion of P10 with EcoRI & SpeI = F41	Digestion of P10 with XbaI & PstI = F42
	as expected, lower band was cut out	as expected, lower band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030)

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

PCR of pActin (P143)

Investigator: Florian, Jeff

Aim of the experiment: PCR of pActin (P143).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P143:O62 (pActin_for))
1 µl	10 µM Reverse Primer (P143:O63 (pActin_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P143)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	51 °C	60 s
	68 °C	80 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Results:

After analytical gelelectrophoresis there was no product visible. PCR did't work!

Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))

Investigator: Jeff

Aim of the experiment: Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).

Procedure:

Ligation batch for F17+F40:

volume	reagent
1.63 µl	F17 (61.4 ng/µl, 2086 bp)
1.88 µl	F40 (22.8 ng/µl, ~297 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
13.49 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F42+F38:

volume	reagent
1.36 µl	F42 (73.5 ng/µl, 2070 bp)
3.42 µl	F38 (9.2 ng/µl, 217 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
12.22 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F41+F39:

volume	reagent
1.11 µl	F41 (89.9 ng/µl, 2070 bp)
13.74 µl	F39 (16.0 ng/µl, 1517 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
2.15 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Thursday, May 30th

Gradient PCR of pActin (P143)

Investigator: Ingmar

Aim of the experiment: PCR of pActin

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer O62 (pActin_for)
1 µl	10 µM Reverse Primer O63 (pActin_rev)
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA P143
35.75 µL	ddH₂O Water
=50 µL	TOTAL

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq GC Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer O62 (pActin_for)
1 µl	10 µM Reverse Primer O63 (pActin_rev)
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA P143
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	47 °C	60 s
	68 °C	120 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

A temperature gradient was applied beginning at 47°C and ranging to 51°C. For each integer (47, 48, 49, 50 and 51°C) a single PCR batch was used.

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Analytical gelelectrophoresis Procedure:

4 µl of each PCR product were mixed seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

1 kbp ladder	F45 GC buffer	F46 GC buffer	F47 GC buffer	F47 GC buffer	F49 GC buffer	normal buffer	normal buffer	normal buffer	normal buffer	normal buffer	1 kbp ladder
	PCR products has expected length	PCR products has expected length	PCR products has expected length	PCR products has expected length	PCR products has expected length	PCR products has expected length, some byproducts	PCR products has expected length, some byproducts	PCR products has expected length, some byproducts	PCR products has expected length, some byproducts	PCR products has expected length, some byproducts

Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25)

Investigator: Jeff, Flo

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25)

Investigator: Flo, Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25).

Procedure:

Analytical digestion of ligation products of F17+F28 (FluA in pSB1C3 RFC25) with XbaI & AgeI.

volume	reagent
2.5 µl P150/P151/P152/P153
2 µl	CutSmart Buffer (10x)
0.25 µl	XbaI
0.25 µl	AgeI-HF
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder#	Digestion of P150 with XbaI&AgeI-HF	Digestion of P151 with XbaI&AgeI-HF	Digestion of P152 with XbaI&AgeI-HF	Digestion of P153 with XbaI&AgeI-HF
	as expected	as expected	as expected	as expected

Preparative gelelectrophoresis of oligohybridization products F31 & F32

Investigator: Flo, Jeff

Aim of the experiment: Preparative gelelectrophoresis of oligohybridization products F31 & F32.

Procedure:

50 µl of the 1:10 dilution of F31 and F32 were mixed with 5 µl of DNA loading buffer (10x).

Preparative gelelectrophoresis was performed 1.5 h on a 2% agarose gel at 90 V.

1 kbp ladder

F31

F32

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Gelpurified F31 and F32 were labelled as F43 and F44.

Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Ligation of F42+F43 and F17+F32

Investigator: Rosario

Aim of the experiment: Ligation of F42+F43 and F17+F32.

Procedure:

Ligation batch for F42+F43:

volume	reagent
1.36 µl	F42 (73.5 ng/µl, 2070 bp)
1.51 µl	F43 (5.4 ng/µl, 34 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
14.13 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F17+F32:

volume	reagent
1.63 µl	F17 (61.4 ng/µl, 2086 bp)
0.25 µl	F32 (2510.8 ng/µl, 49 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
15.12 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature&

Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS)

Investigator: Louise

Aim of the experiment: Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS).

Procedure:

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

5 colonies of EreB SDMIII (P149) and 2 colonies of BBa_K801030 (SV40 NLS) were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Friday, May 31st

Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)

Investigator: Louise, Jeff, Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of P149 (QCIII of EreB) with EcoRI-HF and PstI-HF

Investigator: Louise, Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF to check if QCII was successful

Procedure:

Analytical digestion of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF

volume	reagent
2.5 µl	P149-P158/P133 (control before QCIII)
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder#	Digestion of P133 with EcoRI-HF and PstI-HF	Digestion of P154 with EcoRI-HF and PstI-HF	Digestion of P155 with EcoRI-HF and PstI-HF	Digestion of P156 with EcoRI-HF and PstI-HF	Digestion of P157 with EcoRI-HF and PstI-HF	Digestion of P158 with EcoRI-HF and PstI-HF
	as expected	as expected	as expected	as expected	as expected	as expected

Sequencing of P157 and P160

Investigators: Louise, Jeff, Katrin

Aim of the experiment: Sequencing of QCIII product (EreB) and BBa_K801030 (SV40 NLS)

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
P157	Miniprep of transformation of QCIII product of P149 (QC - SDMIII - of EreB to remove forbidden PstI)	FR01002289 (VR)	FR01002290 (VF2)
P160	Miniprep of transformation of BBa_K801030 (SV40 NLS)	FR01002291 (VR)	FR01002292 (VF2)

Ligation of F29+F30

Investigator: Katrin

Aim of the experiment: Ligation of F29+F30

Procedure:

Ligation batch

volume	reagent
4.22 µl	F29 (23.7 ng/µl, 2110 bp))
10.48 µl	F30 (9,5 ng/µl, about 700 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
2.3 µl	ddH₂O
=20 µl	TOTAL

No negative control was prepared because there was not enough vector backbone left Ligation was performed at 37 °C overnight

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124 (Laccase)

Investigator: Louise, Katrin

Aim of the experiment: Removal of forbidden restiction site from P124 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P124 - 1:10 dilution)	need to get between 5 and 50 ng/µl
1 µl	1:10 dilution of O30 (for P124, 10 µM)
1 µl	1:10 dilution of O31 (for P124, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P124

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
		4 °C	hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Preparative digestion of pActin(F46) with XbaI & PstI

Investigator: Ingmar

Aim of the experiment: Preparative digestion of pActin(F46) in order to ligat this fragment in pSB1C3 later on.

Procedure:

Batch for preparative digestion of pActint(F46) with XbaI and PstI

volume	reagent
25 µl	F46 PCR product of pActin
5 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
1 µl	PstI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batchesand loaded on a 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 90 min.

The band was extracted by QIAquick Gel Extraction Kit, QIAGEN. The resulting fragment was named: F50

Ligation of F42+F50 (pActin in pSB1C3)

Investigator: Ingmar

Aim of the experiment: Ligation of F42+F50 (pActin in pSB1C3(RFC25).

Procedure:

Ligation batch

volume	reagent
1.37 µl	F42
13.13 µl	F50
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
2.5 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt)

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt).

Procedure:

The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 5 mL of LB-medium + 5 µL chloramphenicol(1000x).

3 colonies of each agar plate were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32

Investigator: Louise, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Saturday, June 1st

Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA were added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Transformation of E. coli XL1 blue with QCIII product of Laccase (SDMIII of P124)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with QCIII product of Laccase

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.

Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSB1C3), P164-166 (F42+F38, t35s in pSB1C3), P167-169 (F17+F40, PIF3 in pSB1C3)

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSC1C3) with EcoRI-HF and SpeI-HF, P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF and P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI

Procedure:

Analytical digestion of P161-163 (F39 + F41, npt-casette in pSC1C3), P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF

volume	reagent
2.5 µl	P161-P166
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI-HF
0.25 µl	SpeI-HF
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Analytical digestion of P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI

volume	reagent
2.5 µl	P167-P169
2 µl	CutSmart Buffer (10x)
0.25 µl	AgeI-HF
0.25 µl	XbaI
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder#	Digestion of P161 with EcoRI-HF and SpeI-HF	Digestion of P162 with EcoRI-HF and SpeI-HF	Digestion of P163 with EcoRI-HF and SpeI-HF	Digestion of P164 with EcoRI-HF and SpeI-HF	Digestion of P165 with EcoRI-HF and SpeI-HF	Digestion of P166 with EcoRI-HF and SpeI-HF	Digestion of P167 with AgeI-HF and XbaI	Digestion of P168 with AgeI-HF and XbaI	Digestion of P169 with AgeI-HF and XbaI
	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected

Sunday, June 2nd

Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F44 and F42+F32

Investigator: Andi

Aim of the experiment: Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F32 and F42+F32.

Procedure:

The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

4 colonies of each agar plate were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Week 7

Monday, June 3rd

Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10)

Investigator: Andi, Johanna, Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10).

Procedure:

Mastermix for analytical digestion of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10) with XbaI+PstI.

volume	reagent
18 µl	CutSmart Buffer (10x)
2.25 µl	EcoRI-HF
2.25 µl	SpeI-HF
135 µl	ddH2O
=157.5 µl	TOTAL

17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P170-P177)

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Analytical digestion of P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10):

volume	reagent
2.5 µl	P178-P181
2 µl	CutSmart Buffer (10x)
0.25 µl	NgoMIV
0.25 µl	PstI-HF
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder	Digestion of P170 with EcoRI-HF and PstI-HF	Digestion of P162 with EcoRI-HF and PstI-HF	Digestion of P163 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF	Digestion of P164 with EcoRI-HF and PstI-HF
	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible	as expected, insert too small to be visible

1 kbp ladder DNA ladder	Digestion of P142 with NgoMIV and PstI-HF	Digestion of P178 with NgoMIV and PstI-HF	Digestion of P179 with NgoMIV and PstI-HF	Digestion of P180 with NgoMIV and PstI-HF	Digestion of P181 with NgoMIV and PstI-HF
	Ctrl (before QuickChange III)	as expected, plasmid is only linearized, all NgoMIV sites are removed	as expected, plasmid is only linearized, all NgoMIV sites are removed	as expected, plasmid is only linearized, all NgoMIV sites are removed	as expected, plasmid is only linearized, all NgoMIV sites are removed

Tuesday, June 4th

Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag)

Investigators: Louise, Johanna

Aim of the experiment: Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode1	Barcode2
P162	Miniprep of ligation product F39 + F41 (npt-casette in pSB1C3)	FR01002331 (VR)	FR01002330 (VF2)
P165	Miniprep of ligation product F42+F38 (t35s in pSB1C3)		FR01002332 (VF2)
P168	Miniprep of ligation product F17+F40 (PIF3 in pSB1C3)		FR01002333 (VF2)
P179	Miniprep of ligation product Laccase QCIII (P142)	FR01002335 (VR)	FR01002334 (VF2)
P177	Miniprep of ligation product MiniMCSII F42+F43		FR01002336 (VF2)
P170	Miniprep of ligation product F17+F32 (SpyTag)		FR01002328 (VF2)

Preparative digestion and gelelectrophoresis of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI

Investigator: Louise, Johanna

Aim of the experiment: Preparative digestion of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI.

Procedure:

Batch for preparative digestion of P39(middle-linker) with PstI and AgeI-HF

volume	reagent
15 µl	Plasmid DNA P39
4 µl	CutSmart Buffer
1 µl	AgeI-HF(20 U/µl)
1 µl	PstI (20 U/µl)
19 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P20(GFP-Psb1C3) with NgoMIV and PstI

volume	reagent
20 µl	Plasmid DNA P20
4 µl	CutSmart Buffer
1 µl	NgoMIV (20 U/µl)
1 µl	PstI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of F45(pActin) with XbaI and PstI

volume	reagent
25 µl	Fragment DNA F45
5 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
1 µl	PstI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 90 min.

1 kbp DNA ladder	Digestion of P20 with NgoMIV & PstI = F53	Digestion of P39 with AgeI & PstI = F52	Digestion of F45 with XbaI & PstI = F51
	as expected, lower band was cut out	as expected, band was cut out	as expected, lower band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)

Investigator: Andi, Jeff

Aim of the experiment: Removal of forbidden restiction site SDM IV from P179 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P179 - 1:6 dilution)	need to get between 5 and 50 ng/µl
1 µl	1:10 dilution of O32 (for P179, 10 µM)
1 µl	1:10 dilution of O33 (for P179, 10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters - P179

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
		4 °C	hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Preparative digestion and gelelectrophoresis of P182 (Gensynthesis 1) with XbaI and AgeI-HF, P183 (Gensynthesis 2) with XbaI and AgeI-HF

Investigator: Andi, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P182 (Gensynthese 1) with XbaI and AgeI-HF, P183 (Gensynthese 2) with XbaI and AgeI-HF

Procedure:

Batch for preparative digestion of P182 with XbaI and AgeI-HF

volume	reagent
15 µl	Plasmid DNA P182
4 µl	CutSmart Buffer
1 µl	AgeI-HF(20 U/µl)
1 µl	XbaI (20 U/µl)
19 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P183 with XbaI and AgeI- HF

volume	reagent
15 µl	Plasmid DNA P183
4 µl	CutSmart Buffer
1 µl	AgeI-HF (20 U/µl)
1 µl	XbaI (20 U/µl)
19 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1.5% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 70 V for 60 min.

1 kbp DNA ladder	Digestion of Gensynthesis 1 with XbaI & AgeI	100 bp DNA ladder	Digestion of Gensynthesis 2 with XbaI & AgeI	1 kbp DNA ladder
	as expected, fragments (78 bp and 530 bp) were cut out		as expected, fragments (111 bp, 206 bp and 359 bp) were cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF

Investigator: Andi, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF

Procedure:

Batch for preparative digestion of P9 with XbaI and AgeI-HF

volume	reagent
15 µl	Plasmid DNA P9
4 µl	CutSmart Buffer
1 µl	AgeI-HF(20 U/µl)
1 µl	XbaI (20 U/µl)
19 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

The resulting fragments were named: ?????

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 70 V for 60 min.

Gelfragmentation was like expected, both bands were cut out (upper band = PhyB, lower band = pSB1C3 RFC25)

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase)

Investigator: Andi, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2x 10 µl of DNA was added seperately into 2x 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Transformation of E. coli XL1 blue with P39

Investigator: Louise, Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P39

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added seperately into 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Preparative digestion and gelelectrophoresis of P39 with AgeI & PstI, P20 with NgoMIV & PstI

Where is the snapshot of the gel =DDDD

Investigator: Johanna, Louise

Aim of the experiment:Preparative digestion of P39 with AgeI & PstI, P20 with NgoMIV & PstI to fusion Middle linker of P39 with GFP of P20.

Procedure:

Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.

volume	reagent
20 µl	P39
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
2 µl	AgeI-HF
2 µl	PstI
11.6 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.

volume	reagent
20 µl	P45
4 µl	NEBuffer 4
0.4 µl	BSA (100x)
2 µl	NgoMIV
2 µl	PstI
11.6 µl	ddH₂O
=40 µl	TOTAL

Incubation for 150 min at 37 °C.

Preparative gelelectrophoresis was performed at 90 V for 1 h.

File:FOTOFOTO.png

1 kbp ladder	P20 digested with NgoMIV&PstI (=F30)	P39 digested with NgoMIV&PstI (=F30)
	lower band was cut out	band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51

Investigator: Jeff, Andi, Johanna, Louise

Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.

Procedure:

Ligation batch for F54+F59 :

volume	reagent
2.2 µl	F54 (5.9 ng/µl, 90 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
13.3 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F55+F59:

volume	reagent
11.6 µl	F55 (6.4 ng/µl, 520 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
3.9 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F56+F59:

volume	reagent
3.3 µl	F56 (4.7 ng/µl, 110 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
12.2 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F57+F59:

volume	reagent
3.1 µl	F57 (9.1 ng/µl, 200 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
12.4 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F58+F59:

volume	reagent
4.4 µl	F58 (12.9 ng/µl, 400 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
11.1 µl	ddH₂O
=20 µl	TOTAL

Negative Control Ligation batch for F59:

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
15.5 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F52+F53:

volume	reagent
10 µl	F52 (9.9 ng/µl, XXX bp)
7 µl	F53 (12.2 ng/µl, XXX bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F42+F51:

volume	reagent
1.36 µl	F42 (XXX ng/µl, XXX bp)
15.64 µl	F51 (8.2 ng/µl, 1237 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Cycled ligation has been performed after following protocol in a thermocycler:

99 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

Lid temperature = 37 °C

Plating of received E. coli containing biobricks

Investigator: Jeff, Andreas

Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.

Operational sequence:

Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.

The biobricks were:

Name	Function
BBa_J61032	Alkaline Phosphatase
BBa_K426020	Self lysis device
BBa_K729004	Nuclease from S. Aureus
BBa_K365001	PIF6
BBa_K648011	RBS B0034 with cI repressor C0051 under the control of constitutive promoter J23113

Wednesday, June 5th

Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53

Investigator: Florian, Rosario, Louise, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics.

Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020)

Investigator: Rosario, Florian, Louise, Jeff

Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020).

Procedure:

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampicillin(1000x).

2 colonies of each plate were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.

Thursday, June 6th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011

Investigator: Ingmar

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
P39	P184
P39	P185
P20	P186
P20	P187
BBa_J61032	P188
BBa_J61032	P189
BBa_K426020	P190
BBa_K426020	P191
BBa_K729004	P192
BBa_K729004	P193
BBa_K365001	P194
BBa_K365001	P195
BBa_K648011	P196
BBa_K648011	P197
Genesynthesis 2	P198
Genesynthesis 2	P199
Genesynthesis 1	P200
Genesynthesis 1	P201

Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011)

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011).

Procedure:

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI-HF
0.25 µl	SpeI-HF
15 µl	ddH2O
2.5 µl	Plasmid
=20 µl	TOTAL

Mastermix (12x)

volume	reagent
24 µl	CutSmart Buffer (10x)
3 µl	EcoRI-HF
03 µl	SpeI-HF
180 µl	ddH2O
=210 µl	TOTAL

17.5 µl Mastermix + 2.5 µl Plasmid each

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V

1 kb Marker	P188 Alk.Phos.	P189 Alk.Phos.	P190 self lysis device	P191 self lysis device	P192 Nuclease	P193 Nuclease	P194 PIF6	P195 PIF6	P196 RBS	P197 RBS
	successful	successful	successful	successful	successful	successful	successful	successful	successful	successful

Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56

Procedure:

The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampecillin (1000x).

2 colonies of each agar plate were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overday.
The ligation of F42 and F51 was not successful and has to be repeated. One reason for the failure could be secondary structures of the promoter. Therefore the new ligation batch should be heated to 95°C for a while before adding the T4 ligase.

PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43)

Investigator: Jeff

Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43).

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P192: O64 (NucA_fw); P194: O42 (PIF6_fw))
1 µl	10 µM Reverse Primer (P192: O65 (NucA_rv); P194: O43 (PIF6_rv))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	1:1000 dilution of plasmid DNA (P192; P194)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The gradient PCR program was performed after following scheme with following conditions:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	55 °C	60 s
	68 °C	30 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

PCR product of P192 = F61; PCR product of 194 = F62

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)

Investigator: Jeff

Aim of the experiment: Removal of forbidden restiction site from P179 (Laccase).

Procedure: Quickchange - PCR

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid template (P179 - 1:10 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O32 (10 µM)
1 µl	1:10 dilution of O33 (10 µM)
18.5 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P179

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
		4 °C	hold

After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour

Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179

Investigator: Rosario, Andi, Jeff

Aim of the experiment: Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179.

Procedure:

4 µl of F61 or F62 or the DpnI digestion of the SDMIV product of P179 were mixed with 0.444 µl of DNA loading buffer (10x)

Analytical gelelectrophoresis was performed at 90 V for 1 h on a 1% agarose gel.

1 kbp DNA ladder	PCR product F61	PCR product F62	DpnI digestion of QCIV of P179
	successful	successful	successful

Ligation of F42+F51

Investigator: Ingmar

Aim of the experiment: Ligation of F42+F51.

Procedure:

Ligation batch for F42+F51:

volume	reagent
0.68 µl	F42 (73.5 ng/µl, 2070 bp)
15.64 µl	F51 (10.93 ng/µl, 1237 bp)
5.39 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.
The reaction batch was heated up to 95°C for 5 min before the enzyme and buffer were added.
The ligation was performed for two hours at room temperature.

Lid temperature = 37 °C

Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10) and DpnI digestion of the SDMIV product of P179 (Laccase)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10 )and DpnI digestion of the SDMIV product of P179 (Laccase).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sequencing of Biobricks P188/189 (alk.Phos., 1475 bp), P190/191 (self lysis device, 2910 bp), P192/193 (Nuclease, 561 bp), P194/195 (PIF6, 300 bp), P196/197 (RBS, 918 bp)

Investigators: Johanna

Aim of the experiment: Sequencing of the Biobrick MiniPreps

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM):

Label	Konz. ng/µl	Vol. BB µl	Vol H2O µl
P188	160.6	5	10
P189	123.6	7.5	7.5
P190	319.2	3	12
P191	127.0	10	5
P192	336.9	3	12
P193	230.6	4	11
P194	156.6	5	10
P195	197.4	4	11
P196	90.3	10	5
P197	230.2	4	11

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P188	alk.Phos.	FR01002327 (O3)	FR01002326 (O4)
P189	alk.Phos.	FR01002325 (O3)	FR01002320 (O4)
P190	self lysis device	FR01002319 (O3)	FR01002318 (O4)
P191	self lysis device	FR01002317 (O3)	FR01002316 (O4)
P192	Nuclease	FR01002315 (O3)	- (O4)
P193	Nuclease	FR01002314 (O3)	- (O4)
P194	PIF6	FR01002313 (O3)	- (O4)
P195	PIF6	FR01002312 (O3)	- (O4)
P196	RBS	FR01002324 (O3)	FR01002323 (O4)
P197	RBS	FR01002322 (O3)	FR01002321 (O4)

Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII)

Investigator: Rosario, Andi, Florian, Johanna, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII.

Procedure:

Batch for preparative digestion of F61 (Thermonuclease) with XbaI & AgeI:

volume	reagent
25 µl	PCR product F61
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of F62 (PIF6) with XbaI & AgeI:

volume	reagent
25 µl	PCR product F62
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P165 (t35S) with XbaI:

volume	reagent
20 µl	Plasmid DNA P165
4 µl	CutSmart Buffer
1 µl	XbaI (20 U/µl)
15 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of F31 (miniMCSII):

volume	reagent
10 µl	Oligohybridization F31
5 µl	CutSmart Buffer
1 µl	SpeI-HF (20 U/µl)
34 µl	ddH₂O
=40 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on an 2% agarose gel for preparative gelelectrophoresis (1% agarose gel for digestion of P165).

Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	Digestion of F31 with SpeI-HF	Digestion of F61 with XbaI & AgeI-HF	Digestion of F62 with XbaI & AgeI-HF
	Fragment length as expected; band was cut out	Fragment length as expected; band was cut out	Fragment length as expected; band was cut out

1 kbp DNA ladder	Digestion of P165 with XbaI
	Fragment length as expected; band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Dephosphorylation of preparative digestion of P165 with FastAP

Investigator: Rosario, Jeff

Aim of the experiment: Dephosphorylation of preparative digestion of P165 with FastAP to prevent re-ligation.

Procedure:

After preparative digestion, gelelectrophoresis and gelextraction of P165, the digestion product was purified with QIAquick PCR Purification Kit, Qiagen.

Batch for the dephosphorylation of digestion product of P165

volume	reagent
30 µl	Digestion product of P165
4 µl	10X Fast AP buffer
2 µl	FastAP Thermosensitive Alkaline Phosphatase
4 µl	ddH₂O
=40 µl	TOTAL

The reaction batch was spun briefly and incubated at 37 °C for 10 min.

The reaction was stopped by heating to 75 °C for 5 min.

The dephosphorylized digestion product of P165 product was purified with QIAquick PCR Purification Kit, Qiagen.

The resulting product was labelled as F63

Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51

Investigator: Jeff, Rosario

Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.

Procedure:

Ligation batch for F59+F65 (Thermonuclease in pSB1C3 RFC25):

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
4.56 µl	F65 (14.1 ng/µl, 447 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
10.94 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F59+F66:

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
3.78 µl	F66 (11.3 ng/µl, 297 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
11.72 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F63+F64:

volume	reagent
12.05 µl	F63 (8.3 ng/µl, 2287 bp)
4.95 µl	F64 (4.3 ng/µl, 22 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

Ligation was performed at 16 °C overnight.

Friday, June 7th

Picking of QC IV Laccase

Investigator: Johanna

Aim of the study: Picking of QC IV Laccase (concentrated) and QC IV Laccase (diluted)

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)

3 colonies were picked for every Transformation product.

Preparation of Knop-Medium for Moss

Investigator: Andi

Aim of the experiment: Preparation of Knop-Medium for Moss

Procedure:

All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
Volume of prepared Medium: 2.6L
10 ml of each stock were converted into a 5L Erlenmeyer flask and filled up to 2.6L with destilled water (ELGA); 32.5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH/NaOH
200ml of the prepared medium was filled into three 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium; the rest (2L) was left in the 5L EM flask
All flasks have been autoclaved

Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
F52+F53 I	P202
F52+F53 II	203
F59+F54 I	P204
F59+F54 II	P205
F59+F55 I	P206
F59+F55 II	P207
F59+F56 I	P208
F59+F56 II	P209
F59+F57 I	P210
F59+F57 II	P211
F59+F58 I	P212
F59+F58 II	P213

Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of ligation product were added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of each tube were plated on agarose plates containing antibiotics (CamR).

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sequencing of P204 (Ligation Product F59+F54), P206 (Ligation Product F59+F55), P208 (Ligation Product F59+F56), P210 (Ligation Product F59+F57), P212 (Ligation Product F59+F58)

A monster is born!

Investigators: Louise

Aim of the experiment: Sequencing of Ig-Kappa (P204), Nanoluciferase (P206), SigP (P208), Transmembrand. (P210) and SpyCatcher (P212) Ligations in pSB1C3 RFC25

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Name	Barcode (O3)
P204	Ig-Kappa-SigP + pSB1C3 RFC25	FR01002304
P206	Nanoluciferase + pSB1C3 RFC25	FR01002303
P208	SERK-SigP + pSB1C3 RFC25	FR01002302
P210	Transmembrand + pSB1C3 RFC25	FR01002301
P212	SpyCatcher + pSB1C3 RFC25	FR01002300

Analytical digestion and gelelectrophoresis of P202-P213

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P202-P213

Procedure:

Mastermix for analytical digestion of P202-P213 with XbaI+AgeI

volume	reagent
30 µl	CutSmart Buffer (10x)
3.75 µl	XbaI
3.75 µl	SpeI-HF
225 µl	ddH2O
=262.5 µl	TOTAL

17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P202-P203)

Incubation for 90 min at 37 °C.

Inaddition, P203 was digested for 4 and for 8 seconds in the microwave, a negative control was incubated at room temperature

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp ladder DNA ladder	Digestion of P202 with XbaI und AgeI (GFP + linker)	Digestion of P203 with XbaI und AgeI (GFP + linker)	Digestion of P204 with XbaI und AgeI	Digestion of P205 with XbaI und AgeI	Digestion of P206 with XbaI und AgeI	Digestion of P207 with XbaI und AgeI	Digestion of P208 with XbaI und AgeI	Digestion of P209 with XbaI und AgeI	Digestion of P210 with XbaI und AgeI
	as expected	as expected	not as expected	not as expected	not as expected	not as expected	not as expected	not as expected	as expected (SERK-TMD)

1 kbp ladder DNA ladder	Digestion of P211 with XbaI und AgeI	Digestion of P212 with XbaI und AgeI	Digestion of P213 with XbaI und AgeI	Digestion of P203 with XbaI und AgeI (microwave 4 seconds)	Digestion of P203 with XbaI und AgeI (microwave 8 seconds)	Digestion of P203 with XbaI und AgeI (control)
	as expected (SERK-TMD)	not as expected	as expected	interesting	interesting	interesting

Saturday, June 8th

Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
QC IV Laccase clone 1	P214
QC IV Laccase clone 2	P215
QC IV Laccase clone 3	P216
QC IV Laccase clone 4	P217
QC IV Laccase clone 5	P218
QC IV Laccase clone 6	P219

Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)

Investigator: Rosario

Aim of the study: Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3) Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)

2 colonies were picked for every Transformation product.

Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67)

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67) with AgeI/SpeI

Procedure:

Batch for preparative digestion of F47 (pActin, fragment named F68) with XbaI/SpeI

volume	reagent
25 µl	PCR product F47
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI

volume	reagent
20 µl	plasmid P202
5 µl	CutSmart Buffer
1 µl	SpeI-HF(20 U/µl)
1 µl	NgoMIV (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI

volume	reagent
20 µl	Plasmid DNA P210
5 µl	CutSmart Buffer
1 µl	AgeI-HF (20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis

Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of F47 (pActin, fragment named F68) with XbaI/SpeI
	Fragment length as expected; band was cut out

1 kbp DNA ladder	digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
	Fragment length as expected; band was cut out

1 kbp DNA ladder	digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
	Fragment length as expected; band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentration determined with Nanodrop:

F68	pActin	10,4 ng/µl
F69	GFP+linker	13,0 ng/µl
F67	SERK-TMD	14,3 ng/µl

Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI (backbones for ligation)

Procedure:

Batch for preparative digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI

volume	reagent
20 µl	Plasmid P7
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI

volume	reagent
20 µl	plasmid P8
5 µl	CutSmart Buffer
1 µl	SpeI-HF(20 U/µl)
1 µl	NgoMIV (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

before gelelectrophoresis, P7 was dephosphorylated

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 0.5% agarose gel for preparative gelelectrophoresis

Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI	digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
	Fragment lengths as expected; lower band was cut out	Fragment lengths as expected; lower band was cut out

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentration determined with Nanodrop:

F75	dephosphorylated Backbone (P7)	39,4 ng/µl
F76	dephosphorylated Backbone (P8)	52,0 ng/µl

Dephosphorylation of F75

Investigator: Jeff, Katrin

Aim of the experiment: Dephosphorylation of cut vector pSB1C3 prevent re-ligation

Procedure:

the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer

Batch for the dephosphorylation of F75

volume	reagent
30 µl	Plasmid DNA F75 from digestion
3 µl	10X Fast AP buffer
1.3 µl	FastAP Thermosensitive Alkaline Phosphatase
=34.3 µl	TOTAL

the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
the reaction was stopped by heating to 75 °C for 5 minutes

Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI

Investigator: Jeff, Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI

Procedure:

Batch for preparative digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI

volume	reagent
20 µl	Plasmid P7
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII

volume	reagent
20 µl	Plasmid P7
5 µl	CutSmart Buffer
1 µl	XbaI(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Incubation for 3 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 2% agarose gel for preparative gelelectrophoresis

Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI	digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
	Fragment lengths as expected; the two lower bands were cut out (upper:F70, lower: F71)	Fragment lengths as expected; all bands were cut out (upper: F72, middle: F73, lower: F74)

Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentrations measured with Nano-Drop

Fragment	Name	Length	Concentration
F70	Nanoluciferase	510 bp	9.2 ng/µl
F71	IgKappa-SigP	67 bp	3.1 ng/µl
F72	SpyCatcher	339 bp	10.8 ng/µl
F73	SERK-TMD	186 bp	8.6 ng/µl
F74	SERK-SigP	100 bp	4.8 ng/µl

Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)

Investigator: Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)

Procedure:

Mastermix for analytical digestion of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)

17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P214-219, P179)

Incubation for 90 min at 37 °C.

digestion for 4 x 10 seconds in the microwave (600 watt), with two minute breaks in between the microwaving

Analytical gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of P214 (Miniprep of Laccase QC IV clone 1) with NgoMVI/AgeI	digestion of P215 (Miniprep of Laccase QC IV clone 2) with NgoMVI/AgeI	digestion of P216 (Miniprep of Laccase QC IV clone 3) with NgoMVI/AgeI	digestion of P217 (Miniprep of Laccase QC IV clone 4) with NgoMVI/AgeI	digestion of P218 (Miniprep of Laccase QC IV clone 5) with NgoMVI/AgeI	digestion of P219 (Miniprep of Laccase QC IV clone 6) with NgoMVI/AgeI	digestion of P179 (Miniprep of ligation product Laccase QCIII clone 2) with NgoMVI/AgeI
	as expected	as expected	as expected	as expected	as expected	as expected	as expected

Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix)

Investigator: Jeff

Aim of the experiment: Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix).

Procedure:

volume	reagent
2.54 µl	F75 (39.4 ng/µl, ? bp)
8.30 µl	F69 (13.0 ng/µl, ? bp)
6.16 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

volume	reagent
7.00 µl	F67 (14.3 ng/µl, ? bp)
7.62 µl	F69 (13.0 ng/µl, ? bp)
2.38 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Incubation: 1 h 22°C

Transformation of E. coli XL1 blue with F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F75+F69(Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Sunday, June 9th

Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64

Investigator: Katrin

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
F59+F65 clone 1	P220
F59+F65 clone 2	P221
F59+F66 clone 1	P222
F59+F66 clone 2	P223
F59+F56 clone 1	P224
F59+F56 clone 2	P225
F59+F55 clone 1	P226
F59+F55 clone 2	P227
F59+F57 clone 1	P228
F59+F57 clone 2	P229
F59+F58 clone 1	P230
F59+F58 clone 2	P231
F63+F64	P232
F42+F51	P233
F59+F54 clone 1	P234
F59+F54 clone 2	P235

Analytical digestion and gelelectrophoresis of P220-P235 ((P220-231, P233-P235 with XbaI/SpeI, P232 with MfeI/Pst1)

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P220-P235 (Minipreps)

Procedure:

Mastermix for analytical digestion of P220-231, P233-P235 with XbaI, SpeI

volume	reagent
34 µl	CutSmart Buffer
4.25 µl	XbaI(20 U/µl)
4.25 µl	SpeI-HF (20 U/µl)
255 µl	ddH₂O
=266.5 µl	TOTAL

17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA

reaction batch for analytical digestion of P232 with MfeI/PstI

volume	reagent
2.5 µl	P232
2 µl	CutSmart Buffer
0.25 µl	MfeI(20 U/µl)
0.25 µl	PstI-HF(20 U/µl)
15 µl	ddH₂O
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min
for P224, P225, P228, P229, P230, P231, P234, P235,a 2% agarose gel was used, gelelectrophoresis was performed at 90 V for 45 min

1 kbp DNA ladder	100 bp DNA ladder	digestion of P224 (SERK-SigP, clone 1) with XbaI, SpeI	digestion of P225 (SERK-SigP, clone 2) with XbaI, SpeI	digestion of P228 (SERK TMD, clone 1) with XbaI, SpeI	digestion of P229 (SERK TMD, clone 2) with XbaI, SpeI	digestion of P230 (SpyCatcher, clone 1) with XbaI, SpeI	digestion of P231 (SpyCatcher, clone 2) with XbaI, SpeI	digestion of P234 (Ig-K-SigP, clone 1) with XbaI, SpeI	digestion of P235 (Ig-K-SigP, clone 2) with XbaI, SpeI
		not as expected	not as expected	as expected	as expected	as expected	as expected	fragment not visible due to small size	not as expected

1 kbp DNA ladder	digestion of P220 (Thermonuclease, clone 1) with XbaI, SpeI	digestion of P221 (Thermonuclease, clone 2) with XbaI, SpeI	digestion of P222 (PIF6, clone 1) with XbaI, SpeI	digestion of P223 (PIF6, clone 2) with XbaI, SpeI	digestion of P226 (Luciferase, clone 1) with XbaI, SpeI	digestion of P227 (Luciferase, clone 2) with XbaI, SpeI	digestion of P232 (miniMCS) with MfeI/PstI	digestion of P233 (pActin) with XbaI, SpeI
	not as expected	not as expected	as expected	as expected	as expected	as expected	not as expected	as expected

Picking of F67+F69 (SERK-TMD+linker GFP)

Investigator: Katrin

Aim of the study: Picking of F67+F69 (SERK-TMD+linker GFP)

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (5 µl CamR and 5 ml LB-medium)

3 colonies were picked for every Transformation product.

There were no colonies on the plates for ligations F68+F75 (pActin, pSB1C3),

(probably due to dephosphorylation)

Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI

Investigator: Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI

Procedure:

Batch for preparative digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI

volume	reagent
20 µl	P220
5 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P160 (NLS, fragment named F77) with AgeI/SpeI

volume	reagent
20 µl	plasmid P160
5 µl	CutSmart Buffer
1 µl	SpeI-HF(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI

volume	reagent
20 µl	plasmid P40
5 µl	CutSmart Buffer
1 µl	SpeI-HF(20 U/µl)
1 µl	NgoMIV (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P7 (PhyB, fragment named F78) with AgeI/SpeI

volume	reagent
20 µl	plasmid P40
5 µl	CutSmart Buffer
1 µl	SpeI-HF(20 U/µl)
1 µl	AgeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches containing P220 and P40 after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis

the vector backbones where only a few base pairs were cut out were purified via PCR-purification

Preparative gelelectrophoresis was performed at 90 V for 60 min.

digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI	1 kbp DNA ladder	digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
not as expected, band was discarded		as expected, lower band (108 bp) was cut out

the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)

Investigator: Katrin

Aim of the study: PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)

in order to get rid of the few base pairs that were cut out during digestion, the digestion batches were purified

with QIAquick PCR Purification Kit, Qiagen

the yiedls were 56.2 ng/µl for F77 and 277,6 ng/µl for F78

Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Investigator: Katrin Aim of the experiment: Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Procedure:

Ligation batch for F78+F79(PhyB+linker)

volume	reagent
0.36 µl	F78 (277.6 ng/µl, 4807 bp)
1.77 µl	P79 (3.8 ng/µl, 108 bp)
14.87 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F59+F71 (pSB1C3+Ig-K)

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
3.12 µl	P71 (3.1 ng/µl, 67 bp)
12.38 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K) and negative controls F78 and F59

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

PCR of P192 (Thermonuclease, O64 & O65)

Investigator: Katrin

Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65)

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer O64 (NucA_fw)
1 µl	10 µM Reverse Primer O65 (NucA_rv)
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	1:1000 dilution of plasmid DNA (P192)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The gradient PCR program was performed after following scheme with following conditions:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	55 °C	60 s
	68 °C	30 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

Week 8

Monday, June 10rd

Analytical Gel of PCR of Thermonuclease (P192)

Investigator: Florian

Aim of the study: Analysis of PCR of Thermonuclease (P192); supposed letgth: 447 bp

Lane:	DNA ladder 1kb	P192 PCR product	DNA ladder 1kb
Result:	-	as expected	-

PCR Purification of Thermonuclease (P192)

Investigator: Andi

Aim of the study: PCR Purification of Thermonuclease (P192)

After the PCR the batch was purified with QIAquick PCR Purification Kit, Qiagen
Eluation in 35 µl EB buffer -> F80

Miniprep of F67+F69 Ligation (Linker+GFP in Suffix of TMD)

Investigator: Johanna

Aim of the study: Preparation of DNA of the F67+F69 Ligation from 09.06.13 (Linker+GFP in Suffix of TMD)

Procedure: according to MiniPrep Kit QIAGEN; measurement of concentration (NanoDrop)

Eluation in 35 µl EB buffer each

Plamsid	Concentration
P236	108.0 ng/µl
P237	116.0 ng/µl
P238	165.4 ng/µl

Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)

Investigator: Johanna

Aim of the study: Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)

3 colonies were picked for every Transformation product.

Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)

Investigator: Johanna

Aim of the study: Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)

Procedure:Eurofins SmartSeq Kit protocol (15 µl plasmid DNA (50 - 100 ng), 2 µl sequencing primer).

#	P150	P233	P213	P236	P237
Name	FluA	pActin	Spycatcher	Linker+GFP+TMD clone1	Linker+GFP+TMD clone2
O3 (forward)	FR01002308	FR01002307	FR01002305	FR01002299	FR01002298
O4 (reverse)	n.a.	FR01002306	n.a.	FR01002297	FR01002311

PCR of BPul Laccase P219 + Analytical Gelelectrophoresis

Investigator: Andi, Johanna

Aim of the experiment: PCR of Bpul Laccase P219.

Procedure:

Operational sequence:

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P219:O24 (Bpul_for))
1 µl	10 µM Reverse Primer (P143:O25 (Bpul_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P219)- 1:1000 dilution
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	55 °C	60 s
	68 °C	90 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting fragment was called F82
Further on an analytif Gelelectrophoresis was performed to be sure that the expected fragment was amplified.

DNA ladder 1kb

P219 PCR product

As we can see, the fragment has the expected length of about 1600 BP.

Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI

Investigator: Florian, Johanna

Aim of the experiment: Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.

Procedure:

Batch for preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.

volume	reagent
25 µl	F80
5 µl	CutSmart Buffer
1 µl	AgeI (20 U/µl)
1 µl	XbaI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Incubation for 2.5 h at 37 °C.

The digested fragment was purified via PCR-purification (Qiagen-kit)

A yield of 26.2 ng/µl could be measured for F81

Ligation of F59 + F81 (Thermonuclease in pSB1C3)

Investigator: Florian, Johanna

Aim of the experiment: Ligation of F59 + F81 (Thermonuclease in pSB1C3).

Procedure:

Ligation batch for F59 + F81 (Thermonuclease in pSB1C3)

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
2.45 µl	P81 (26.2 ng/µl, 447 bp)
13.55 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
0.5 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3) and the negative control F59.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Addition of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspensions were plated on plates containing antibiotics (CamR).

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).

Preparative digestion and purification of F82 (PCR product of Bpul Laccase) with XbaI/AgeI-HF and PCR purification kit of Qiagen

Investigator: Andi, Johanna

Aim of the experiment: Preparative digestion and purification of F82 (Bpul Laccase) with XbaI+AgeI and PCR purification kit of Qiagen

Procedure:

Batch for preparative digestion of F82 with XbaI/AgeI

volume	reagent
25 µl	F82
5 µl	CutSmart Buffer
1 µl	AgeI-HF(20 U/µl)
1 µl	XbaI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Incubation for 2.5 h at 37 °C.

the digested fragment was purified via PCR-purification

the resulting fragment was called F83 (F82 digested with XbaI+AgeI)

Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)

Investigator: Andi, Johanna

Aim of the experiment: Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)

Procedure:

Ligation batch for F83+F59 (psB1C3+Bpul Laccase)

volume	reagent
15.5 µl	F83 (9.2 ng/µl, 1600 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.
The ligation was performed over night at 16°C.

Tuesday, June 11th

Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83

Investigator: Louise, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa)

Investigator: Louise, Johanna

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
F78+F79 (PhytochromB +Linker) clone 1	P239
F78+F79 (PhytochromB +Linker) clone 2	P240
F78+F79 (PhytochromB +Linker) clone 3	P241
F59+F71 (IgKappa) clone 4	P242
F59+F71 (IgKappa) clone 5	P243
F59+F71 (IgKappa) clone 6	P244

Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI

Investigator: Louise, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	AgeI
0.25 µl	XbaI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P293-P244)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min
For P242-P244 a 2% agarose gel was used and for P293-P241 a 0.5% agarose gel was used.

Lane:	Digestion of P242 (IgK) with XbaI & AgeI, clone 1	Digestion of P243 (IgK) with XbaI & AgeI, clone 2	Digestion of P244 (IgK) with XbaI & AgeI, clone 3	DNA ladder 100bp
Result:	as expected	as expected	as expected	-

Lane:	Digestion of P239 (PhyB+Linker) with XbaI & AgeI, clone 1	Digestion of P240 (PhyB+Linker) with XbaI & AgeI, clone 2	Digestion of P241 (PhyB+Linker) with XbaI & AgeI, clone 3	DNA ladder 1kb
Result:	failed	as expected	as expected	-

Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI

Investigator: Johanna, Rosario, Flo

Aim of the experiment: Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI

Procedure:

Batch for preparative digestion of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI

volume	reagent
20 µl	P204
5 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P157 (EreB, fragment named F86) with NgoMIV/SpeI

volume	reagent
20 µl	P157
5 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI

volume	reagent
20 µl	P208
5 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P213 (Spycatcher, fragment named F88) with NgoMIV/SpeI

volume	reagent
20 µl	P213
5 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Batch for preparative digestion of P170 (Spytag, frament named F89) with NgoMIV/SpeI

volume	reagent
20 µl	P170
5 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Incubation for 2.5 h at 37 °C.

5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel (P204, P157, P208, P213) and 2 % agarose gel (P170) for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of P204 (IgKappa-SigP) with AgeI/SpeI	100 bp DNA ladder (Generuler)	digestion of P157 (EreB) with NgoMIV/SpeI
	as expected, lower band was cut out		as expected, lower band was cut out

1 kbp DNA ladder	digestion of P208 (Nanoluc) with NgoMIV/SpeI	100 bp DNA ladder (Generuler)	digestion of P213 (Spycatcher) with NgoMIV/SpeI
	as expected, lower band was cut out		as expected, lower band was cut out

100 bp DNA ladder (Generuler)	digestion of P170 (Spytag) with NgoMIV/SpeI
	as expected, lower band was cut out

the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)

Investigator: Johanna

Aim of the experiment: Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)

Procedure:

Ligation batch for F90+F86 (IgKappa-SigP + EreB)

volume	reagent
5.7 µl	F86 (31.0 ng/µl, 1254 bp)
6.5 µl	F90 (15.3 ng/µl, 2144 bp)
4.8 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F90+F87 (IgKappa-SigP + Nanoluciferase)

volume	reagent
7.0 µl	F87 (10.2 ng/µl, 510 bp)
6.5 µl	F90 (15.3 ng/µl, 2144 bp)
3.5 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F90+F88 (IgKappa-SigP + SpyCatcher)

volume	reagent
6.3 µl	F88 (8.7 ng/µl, 339 bp)
6.5 µl	F90 (15.3 ng/µl, 2144 bp)
4.2 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F90+F89 (IgKappa-SigP + SpyTag)

volume	reagent
10.5 µl	F89 (8.7 ng/µl, 39 bp)
6.5 µl	F90 (15.3 ng/µl, 2144 bp)
0.0 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control of F 90 (IgKappa-SigP) was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.

PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!

Investigator: Andi

Aim of the experiment: PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!

Procedure:

Operational sequence:

The PCR for P143 (pActin) was performed three times under different conditions

First batch - PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P143:O62 (pAct_for))
1 µl	10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P143)- 1:1000 dilution
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Second batch - PCR reaction mixture

volume	reagent
10 µl	5x OneTaq GC Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P143:O62 (pAct_for))
1 µl	10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P143)- 1:1000 dilution
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Third batch - PCR reaction mixture

volume	reagent
10 µl	5x Herculase Fusion Polymerase reaction buffer
2.5 µl	10 mM dNTPs
1.25 µl	10 µM Forward Primer (P143:O62 (Bpul_for))
1.25 µl	10 µM Reverse Primer (P143:O63 (Bpul_rev))
1 µL	Herculase II Fusion Polymerase
2 µl	Plasmid DNA (P143)- 1:1000 dilution
0.5 µl	DMSO
35.75 µL	ddH₂O Water
=50 µL	TOTAL

The content of all batches has been mixed with a pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	51 °C	60 s
	68 °C	90 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

The PCR for P192(Thermonuclease) was performed three times under different conditions

First batch - PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Reaction Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl	10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P192)- 1:1000 dilution
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Second batch - PCR reaction mixture

volume	reagent
10 µl	5x OneTaq GC Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl	10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P192)- 1:1000 dilution
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Third batch - PCR reaction mixture

volume	reagent
10 µl	5x Herculase II DNA Fusion Polymerase reaction Buffer
2.5 µl	10 mM dNTPs
1.25 µl	10 µM Forward Primer (P192:O64 (NucA_fw))
1.25 µl	10 µM Reverse Primer (P192:O65(NucA_rev))
1 µL	Herculase II Fusion DNA Polymerase
3 µl	Plasmid DNA (P192)- 1:1000 dilution
0.5 µL	DMSO
30.5 µL	ddH₂O Water
=50 µL	TOTAL

The PCR program of the Thermonuclease (P192) was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	54 °C	60 s
	68 °C	40 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

Mix the content of the batches with pipette

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The concentrations of all PCR products have been measured

P143 - Batch #1 - PCR product - 5.9 ng/µl

P143 - Batch #2 - PCR product - 40.5 ng/µl

P143 - Batch #3 - PCR product - 12.4 ng/µl

P192 - Batch #1 - PCR product - 39.7 ng/µl

P192 - Batch #2 - PCR product - 23.1 ng/µl

P192 - Batch #3 - PCR product - 82.5 ng/µl

As we can see, the highest concentration of PCR product seems to be in P143 - Batch #2 and P192 - Batch #3.

Further on an analytic Gelelectrophoresis was performed to be sure that the expected fragment was amplified.

DNA ladder 1kb

P143 - Batch #1 - PCR product

P143 - Batch #2 - PCR product

P143 - Batch #3 - PCR product

P192 - Batch #1 - PCR product

P192 - Batch #2 - PCR product

P192 - Batch #3 - PCR product

As we can see, the fragment has the expected length of about 1600 BP referring to the PCR product of P143 and about XXX BP referring to the product of P192.
Further on the analytical gel confirms the output of the nano drop.

Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI

Investigator: Andi

Aim of the experiment: Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI

Procedure:

Batch for preparative digestion of PCR reaction batch #2 of P143 (pActin) with XbaI/PstI.

volume	reagent
24 µl	PCR reaction batch #2 of P143
5 µl	CutSmart Buffer
1 µl	PstI #1 (20 U/µl)
1 µl	PstI #2 (20 U/µl)
1 µl	XbaI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

I used 1 µl of both PstI batches because one seems to be broken
Incubation for 2.5 h at 37 °C.
The digested fragment was purified via PCR-purification (Qiagen-kit)
A yield of 16.9 ng/µl could be measured for the resulting F84

Batch for preparative digestion of PCR reaction batch #3 of P192 (Thermonuclease) with XbaI/AgeI-HF.

volume	reagent
24 µl	PCR reaction batch #2 of P143
5 µl	CutSmart Buffer
1 µl	AgeI-HF (20 U/µl)
1 µl	XbaI (20 U/µl)
19 µl	ddH₂O
=50 µl	TOTAL

Incubation for 2.5 h at 37 °C.
The digested fragment was purified via PCR-purification (Qiagen-kit)
A yield of 46.2 ng/µl could be measured for the resulting F85

Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) and F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI and F59 (psB1C3 digested with AgeI-HF + XbaI)

Investigator: Andi

Aim of the experiment: Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) with F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI-HF) with F59 (psB1C3 digested with AgeI-HF/XbaI)

Procedure:

Ligation batch for F84+F42 (psB1C3+pActin)

volume	reagent
12.6 µl	F84 (16.9 ng/µl, 1500 bp)
1.4 µl	F42 (73.5 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
4 µl	ddH2O
=20 µl	TOTAL

Ligation batch for F85+F59 (psB1C3+Thermonuclease)

volume	reagent
1.6 µl	F85 (46.2 ng/µl, 500 bp)
1.5 µl	F59 (66.7 ng/µl, 2100 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
13.9 µl	ddH2O
=20 µl	TOTAL

Negative control was also prepared for both batches. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.
The ligation was performed over night at 16°C.

Transformation of E. coli XL1 blue with plasmids P157, P208 and P213

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P157, P208 and P213.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Wednesday, June 12th

Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chlorampenicol plates.

Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI

Investigator: Louise, Florian

Aim of the experiment: Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI

Procedure:

Batch for preparative digestion of P240 (PhyB + Linker) with AgeI/SpeI

volume	reagent
20 µl	P240
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P244 (IgKappa) with AgeI/SpeI

volume	reagent
20 µl	P244
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P197 (XylE) with NgoMIV/SpeI

volume	reagent
20 µl	P197
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P150 (FluA) with NgoMIV/SpeI

volume	reagent
20 µl	P150
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P126 (N-TEV) with AgeI/SpeI

volume	reagent
20 µl	P126
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P127 (C-TEV) with NgoMIV/SpeI

volume	reagent
20 µl	P127
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P168 (PIF3) with NgoMIV/SpeI

volume	reagent
20 µl	P168
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P222 (PIF6) with NgoMIV/SpeI

volume	reagent
20 µl	P222
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis

Preparative gelelectrophoresis was performed at 90 V for 60 min.

1 kbp DNA ladder	digestion of P126 (N-TEV) with AgeI/SpeI	digestion of P127 (C-TEV) with NgoMIV/SpeI	digestion of P150 (FluA) with NgoMIV/SpeI
	as expected, upper band was cut out	as expected, lower band was cut out	as expected, lower band was cut out

1 kbp DNA ladder	digestion of P168 (PIF3) with NgoMIV/SpeI	digestion of P197 (XylE) with NgoMIV/SpeI	digestion of P222 (PIF6) with NgoMIV/SpeI
	as expected, lower band was cut out	as expected, lower band was cut out	as expected, lower band was cut out

100 bp DNA ladder (Generuler)	digestion of P244 (IgKappa) with AgeI/SpeI	digestion of P240 (PhyB + Linker) with AgeI/SpeI	1 kbp DNA ladder
	as expected, upper band was cut out	as expected, upper band was cut out

the DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN.

the fragments received the following names:

P126 (N-TEV)	F93
P127 (C-TEV)	F94
P150 (FluA)	F95
P168 (PIF3)	F96
P197 (XylE)	F97
P222 (PIF6)	F98
P240 (PhyB + Linker)	F91
P244 (IgKappa)	F92

Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension were plated on chloramphenicol plates.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.

Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3)

Investigator: Jeff

Aim of the study: Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3).

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)

2 colonies were picked for every ligation product and 1 colony was picked for every transformation product.

Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Investigator: Jeff, Flo, Louise, Christopher

Aim of the experiment: Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Procedure:

Ligation batch for F92+F89 :

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2153 bp)
16.14 µl	F89 (6.8 ng/µl, 39 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
0.0 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F92+F87 :

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2153 bp)
7.0 µl	F87 (10.2 ng/µl, 510 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
9.14 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F92+F86:

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2153 bp)
5.7 µl	F86 (31.0 ng/µl, 1260 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
10.44 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F92+F88 :

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2153 bp)
5.4 µl	F88 (8.7 ng/µl, 339 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
10.74 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F92+F97:

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2153 bp)
6.6 µl	F97 (19.4 ng/µl, 918 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
9.5 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F90+F97:

volume	reagent
6.5 µl	F90 (15.3 ng/µl, 2144 bp)
6.6 µl	F97 (19.4 ng/µl, 918 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
3.9 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F91+F94:

volume	reagent
0.63 µl	F91 (159 ng/µl, 4129 bp)
4.6 µl	F94 (5.6 ng/µl, 354 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
11.77 µl	ddH₂O
=20 µl	TOTAL

Ligation batch for F93+F79:

volume	reagent
3.6 µl	F93 (27.4 ng/µl, 2440 bp)
3.5 µl	F79 (3.8 ng/µl, 108 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
9.9 µl	ddH₂O
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

The ligation was performed at room temperature for 1 hour

Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls

Investigator: Jeff, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Thursday, June 13th

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Investigator: Andi

Aim of the experiment: Removal of forbidden restriction site from P251 (Laccase) and P163 (NPT-Cassette).

Procedure: Quickchange - PCR

Reaction batch for P219 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P251 - 1:20 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O74 (10 µM)
1 µl	1:10 dilution of O75 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P163 - 1:60 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O70 (10 µM)
1 µl	1:10 dilution of O71 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P250 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P250 - 1:20 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O74 (10 µM)
1 µl	1:10 dilution of O75 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P217 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P217 - 1:100 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O74 (10 µM)
1 µl	1:10 dilution of O75 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
		4 °C	hold

After the PCR, the Quickchange product was stored in the fridge.

PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8) and analytical gel electrophoresis

Investigator: Louise, Johanna, Christopher

Aim of the experiment: PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8).

Procedure:

Operational sequence:

PCR reaction mixture for AlcR

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (O78 (AlcR_fw))
1 µl	10 µM Reverse Primer (O79 (AlcR_rv))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P61)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

PCR reaction mixture for IRES

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer (O68 (IRES_fw))
1 µl	10 µM Reverse Primer (O69 (IRES_rv))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P61)
35.75 µL	ddH₂O Water
=50 µL	TOTAL

Mix with pipette

The gradient PCR program was performed after following scheme with following conditions (T_m=56 °C; ΔG=4 °C; AlcR in row 11(=60 °C); IRES in row 1 (=52.0 °C)):

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	T_m=56 °C; ΔG=4 °C	60 s
	68 °C	160 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Resulting products was labeled as F99 (AlcR) and F100 (IRES).

Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES)

Investigator: Louise, Christopher

Aim of the experiment: Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES).

Procedure:

4 µl of F99 and F100 were mixed with 0.44 µl DNA loading buffer (10x)

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

1 kbp DNA ladder	F100	F99
	as expected	as expected

Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)

Investigator: Rosario

Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
P157	P245
P208	P246
P213	P247
F59+F81 I	P248
F59+F81 II	P249
F59+F83 I	P250
F59+F83 II	P251

Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (Laccase) with EcoRI and SpeI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (LAccase) with EcoRI and SpeI

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	SpeI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P248-P251)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min
For P248-P251 a 1% agarose gel was used.

Lane:	DNA marker 1kb	Digestion of P248 with EcoRI & SpeI	Digestion of P249 with EcoRI & SpeI	Digestion of P250 with EcoRI & SpeI	empty	Digestion of P251 with EcoRI & SpeI
Result:	-	as expected	as expected	no insert (Laccase)	-	no insert (Laccase)

Transformation of E. coli XL1 blue with P204

Investigator: Johanna

Aim of the experiment: Transformation of E. coli XL1 blue with P204

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Sequencing of P61 (pSH21/pAutoRex8)

Investigator: Rosario, Katrin

Aim of the study: Sequencing of P61 (pSH21/pAutoRex8)

Procedure:

Eurofins SmartSeq Kit protocol (15 µl plasmid DNA (50 - 100 ng), 2 µl sequencing primer).

#	P61
Name	pSH 21 (AlcR/IRES)
O88 (primer 1)	FR02305828
O89 (primer 2)	FR02305829

Picking of F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79, P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa), F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Investigator: Rosario, Katrin

Aim of the study: Picking of F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79, P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa), F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
There were no colonies on plates F42+F84 and xy
There were a lot of colonies on NK 90 but there were significantly more colonies on the plates with ligation products
2 colonies were picked for every ligation product.

Friday, June 14th

Picking of transformed Plasmid E. coli XL1 blue with P204, F92+F86 and F42+F84

Investigator: Johanna

Aim of the study: Picking of P204, F92+F86 and F42+F84

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl CamR, 4 ml LB-medium)

3 colonies were picked for every transformation product.

Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue

Investigator: Rosario, Johanna, Andi

Aim of the experiment:Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
F59+F85	P252
F59+F85	P253
F90+F86	P254
F90+F86	P255
F90+F87	P256
F90+F87	P257
F90+F88	P258
F90+F88	P259
F90+F89	P260
F90+F89	P261
F90+F97	P262
F90+F97	P263
F91+F94	P264
F91+F94	P265
F92+F87	P266
F92+F87	P267
F92+F97	P268
F92+F97	P269
F92+F88	P270
F92+F88	P271
F92+F89	P272
F92+F89	P273
F93+F79	P274
F93+F79	P275
P222	P276
P222	P277
P240	P278
P240	P279
P126	P280
P126	P281
P244	P282
P244	P283
P127	P284
P127	P285

Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.

The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.

Sequencing of P276 (Retrafo of P222, fw), P282 (Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv)

Investigators: Louise

Aim of the experiment: Sequencing of P276(Retrafo of P222, fw), P282(Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P276	PIF6	FR02305830 (O3)	-
P282	IgKappa-SigP in pSB1C3	FR02305831 (O3)
P278	PhyB-36AALinker in pSB1C3	FR02305832 (O3)	FR02305833 (O4)
P217	Laccase QCIV	FR02305826 (O3)	FR02305827 (O4)

Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI

Investigator: Rosario, Johanna, Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI

Procedure:

Batches for every Plasmid of P252-P275 contained

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	SpeI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P252-P275)
=20 µl	TOTAL

The batches for the Quickchangeproduct were prepared with 6 µl of Quickchangeproduct and 2 µl DNA loading buffer.

Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
For P252-P275 a 1% agarose gel was used.

Lane:	DNA marker 1kb	Digestion of P252 with EcoRI & SpeI	Digestion of P253 with EcoRI & SpeI	Digestion of P254 with EcoRI & SpeI	Digestion of P255 with EcoRI & SpeI	Digestion of P256 with EcoRI & SpeI	Digestion of P257 with EcoRI & SpeI	Digestion of P258 with EcoRI & SpeI
Result:	-	corrupt	as expected	corrupt	corrupt	corrupt	as expected	corrupt

Lane:	DNA marker 1kb	Digestion of P259 with EcoRI & SpeI	Digestion of P260 with EcoRI & SpeI	Digestion of P261 with EcoRI & SpeI	Digestion of P262 with EcoRI & SpeI	Digestion of P263 with EcoRI & SpeI	Digestion of P264 with EcoRI & SpeI	Digestion of P265 with EcoRI & SpeI
Result:	-	as expected	as expected (sequencing necessary)	as expected (sequencing necessary)	bad miniprep	as expected	not sure (has to be repeated with control P278)	not sure (has to be repeated with control P278)

Lane:	DNA marker 1kb	Digestion of P266 with EcoRI & SpeI	Digestion of P267 with EcoRI & SpeI	Digestion of P268 with EcoRI & SpeI	Digestion of P269 with EcoRI & SpeI	Digestion of P270 with EcoRI & SpeI	Digestion of P271 with EcoRI & SpeI	Digestion of P272 with EcoRI & SpeI
Result:	-	as expected	as expected	as expected	as expected	as expected	as expected	as expected (has to be sequenced)

Lane:	DNA marker 1kb	Digestion of P273 with EcoRI & SpeI	Digestion of P274 with EcoRI & SpeI	Digestion of P275 with EcoRI & SpeI	Digestion of QC I product of P251 with DpnI	Digestion of QC I product of P163I with DpnI	Digestion of QC V product of P163II with DpnI	Digestion of QC V product of P217 with DpnI
Result:	-	as expected (has to be sequenced)	as expected (has to be sequnced)	as expected (has to be sequnced)	corrupt, no QC product	corrupt, no QC product	corrupt, no QC product	as expected, QC product

Preparative Digestion of IRES (F100) and AlcR (F99) with EcoRI and SpeI

volume	reagent
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI
1 µl	SpeI
9 µl	ddH2O
25 µl	Fragment DNA (F99 or F100)
=40 µl	TOTAL

Incubation for 150 min at 37 °C
Purification by QIAGEN PCR Purification Kit
new Names: F99 -> F101 and F100 -> F102

Oligohybridization of Strep-TEV (O90 + O91)

Investigator: Rosario

Aim of the experiment: Oligohybridization of Strep-TEV (O90 + O91)

Procedure:

25 µL of 100 pmol/µl of Strep-TEV_fw and 25 µL of 100 pmol/µl Strep-TEV_rev

Heating up to 95 °C for 5 min

Cooling at RT in a styropor box overnight

Saturday, June 15th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3)

Investigator: Jeff

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube	Concentration in ng/µl
ReTraFo of P204 (IgKappa-SigP in pSB1C3)	P286	105.7
ReTraFo of P204 (IgKappa-SigP in pSB1C3)	P287	303.6
ReTraFo of P204 (IgKappa-SigP in pSB1C3)	P288	268.1
F92+F86 (IgKappa-SigP_EreB in pSB1C3)	P289	121.2
F92+F86 (IgKappa-SigP_EreB in pSB1C3)	P290	402.3
F92+F86 (IgKappa-SigP_EreB in pSB1C3)	P291	405.2

Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus)

Investigator: Jeff

Aim of the experiment: Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus).

Procedure:

Ligation batch for F41+F101 (AlcR), 50 ng vector used, 1:1:

volume	reagent
0.56 µl	F41 (89.9 ng/µl, 2086 bp)
5.23 µl	F101 (11.3 ng/µl, 2466 bp)
11.21 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
0.5 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F41+F102 (IRES of poliovirus), 100 ng vector used, 3:1:

volume	reagent
1.11 µl	F41 (89.9 ng/µl, 2086 bp)
7.92 µl	F102 (11.4 ng/µl, 628 bp)
7.97 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
0.5 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

The ligation was performed for 1 hour at room temperature.

Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI.

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI-HF
0.25 µl	SpeI-HF
15 µl	ddH2O
2.5 µl	Plasmid DNA (P289-P291, P245)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

Lane:	DNA ladder 100bp	Digestion of P289 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 1	Digestion of P290 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 2	Digestion of P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 3	Digestion of P245 (EreB in pSB1C3) with EcoRI & SpeI
Result:		as expected	as expected	as expected	as expected (control)

Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of ligation product was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sunday, June 16th

QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P217 (Laccase), P163 (NPT-Cassette)

Investigator: Andi

Aim of the experiment: Removal of forbidden restriction site from P217 (Laccase) and P163 (NPT-Cassette).

Procedure: Quikchange - PCR

Reaction batch for P217 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P217 - 1:100 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O74 (10 µM)
1 µl	1:10 dilution of O75 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P163 - 1:50 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O70 (10 µM)
1 µl	1:10 dilution of O71 (10 µM)
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	5 min
		4 °C	hold

After the PCR, the Quickchange product was stored in the fridge.

Picking of transformed Plasmid E. coli XL1 blue with QC of P217, P163, F41+F101 and F41+F102

Investigator: Andi, Jeff

Aim of the study: Picking of QC of P217, P163, F41+F101 and F41+F102

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)

1 colonies were picked for every transformation product.

Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV)

Investigator: Andi, Jeff

Aim of the experiment: Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV).

Procedure:

Ligation batch for F59+F74 (SERK-SigP), 100 ng vector used, insert:vector = 3:1

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
3 µl	F74 (4.8 ng/µl, 100 bp)
12.5 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F59+F103 (Strep-TEV), 100 ng vector used, insert:vector = 3:1

volume	reagent
1.5 µl	F59 (66.7 ng/µl, 2086 bp)
15.5 µl	F103 (504.9 ng/µl, 54 bp)
7.97 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Investigator: Andi, Jeff

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013) and P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of ligation product/ 2 µl of plasmid (P7) was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Week 9

Monday, June 17th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES) Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
QC I of P163	P292
QC V of P217	P293
F41+F101 I	P294
F41+F101 II	P295
F41+F101 III	P296
F41+F102 I	P297
F41+F102 II	P298
F41+F102 III	P299

Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Investigator: Rosario

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)

1 colony was picked for every transformation product.

Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI

Investigator: Johanna, Flo

Aim of the experiment: Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI

Procedure:

Batch for preparative digestion of P264 (PhyBlinker+cTev) with AgeI/SpeI

volume	reagent
20 µl	P264
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P299 (IRES) with NgoMIV/SpeI

volume	reagent
20 µl	P299
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P274 (nTevlinker)with AgeI/SpeI

volume	reagent
20 µl	P274
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P111 (TEV) with AgeI/SpeI

volume	reagent
20 µl	P111
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P248 (Nuclease) with NgoMIV/SpeI

volume	reagent
20 µl	P248
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane:	Digestion of P111 with AgeI & SpeI	1 kbp DNA ladder	Digestion of P248 with NgoMIV & SpeI	Digestion of P264 with AgeI & SpeI
Result:	as expected		as expected	as expected

Lane:	Digestion of P274 with AgeI & SpeI	1 kbp DNA ladder	Digestion of P299 with NgoMIV & SpeI
Result:	low concentration, discarded		not digested, discarded

The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI-HF
0.25 µl	SpeI-HF (PstI-HF for P292)
15 µl	ddH2O
2.5 µl	Plasmid DNA (P292-P299)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

Lane:	1 kbp DNA ladder	Digestion of P292 (QC I P163 (NPT-casette)) with EcoRI & PstI	Digestion of P293 (QC V P217 (Laccase)) with EcoRI & SpeI	Digestion of P294 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 1	Digestion of P295 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 2	Digestion of P296 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 3	Digestion of P297 (F41+F102 (IRES)) with EcoRI & SpeI, clone 1	Digestion of P298 (F41+F102 (IRES)) with EcoRI & SpeI, clone 2	Digestion of P299 (F41+F102 (IRES)) with EcoRI & SpeI, clone 3	1 kbp DNA ladder
Result:		corrupt	corrupt	as expected	as expected	as expected	as expected	as expected	as expected

DpnI digestion of P163QCI and P217QCV

Investigator: Flo, Jeff

Aim of the experiment: DpnI digestion of P163QCI and P217QCV.

Procedure:

1 µl of DpnI was added to the QuikChange products of P163QCI and P217QCV.

The mixtures were incubated for 1 h at 37 °C; afterwards they were ready for transformation.

Analytical gelelectrophoresis of DpnI digestion of P163QCI and P217QCV

Investigator: Jeff, Flo

Aim of the experiment: Analysis of the DpnI digestion of P163QCI and P217QCV

Procedure:

4.5 µl of the QuikChange products of P163 and P217, named P163QCI and P217QCV were mixed together with 0.5 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 30 min.

Lane:	1 kbp DNA ladder	DpnI digestion of P163QCI.1	DpnI digestion of P163QCI.2	DpnI digestion of P217QCV.1	DpnI digestion of P217QCV.2
Result:		empty	empty	empty	empty

Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease)

Investigator: Jeff, Flo

Aim of the experiment: Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease).

Procedure:

Ligation batch for F77+F105 (NLS in pSB1C3 + mature thermonuclease), 100 ng vector used, insert:vector = 3:1

volume	reagent
1.78 µl	F77 (56.2 ng/µl, 2143 bp)
5.22 µl	F105 (12 ng/µl, 447 bp)
10 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed overnight at 16 °C.

Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations

Investigator: Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange product/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv)

Investigators: Rosario

Aim of the experiment: Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P248	NucA	FR02305834 (O3)	-
P275	N-TEV_36AAlinker	FR02305835 (O3)
P299	IRES	FR02305825 (O3)	-
P292	QC I NPT-casette	FR02305824 (O3)	FR02305823 (O4)
P293	QC V Laccase	FR02305822 (O3)	FR02305821 (O4)
P290	IgKappa_EreB	FR02305820 (O3)	FR02305819 (O4)

Tuesday, June 18th

Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275

Investigator: Rosario

Aim of the study: Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275.

Operational sequence:

Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)

2 colonies were picked for every transformation product.

Transformation of E. coli XL1 blue with F77+F105 (NLS in pSB1C3 + mature thermonuclease) and P299 Retrafo

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB)

Investigator: Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
P7 (PhyB) clone 1	P300
P7 (PhyB) clone 2	P301
F59+F74 (Serk-Sigp in PsB1C3) clone 1	P302
F59+F74 (Serk-Sigp in PsB1C3) clone 2	P303
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 1	P304
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 2	P305

Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI

Investigator: Louise, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI.

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	AgeI
0.25 µl	XbaI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P300-P305)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane:	Digestion of P300 with XbaI/AgeI	Digestion of P301 with XbaI/AgeI	Digestion of P302 with XbaI/AgeI	Digestion of P303 with XbaI/AgeI	Digestion of P304 with XbaI/AgeI	Digestion of P305 with XbaI/AgeI	1 kb DNA Ladder	100 bp DNA Ladder
Result:	as expected	as expected	as expected	as expected	as expected	as expected

Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI

Investigator: Louise, Flo, Jeff, Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI.

Procedure:

Batch for preparative digestion of P212 (SERK-TMD) with NgoMIV/SpeI

volume	reagent
20 µl	P212
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI

volume	reagent
20 µl	P238
4 µl	CutSmart Buffer
1 µl	NgoMIV(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P303 (SERK-SigP) with AgeI/SpeI

volume	reagent
20 µl	P303
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P304 (StrepTEV-Linker) with AgeI/SpeI

volume	reagent
20 µl	P304
4 µl	CutSmart Buffer
1 µl	AgeI(20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and P238, P303 and P304 were loaded on a 1% agarose gel, while P212 was loaded on a 2% agarose gel for preparative gelelectrophoresis.

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane:	Digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI	1 kbp DNA ladder	Digestion of P303 (SERK-SigP) with AgeI/SpeI	Digestion of P304 (StrepTEV-Linker) with AgeI/SpeI
Result:	as expected		as expected	as expected

Lane:	1 kbp DNA ladder	Digestion of P212 (SERK-TMD) with NgoMIV/SpeI	100 bp DNA Ladder
Result:		as expected

The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN

Ligation of F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff, Flo

Aim of the experiment: Ligation of F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1).

Procedure:

Ligation batch for F109+F86 (SERK-SigP + EreB), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
5.57 µl	F86 (31.0 ng/µl, 1254 bp)
10.51 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F87 (SERK-SigP + NanoLuc), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
6.89 µl	F87 (10.2 ng/µl, 510 bp)
9.19 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F88 (SERK-SigP + SpyCatcher), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
5.37 µl	F88 (8.7 ng/µl, 339 bp)
10.71 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F89 (SERK-SigP + SpyTag), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
12.0 µl	F89 (6.8 ng/µl, 39 bp)
4.08 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F97 (SERK-SigP + XylE), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
6.52 µl	F97 (19.4 ng/µl, 918 bp)
9.56 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F107 (SERK-SigP + SERK-TMD), 100 ng vector used, insert:vector = 3:1

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
5.38 µl	F107 (4.8 ng/µl, 186 bp)
10.70 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), 100 ng vector used, insert:vector = 3:1

volume	reagent
1.38 µl	F110 (72.4 ng/µl, 2143 bp)
5.53 µl	F108 (23.6 ng/µl, 933 bp)
10.09 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

QuikChange mutagenesis to remove forbidden restriction sites - SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)

Investigator: Jeff

Aim of the experiment: Removal of the last existing forbidden restriction site from P217 (Laccase) and the first one from P162 (NPT-Cassette).

Procedure: Quikchange - PCR

Reaction batch for P217 (Laccase)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid template (P217 - 1:100 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O74 (=10 µM)
1 µl	1:10 dilution of O75 (=10 µM)
18.5 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P163 (NPT-Cassette)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid template (P162 - 1:50 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O70 (10 µM)
1 µl	1:10 dilution of O71 (10 µM)
18.5 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
		4 °C	hold

After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)

Investigator: Jeff

Aim of the experiment:

Procedure:

4.5 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 40 min at an 1% agarose gel.

Lane:	1 kbp DNA ladder	DpnI digestion of P163QCI	DpnI digestion of P217QCV
Result:		as expected	as expected

Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/10 µl of ligation products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P294 (AlcR)

Investigators: Rosario

Aim of the experiment: Sequencing of P294 (AlcR).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P294	AlcR	FR02305817 (O3)	FR02305818(O4)

Wednesday, June 19th

QuikChange mutagenesis to remove forbidden restriction sites - SDMI of P294 (AlcR)

Investigator: Florian - Master of QuikChange 2.0

Aim of the experiment: Removal of the first forbidden restriction site from P294 (AlcR).

Procedure: Quikchange - PCR

Reaction batch for P294 (AlcR)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid template (P294 - 1:50 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O80 (=10 µM)
1 µl	1:10 dilution of O81 (=10 µM)
18.5 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P294 (AlcR)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	19	95 °C	30 s
		52 °C	1 min
		68 °C	5 min
		4 °C	hold

After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)

Investigator: Florian

Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR).

Procedure:

4 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

Lane:	1 kbp DNA ladder	DpnI digestion of P294QCI
Result:		only Primers

Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA)

Investigator: Florian

Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.

Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B)

Investigators: Rosario

Aim of the experiment: Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P308	PhyB_36AALinker_C-TEV in pSB1C3	FR02305816 (O3)	FR02305815 (O4)
P310	N-TEV_36AALinker in pSB1C3	FR02305814 (O3)
P303	SERK-SigP in pSB1C3	FR02305813 (O3)	-
P304	Strep-TEV-Linker in pSB1C3	FR02305812 (O3)	-
P305	Strep-TEV-Linker in pSB1C3	FR02305811 (O3)	-
P301	Phytotchrome B	FR02305810 (O3)	FR02305809 (O4)

Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
P160 I	P306
P160 II	P307
P264 I	P308
P264 II	P309
P274 I	P310
P274 II	P311
P275 I	P312
P275 II	P313

Pouring of CamR gel plates

Investigator: Louise, Volker

Aim of the experiment: Pouring of CamR gel plates

Procedure: Preparation of 5 l of LB-Medium with agar was performed after standard recipe

Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Investigator: Rosario

Aim of the experiment: Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Procedure:

Retransformations (P212, P238, P299) 1 clone each
Quickchanges (QCI P162, QCV P217) 2 clones each
Ligation F77+F105 4 clones because of the high number of colonies at F77 negative ctrl
All other Ligations (F110+F108, F109+F86, F109+F87, F109+F88, F109+F89, F109+F97, F109+F107) 2 clones each

Thursday, June 20th

Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)

Investigator: Rosario

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
QC I P162 I	P315
QC I P162 II	P316
QC V P217 I	P317
QC V P217 II	P318
P212 ReTrafo	P319
P238 ReTrafo	P320
P299 ReTrafo	P321
F77+F105 I	P322
F77+F105 II	P323
F77+F105 III	P324
F77+F105 IV	P325
F110+F108 I	P326
F110+F108 II	P327
F109+F86 I	P328
F109+F86 II	P329
F109+F87 I	P330
F109+F87 II	P331
F109+F88 I	P332
F109+F88 II	P333
F109+F89 I	P334
F109+F89 II	P335
F109+F97 I	P336
F109+F97 II	P337
F109+F107 I	P338
F109+F107 II	P339

Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222

Investigator: Johanna, Andi

Aim of the experiment: Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of ddH₂O was added to empty tubes

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol (P170, P111, P160, P275, P244, P240, P212, P208, P217, P204, P213, P222) and ampicillin plates (P314, P114).

Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1)

Investigators: Rosario

Aim of the experiment: Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P308	SERK-TMD_8AAlinker_GFPmut1	FR02305808 (O3)	FR02305807 (O4)

Preparation of media for competent cells

Investigator : Johanna, Louise

Aim of the experiment:Preparation of media for competent cells

Procedure:

Picking of one colony of overnight stem culture of non competent XL1-Blue E.Coli in 50 ml LB-Medium without antibiotics (under the sterile bench)
this was incubate overnight at 37 °C

Batch for 1l of a 0.1 M MgCl₂-solution

amount	reagent
20.33 g	MgCl₂ (M=203.3 g/mol)
1 l	ELGA H₂O

Batch for 500ml of a 50 mM CaCl₂-solution

amount	reagent
3.67 g	CaCl₂ (M=147.02 g/mol)
500 ml	ELGA H₂O

Batch for 50ml of a 50 mM CaCl₂-solution, 15% v/v Glycerin

amount	reagent
0.368 g	CaCl₂ (M=147.02 g/mol)
0.458 g	Glycerin (density=1.26)
50 ml	ELGA H₂O

Autoclave each bottle of solution and leave each in a 4°C room.

QuikChanges QC pActin and QC1 AlcR

Investigator : Andi

Aim of the experiment: QuikChanges QC pActin (P114) and QC1 AlcR (P294)

Procedure: Quickchange - PCR

Reaction batch 1 for P114 (pActin) with Pfu Ultra II

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P114 undiluted)	4.5 ng in 25 µl batch
1 µl	1:10 dilution of O76 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
1 µl	1:10 dilution of O77 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
18 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P114 (pActin)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
3		52 °C	1 min
4		68 °C	6.5 min
5		4 °C	hold

Reaction batch 2 for P114 (pActin) with Q5 Polymerase Mastermix

volume	reagent	Additional information
12.5 µl	2x QS Polymerase Mastermix
1 µl	Plasmid template (P114 - 1:10 dilution)	0.45 ng in 25 µl batch
1.25 µl	1:10 dilution of O76 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
1.25 µl	1:10 dilution of O77 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
9 µl	ddH2O

PCR cycling parameters - P114 (pActin) with Q5 Polymerase

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
3		55 °C	1 min
4		72 °C	3.25 min
5		4 °C	hold

Reaction batch for P294 (AlcR) with Pfu Ultra II

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid template (P294 dilutsion 1:100)	2.79 ng in 25 µl batch
1 µl	1:10 dilution of O76 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
1 µl	1:10 dilution of O77 (=10 µM)	0.5 µl Primer + 4.5 µl ddH2O
18.5 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P294 (AlcR)

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
3		52 °C	1 min
4		68 °C	6.5 min
5		4 °C	hold

After the PCR, the Quickchange products of P114 (pActin) with Q5 Mastermix and P294 (AlcR)were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.

After the PCR, the Quickchange products of P114 (pActin) with PfU II Polymerase was stored in the Cycler at 4 °C over night

Transformation of E. coli XL1-blue with DpnI digested QuikChange product P114 (pActin) and P294 (AlcR)

Investigator: Master of Quickchange, Gel-Penetrator

Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294 (AlcR) QCI and P114 (pActin).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.

PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase

Investigator: Andi, Johanna

Aim of the experiment: PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase

Procedure:

Operational sequence:

Reaction batch with Herculase Polymerase

volume	reagent
10 µl	5x Herculase Fusion Polymerase reaction buffer
2.5 µl	10 mM dNTPs
1.25 µl	10 µM Forward Primer O24 (Bpul_for)
1.25 µl	10 µM Reverse Primer O25 (Bpul_rev)
1 µL	Herculase II Fusion Polymerase
1.5 µl	Plasmid DNA (P317)- 1:500 dilution
0.5 µl	DMSO
32 µL	ddH₂O Water
=50 µL	TOTAL

Reaction batch with Q5 Mastermix

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O24 (Bpul_for)
2.5 µl	10 µM Reverse Primer O25 (Bpul_rev)
1 µl	Plasmid DNA (P317)- 1:500 dilution
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	51 °C	60 s
	68 °C	90 s
Final extension	68 °C	5 min
Hold	4 °C	infinite

After the PCR has finished, the product was hold at 4 °C in the Cycler

Analytical gelelectrophoresis of the DpnI digested products of the performed QuickChange of SDMI of P294 (AlcR)and P114 (pActin)

Investigator: Andi, Johanna

Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)and P114 (pActin.)

Procedure:

9 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

Lane:	100 bp DNA ladder	DpnI digestion of P294QCI	DpnI digestion of P114QCI	100 bp DNA ladder
Result:		expected result	only Primers

Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)

Investigator: Rosario, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	PstI
15 µl	ddH2O
2.5 µl	Plasmid DNA
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

1 kb DNA Ladder	P315	P316	P162 (Control)	P317	P318	P217 (Control)	P322	P323	P324	P325	P248 (Control)
	as expected	as expected	as expected	as expected	as expected	as expected	corrupt	corrupt	corrupt	corrupt	as expected

1 kb DNA Ladder	P334	P335	P170 (Control)	P336	P337	P197 (Control)	P338	P339	P212 (Control)	P320
	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	is repeated on other gel

1 kb DNA Ladder	P320 (Control)	P326	P327
	as expected	as expected	as expected

1 kb DNA Ladder	P328	P329	P157 (Control)	P330	P331	P208 (Control)	P332	P333	P213 (Control)	P326	P327
	as expected	as expected	as expected	as expected	as expected	corrupt	as expected	as expected	corrupt	is repeated on other gel	is repeated on other gel

Friday, June 21st

Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)

Investigator: Master of QuikChange

Aim of the experiment: Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)

Procedure:

9 µl of each of the PCR products were mixed with 1 µl of DNA loading buffer (10x).

9 µl of each of the DpnI digested QuikChange product was mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.

Lane:	1000 bp DNA ladder	PCR product of P317 with Q5 Master Mix	PCR product of P317 with Herculase Polymerase	DpnI digestion of P114
Result:		expected result	expected result	expected result

Preparation of competent cells according to the CaCl₂-method (Cohen et al.,1972)

Investigator: Johanna, Louise

Aim of the experiment: Preparation of competent cells according to the CaCl₂-method (Cohen et al.,1972)

Procedure:

Add Tetracyclin (ratio: 1:1000) to the 2YT-medium
Measure the OD₅₅₀ of it until OD₅₅₀=0.5
Put the culture in Falcon tubes (leaving on ice)
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 40 ml 0,1 M MgCl₂ solution after removing the supernatants
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 20 ml 50mM CaCl₂ solution after removing the supernatants
Incubate the falcon tubes on ice for 30 min
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 2 ml 50mM CaCl₂, 15 % v/v Glycerin solution after removing the supernatants. From this step everything needs to be carry out in a 4°C room.
Mix the solutions of each falcon tube and transfer the content in eppis (each with 100 and accordingly 150 µl)
Store the boxes of eppis at -80 °C

Preparative Digestion of P308 (PhyB-Linker-C-TEV), P299 (IRES), P111 (TEV), P326 (TEV-Linker-TM-GFP), P328 (SERKSigP_EreB), P330 (SERK-SigP_NLuc), P332 (SERK-SigP_SpyCatcher), P334 (SERK-SigP_SpyTag), P336 (SERK-SigP_XylE), P338 (SERK-SigP_SERK-TMD), P8 (pSB1C3) and PCR product of Laccase (F112)

Investigator: Katrin

Aim of the experiment: Peparative Digestion of P308 (PhyB-Linker-C-TEV), P299 (IRES), P111 (TEV), P326 (TEV-Linker-TM-GFP), P328 (SERKSigP_EreB), P330 (SERK-SigP_NLuc), P332 (SERK-SigP_SpyCatcher), P334 (SERK-SigP_SpyTag), P336 (SERK-SigP_XylE), P338 (SERK-SigP_SERK-TMD) and P8 (PhyB)

Procedure:

Batch for preparative digestion of effectors (P328, P330, P332, P334, P336, P338) with AgeI and PstI) (7x Mastermix)

volume	reagent
28 µl	Buffer 4 (10x)
7 µl	AgeI (20 U/µl)
7 µl	PstI (20 U/µl)
98 µl	ddH₂O
=140 µl	TOTAL

20 µl Mastermix + 20 µl Plasmid each

Batches for preparative digestions:

volume	reagent
20 µl	Plasmid
4 µl	Buffer 4 (10x)
1 µl	Enzyme 1
1 µl	Enzyme 2
14 µl	ddH₂O
=40 µl	TOTAL

Plasmid	Name	Enzyme 1	Enzyme 2
P308	PhyB-Linker-C-TEV	SpeI	PstI
P299	IRES	XbaI	PstI
P111	TEV	SpeI	PstI
P326	TEV-Linker-TM-GFP	NgoMIV	PstI
P8	pSB1C3	XbaI	AgeI
F112	Laccase	XbaI	AgeI

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Name:	P308 dig. SpeI + PstI	1kb DNA ladder	P299 dig. XbaI + PstI	P111 dig. SpeI + PstI
cut out:	Backbone		Insert (lower band)	Backbone

Name:	P326 dig. NgoMIV + PstI	1kb DNA ladder	P328 dig. AgeI + Pst	P330 dig. AgeI + PstI
cut out:	Insert (lower band)		Backbone	Backbone

Name:	P332 dig. AgeI + PstI	1kb DNA ladder	P334 dig. AgeI + PstI	P336 dig. AgeI + PstI
cut out:	Backbone		Backbone	Backbone

Name:	P338 dig. AgeI + PstI	1kb DNA ladder		P8 dig. AgeI + XbaI
cut out:	Backbone			Backbone (lower band)

Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3)

Procedure:

Ligation batch for F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB)

volume	reagent
3.52 µl	F117 (28.4 ng/µl, 3413 bp)
4.81 µl	F116 (18.2 ng/µl, 996 bp)
8.67 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc)

volume	reagent
1.89 µl	F118 (52.8 ng/µl, 2663 bp)
6.16 µl	F116 (18.2 ng/µl, 996 bp)
8.95nbsp;µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher)

volume	reagent
2.57 µl	F119 (38.0 ng/µl, 2492 bp)
6.59 µl	F116 (18.2 ng/µl, 996 bp)
7.84nbsp;µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag)

volume	reagent
0.8 µl	F120 (62.5 ng/µl, 2192 bp)
3.75 µl	F116 (18.2 ng/µl, 996 bp)
3.96nbsp;µl	ddH₂O
1 µl	T4 ligase buffer (10x)
0.5 µl	T4 ligase (1 U/µl)
=10 µl	TOTAL

Ligation batch for F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE)

volume	reagent
0.73 µl	F121 (68.3 ng/µl, 3071 bp)
2.56 µl	F116 (18.2 ng/µl, 996 bp)
5.95nbsp;µl	ddH₂O
1 µl	T4 ligase buffer (10x)
0.5 µl	T4 ligase (1 U/µl)
=10 µl	TOTAL

Ligation batch for F123+F124 (Laccase+pSB1C3)

volume	reagent
2.03 µl	F123 (49.3 ng/µl, 2086 bp)
3.80 µl	F124 (58.9 ng/µl, 1555 bp)
11.17nbsp;µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3) and negative controls

Investigator: Jeff, Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3), QCI(Insertion) of pActin and negative controls

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.

Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw)

Investigators: Louise, Katrin

Aim of the experiment: Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw).

Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).

The different genes received the following barcodes:

Label	Name	Barcode1	Barcode2
P303	SERK-SigP	FR02305805 (O3)
P310	N-TEV_36AAlinker		FR02305806 (O4)
P248	Thermonuc.	FR02305804 (O3)
P312	NTEV_36aaLinker	FR02305803 (O3)
P316	npt-casette QCI	FR02305802 (O3)	FR02305801 (O4)
P318	Laccase QCV	FR02305800 (O3)	FR02305799 (O4)
P320	SERK-TM_8aaLinker_GFPmut1	FR02305798 (O3)	FR02305797 (O4)
P326	Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1	FR02305796 (O3)	FR02305795 (O4)
P328	SERK-SigP_EreB	FR02305794 (O3)	FR02305793 (O4)
P330	SERK-SigP_Nanoluc	FR02305792 (O3)
P332	SERK-SigP_Spycatcher	FR02305791 (O3)
P334	SERK-SigP_Spy-tag	FR02305790 (O3)
P336	SERK-SigP_XylE	FR02305789 (O3)	FR02305788 (O4)
P338	SERK-SigP_SERK-TM	FR02305787 (O3)

PCR of P61 to get the IRES of poliovirus 1 Mahoney strain

Investigator: Jeff

Aim of the experiment: PCR of P61 to get the IRES of poliovirus 1 Mahoney strain.

Procedure:

Operational sequence:

Reaction batch with Q5 Mastermix:

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O68 (IRES_polio_fw)
2.5 µl	10 µM Reverse Primer O69 (IRES_polio_rv)
1 µl	Plasmid DNA (P61)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	63 °C	30 s
	72 °C	20 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR

Investigator: Katrin

Aim of the experiment: Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR.

Procedure:

Retransformations 1 clone each
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol (P111, P160) or ampicillin (P114, P314)

Saturday, June 22nd

QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII)

Investigator: Jeff

Aim of the experiment: QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII).

Procedure:

Reaction batch for P316 (npt-casette after QCI)

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P316 - 1:100 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O72 (=10 µM)
1 µl	1:10 dilution of O73 (=10 µM)
19 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch for P342 (AlcR after QCI):

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P342 - 1:100 dilution)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O82 (10 µM)
1 µl	1:10 dilution of O83 (10 µM)
19 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters - P316 (npt-casette after QCI) and P342 (AlcR after QCI):

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
		52 °C	1 min
		68 °C	5 min
3		4 °C	hold

Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:

Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI, gelelectrophoresis of PCR of IRES

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI

Procedure:

Batch

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	PstI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P341, P342; P249)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane:	1 kb DNA Ladder	PCR of IRES	Digestion of P249 with EcoRI and PstI (control)	Digestion of P341 with EcoRI and PstI	Digestion of P342 with EcoRI and PstI
Result:		as expected	as expected	as expected	as expected

Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES

Investigator: Katrin

Aim of the experiment: Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES

Procedure:

Preparative digestion of P310 (N-Tev-linker)

volume	reagent
20 µl	P310
4 µl	CutSmart Buffer (10x)
1 µl	AgeI (20 U/µl)
1 µl	SpeI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Preparative digestion of P160 (SV40 NLS)

volume	reagent
20 µl	P160
4 µl	CutSmart Buffer (10x)
1 µl	AgeI (20 U/µl)
1 µl	PstI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Preparative digestion of P249 (Thermonuclease)

volume	reagent
20 µl	P249
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV (20 U/µl)
1 µl	PstI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Preparative digestion of PCR of IRES)

volume	reagent
25 µl	PCR product
5 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	SpeI (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Name:	P249 dig. NgoMIV + PstI	1kb DNA ladder	P160 dig. AgeI + PstI	P111 dig. SpeI + AgeI
cut out:	Insert(lower band)		Backbone	Backbone

Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IREs+pSB1C3)

Procedure:

Ligation batch for F126+F127 (NLS in pSB1C3+Thermonuclease

volume	reagent
7.2 µl	F126 (13.9 ng/µl, 2143 bp)
4.1 µl	F127 (15.2 ng/µl, 447 bp)
5.7 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F125+F96 (N-TEV-linker in pSB1C3+PIF3)

volume	reagent
6.9 µl	F125 (14.5 ng/µl, 2554 bp)
2.3 µl	F96 (15.1 ng/µl, 297 bp)
7.8nbsp;µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F124+F41 (IREs+pSB1C3)

volume	reagent
1.3 µl	F124 (70.8 ng/µl, 628 bp)
1.2 µl	F41 (81.1 ng/µl, 2086 bp)
14.5nbsp;µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls

Investigator: Volker

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)

Investigator: Katrin Aim of the experiment: Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)

Procedure:

2 clones were picked from each plate
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol or ampicillin (QCI (insertion of P114 (pActin))

Sunday, June 23rd

QuikChange II of pActin (P359, P360)

Investigator: Katrin

Aim of the experiment: QuikChangeII of pActin (P359, P360) Procedure:

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P359, P360)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O76 (=10 µM)
1 µl	1:10 dilution of O77 (=10 µM)
19 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
		52 °C	1 min
		68 °C	6 min
3		4 °C	hold

Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)

Investigator: Katrin

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of P347-P358

Investigator: Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P347-P358

Procedure:

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	PstI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P347-P358)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane:	1 kb DNA Ladder	Digestion of P347 with EcoRI and PstI	Digestion of P348 with EcoRI and PstI	Digestion of P349 with EcoRI and PstI	Digestion of P350 with EcoRI and PstI	Digestion of P351 with EcoRI and PstI	Digestion of P352 with EcoRI and PstI	Digestion of P353 with EcoRI and PstI	Digestion of P354 with EcoRI and PstI
Result:

Lane:	1 kb DNA Ladder	Digestion of P355 with EcoRI and PstI	Digestion of P356 with EcoRI and PstI	Digestion of P357 with EcoRI and PstI	Digestion of P358 with EcoRI and PstI
Result:

Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette

Investigator: Katrin

Aim of the experiment: Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette

Procedure:

1 µl of Dpn1 was added to the reaction, the batch was incubated for one hour at 37°C

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of F125+F98, F125+F96, F128+F41

Investigator: Katrin Aim of the experiment:Picking of F125+F98, F125+F96, F128+F41

Procedure:

2 clones were picked from each plate
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol
F126+F127 was put back into the incubator because there were no colonies visible

Sequencing of P35, P143, P359, P360, P249

Investigators: Katrin

Aim of the experiment: Sequencing of P35, P143, P359, P360, P249

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2
P35	FR02305786 (O4)
P143	FR02305785 (O92)	FR02305784 (O93)
P359	FR02305783 (O62)	FR02305782 (O63)
P360	FR02305781 (O62)	FR02305780 (O63)
P249	FR02305779 (O3)	FR02305778 (O4)

Week 10

Monday, June 24th

Miniprep of ligations F125+F98 (N-TEV-linker in pSB1Cs + PIF6), F125+F96 (N-TEV-linker in pSB1Cs + PIF3) and F128+F41 (PCR of IRES + pSB1C3 backbone) + analytical gel

Investigator: Johanna

Aim of the experiment: Preparation of ligation products F125+F98, F125+F96 and F128+F41

Procedure:

Miniprep according to QIAGEN QIAprep Kit

label	content	conc. in ng/µl
P361	N-TEV-linker (in pSB1C3) + PIF6	163.2
P362	N-TEV-linker in (in pSB1C3) + PIF6	183.5
P363	N-TEV-linker in (in pSB1C3) + PIF3	159.6
P364	N-TEV-linker in (in pSB1C3) + PIF3	200.1
P365	PCR of IRES + (in pSB1C3) backbone	136.0
P366	PCR of IRES + (in pSB1C3) backbone	153.7

Analytical digestion with EcoRI and PstI

reagent	volume
CutSmart buffer	2 µl
EcoRI	0.25 µl
PstI	0.25 µl
ddH2O	15 µl
plasmid DNA	2.5 µl
Total	20 µl

60 min incubation, 37 °C

Gel elektrophoresis on 1% agarose gel

1 kbp DNA ladder	P361	P362	P363	P364	P365	P366
	as expected	as expected	as expected	as expected	as expected	as expected

Dpn1 digestion and transformation of QCII of pActin (insertion) + analytical Gel

Investigator: Jeff

Aim of the experiment: Dpn1 digestion and transformation of QCII of pActin (insertion)

Procedure:

1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new ampicillin plate.

0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid

Electrophoresis on 1% agarose gel

1 kb dna ladder	P359	P360
	successful	successful

Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)

Investigator: Florian

Aim of the experiment: Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)

Procedure: DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The samples received the following barcodes:

Label	Barcode1	Barcode2
P266	FR02305766 fw	FR02305767 rv
P268	FR02305768 fw	FR02305769 rv
P270	FR02305770 fw
P272	FR02305771 fw
P361	FR02305772 fw	FR02305773 rv
P364	FR02305774 fw	FR02305775 rv
P366	FR02305776 fw	FR02305777 rv

Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)

Investigator: Johanna

Aim of the experiment: Retransformation of P270 (IgKappa-SigP_SpyCatcher in pSB1C3), P272 (IgKappa-SigP_SpyTag in pSB1C3), P308 (PhyB_36AALinker_C-TEV in pSB1C3) and P366 (IRES in pSB1C3)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1.5 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of cell suspension were plated on chlorampenicol agar

The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.

Ligation of F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES)

Investigator: Florian, Jeff

Aim of the experiment: Ligation of F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES).

Procedure:

Ligation batch for F92 + F130 (IgKappa-SigP + Laccase)

volume	reagent
0.86 µl	F92 (116.7 ng/µl, 2144 bp)
7.55 µl	F130 (28.3 ng/µl, 1527 bp)
8.59 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109 + F130 (SERK-SigP + Laccase)

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2177 bp)
7.44 µl	F130 (28.3 ng/µl, 1527 bp)
8.64 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES)

volume	reagent
0.82 µl	F113 (122.2 ng/µl, 5281 bp)
2.17 µl	F131 (16.4 ng/µl, 627 bp)
14.01 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F115 + F131 (TEV + IRES)

volume	reagent
1.76 µl	F115 (56.9 ng/µl, 2794 bp)
4.1 µl	F131 (16.4 ng/µl, 627 bp)
11.14 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD)

Investigator: Florian, Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES)

Investigator: Flo, Jeff

Aim of the experiment: Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES).

Procedure:

volume	reagent
14 µl	P308
4 µl	CutSmart Buffer (10x)
1 µl	SpeI-HF (20 U/µl)
1 µl	PstI-HF (20 U/µl)
20 µl	ddH₂O
=40 µl	TOTAL

volume	reagent
14 µl	P348
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV (20 U/µl)
1 µl	SpeI-HF (20 U/µl)
20 µl	ddH₂O
=40 µl	TOTAL

volume	reagent
20 µl	P366
4 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated for 2.5 h at 37 °C.

4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.

Preparative geleletrophoresis was performed at 90 V for 60 min.

Lane	Digestion of P308 with SpeI & PstI	1 kbp DNA ladder	Digestion of P348 with NgoMIV & SpeI	Digestion of P366 with XbaI & PstI
result	2 further unexpected bands; seems that PstI is contaminated with a prefix restriction enzyme; used PstI enzyme batch is discareded; upper band was cut out		as expected; lower band was cut out	as expected; lower band was cut out

Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII

Investigator: Jeff

Aim of the experiment: Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII for plasmid preparation

Procedure:

F126+F127 (NLS+ThermoNuc) 7 clones due to high number of colonies on negative ctrl
AlcR QCII and npt-cassette QCII 2 clones each

Tuesday, June 25th

Sequencing of P344 pASK-Flua(Trippelmutante)

Investigator: Jeff

Aim of the experiment: Sequencing of P344. Concentration was determined by Nanodop to 132 ng/µl

Procedure:

DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer). The samples received the following barcodes:

Label	Barcode1
P344 with F83 (primer from Skerra lab! not fragment 83 from iGEM!)	FR02305765

Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
empty tube was washed with 2 µl water and the volume was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of cell suspension were plated on chlorampenicol agar
The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.

Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease)

Investigator: Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
F126+F127 clone 1	P367
F126+F127 clone 2	P368
F126+F127 clone 3	P369
F126+F127 clone 4	P370
F126+F127 clone 5	P371
F126+F127 clone 6	P372
F126+F127 clone 7	P373
QCII npt clone 1	P374
QCII npt clone 2	P375
QCII AlcR clone 1	P376
QCII AlcR clone 2	P377

Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI

Investigator: Louise, Rosario, Florian

Aim of the experiment:Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI.

Procedure:

Master-Mix for P367-P373 and control P249

volume	reagent
20 µl	CutSmart Buffer (10x)
2.5 µl	EcoRI
2.5 µl	PstI
150 µl	ddH2O
=175 µl	TOTAL

Master-Mix for P374-P375 and control P316

volume	reagent
8 µl	CutSmart Buffer (10x)
1 µl	XbaI
1 µl	PstI
60 µl	ddH2O
=70 µl	TOTAL

Master-Mix for P376-P377 and control P342

volume	reagent
8 µl	CutSmart Buffer (10x)
1 µl	XbaI
1 µl	SpeI
60 µl	ddH2O
=70 µl	TOTAL

Batch for analytical digestion of P367-P377 and controls

volume	reagent
17.5 µl	Master-Mix
2.5 µl	Plasmid DNA
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane	1 kbp DNA ladder	Digestion of P367 with EcoRI and PstI	Digestion of P368 with EcoRI and PstI	Digestion of P369 with EcoRI and PstI	Digestion of P370 with EcoRI and PstI	Digestion of P371 with EcoRI and PstI	Digestion of P372 with EcoRI and PstI	Digestion of P373 with EcoRI and PstI	Digestion of P249 with EcoRI and PstI
result		as expected	as expected	not as expected	not as expected	as expected	as expected	not as expected	as expected

Lane	1 kbp DNA ladder	Digestion of P316 with XbaI and PstI	Digestion of P374 with XbaI and PstI/SpeI	Digestion of P374 with XbaI and PstI/SpeI	Digestion of P375 with XbaI and PstI/SpeI	Digestion of P375 with XbaI and PstI/SpeI
Digestion of P376 with XbaI and SpeI	Digestion of P377 with XbaI and PstI/SpeI	Digestion of P377 with XbaI and PstI/SpeI	Digestion of P342 with XbaI and SpeI
result	not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated	not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated	not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated	not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated	not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
as expected	not fully digested	not fully digested	as expected

Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI

Investigator: Louise, Florian

Aim of the experiment: Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI .

Procedure:

Batch for P26

volume	reagent
20 µl	Plasmid DNA
4 µl	CutSmart Buffer
1 µl	EcoRI
1 µl	KpnI
14 µl	ddH₂O
=40 µl	TOTAL

Batch for P35

volume	reagent
7 µl	Plasmid DNA
4 µl	CutSmart Buffer
1 µl	EcoRI
1 µl	KpnI
27 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and the samples were loaded on a 1% agarose gel.

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane	Digestion of P26 with EcoRI and KpnI	100 bp DNA ladder	Digestion of P26 with EcoRI and KpnI	1 kbp DNA ladder
result	not as expected, no band was cut out		as expected; upper band was cut out, F132

Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)

Investigator: Rosario

Aim of the experiment: Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES).

Procedure:

2 clones were picked for each ligation product and for the biobrick
1 clone was picked for each ReTrafo

Wednesday, June 26th

Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366, lucBB

Investigator: Ingmar

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366 and lucBB.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

The resulting DNA were eluted in tubes labeled as follows:

Content	New Tube
P270 Retrafo	P379
F113+F131	P380
F109+F130	P381
P308 Retrafo	P382
F115+F131	P383
F92+F130	P384
F113+F131	P385
F115+F131	P386
F92+F130	P387
P272 Retrafo	P388
P366	P389
luc BB	P390
F109+F130	P391
luc BB	P392

Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI

Procedure:

volume	reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	XbaI
0.25 µl	PstI
15 µl	ddH2O
2.5 µl	Plasmid DNA (P316,P374,P375)
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane:	1000 bp DNA ladder	Digestion of P316 with XbaI and PstI	Digestion of P374 with XbaI and PstI	Digestion of P375 with XbaI and PstI
Result:		corrupt	corrupt	corrupt

Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)

Investigator: Rosario

Aim of the experiment: Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3).

Procedure:

1 clone was picked for each ReTrafo

Sequencing of P367 (NLS-Thermonuclease), P376 (AlcR QC II), P374 (npt-casette QC II), P348 (Laccase), P350 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P352 (SERK-SigP_SpyCatcher_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P354 (SERK-SigP_NanoLuc_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P356 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-TEV-site-linker_SERK-TMD-8AAlinker_GFPmut1), P26 (DREB1C promoter)

Investigator: Rosario

Aim of the experiment: Sequencing of P367 (NLS-Thermonuclease), P376 (AlcR QC II), P374 (npt-casette QC II), P348 (Laccase), P350 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P352 (SERK-SigP_SpyCatcher_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P354 (SERK-SigP_NanoLuc_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P356 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-TEV-site-linker_SERK-TMD-8AAlinker_GFPmut1), P26 (DREB1C promoter).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2
P367	FR02305740 (O3)
P376	FR02305741 (O3)	FR02305742 (O4)
P374	FR02305743 (O3)	FR02305744 (O4)
P348	FR02305745 (O3)	FR02305746 (O4)
P350	FR02305747 (O3)	FR02305748 (O4)
P352	FR02305749 (O3)	FR02305750 (O4)
P354	FR02305751 (O3)	FR02305752 (O4)
P356	FR02305753 (O3)	FR02305754 (O4)
P358	FR02305755 (O3)	FR02305756 (O4)
P26	FR02305757 (O4)

Thursday, June 27th

Sequencing of P387 (IgkKappa-SigP_Laccase (QCV, RFC25)), P391 (SERK-SigP_Laccase (QCV, RFC25))and P355 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 clone 2)

Investigator: Ingmar

Aim of the experiment: Sequencing of P387 (IgkKappa-SigP_Laccase (QCV, RFC25)), P391 (SERK-SigP_Laccase (QCV, RFC25))and P355 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 clone 2).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2
P387	FR02305758 (O3)	FR02305759 (O4)
P391	FR02305760 (O3)
P355	FR02305761 (O3)	FR02305762 (O4)

Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc) Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

150 µl of competent cells were added to the tubes containing about 1 µl of plasmid DNA and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Rosario, Flo

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin).

Procedure:

Operational sequence:

Reaction batch with Q5 Mastermix:

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl	10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl	Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	62 °C	30 s
	72 °C	45 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Analytical gelelectrophoresis of F133

Investigator: Jeff

Procedure: Analytical gelelectrophoresis of F133 (PCR product of F143 with O96 & O97) to check the quality of the performed PCR.

Procedure:

4.5 µl of F133 were mixed together 0.5 µl of DNA loading buffer (10x)

Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.

Lane:	1000 bp DNA ladder	F133
Result:		as expected; but byproduct visible

Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392 with EcoRI and PstI

Volume	Reagent
2 µl	CutSmart Buffer (10x)
0.25 µl	EcoRI
0.25 µl	PstI
15 µl	ddH2O
2.5 µl	Plasmid DNA
=20 µl	TOTAL

Incubation for 60 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min

Lane:	1000 bp DNA ladder	Digestion of P381 with EcoRI and PstI	Digestion of P391 with EcoRI and PstI	Digestion of P347 with EcoRI and PstI	Digestion of P384 with EcoRI and PstI	Digestion of P387 with EcoRI and PstI
Result:		as expected	as expected	not fully digested	as expected	as expected

Lane:	1000 bp DNA ladder	Digestion of P380 with EcoRI and PstI	Digestion of P385 with EcoRI and PstI	Digestion of P309 with EcoRI and PstI	Digestion of P383 with EcoRI and PstI	Digestion of P386 with EcoRI and PstI	Digestion of P390 with EcoRI and PstI	Digestion of P392 with EcoRI and PstI
Result:		as expected	as expected	as expected	as expected	as expected	as expected	as expected

Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S)

Investigator: Flo, Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S).

Procedure:

Batch for preparative digestion of effectors (P328, P330, P332, P334, P336, P338) with AgeI and PstI)

volume	reagent
20 µl	Plasmid DNA
4 µl	CutSmart Buffer (10x)
1 µl	Enzyme 1 (20 U/µl)
1 µl	Enzyme 2 (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Plasmid	Name	Enzyme 1	Enzyme 2
P391	SERK-SigP_Laccase	AgeI	PstI
P326	Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1	NgoMIV	PstI
P385	PhyB_36AAlinker_C-TEV_IRES	SpeI	PstI
P361	N-TEV_36AAlinker_PIF6	XbaI	PstI
P364	N-TEV_36AAlinker_PIF3	XbaI	PstI
P266	IgKappa-SigP_NanoLuc	EcoRI	AgeI
P388	IgKappa-SigP_SpyTag	AgeI	PstI
P247	SpyCatcher	EcoRI	NgoMIV
P379	IgKappa-SigP_SpyCatcher	AgeI	PstI
P374	npt-casette	EcoRI	XbaI
P165	t35S	EcoRI	SpeI

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane	Digestion of P391 with AgeI & PstI	1kb DNA ladder	Digestion of P326 with NgoMIV & PstI	Digestion of P385 with SpeI & PstI
Results	sideproducts?!; upper band was cut out		as expected, lower band was cut out	as expected

Lane	Digestion of P361 with XbaI & PstI	1kb DNA ladder	Digestion of P364 with XbaI & PstI	Digestion of P266 with EcoRI & AgeI
Results	as expected, lower band was cut out		as expected, lower band was cut out	as expected, lower band was cut out

Lane	Digestion of P388 with AgeI & PstI	1kb DNA ladder	Digestion of P382 with NgoMIV & PstI	Digestion of P247 with EcoRI & NgoMIV
Results	as expected		not expected, wrong plasmid	as expected

Lane	Digestion of P379 with AgeI & PstI	1kb DNA ladder	Digestion of P374 with EcoRI & XbaI	Digestion of P165 with EcoRI & SpeI
Results	as expected		as expected	as expected, but only little DNA

Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) and F143 + F144 (npt-csette + t35S)

Investigator: Flo, Rosario

Aim of the experiment: Ligation of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) and F143 + F144 (npt-csette + t35S).

Procedure:

Ligation batch for F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) (vector:insert = 1:3):

volume	reagent
0.83 µl	F134 (120.6 ng/µl, 1630 bp)
10.29 µl	F135 (17.8 ng/µl, 996 bp)
5.88 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6) (vector:insert = 1:3):

volume	reagent
0.5 µl	F136 (201.2 ng/µl, 5916 bp)
4.6 µl	F137 (8.5 ng/µl, 771 bp)
11.9 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3) (vector:insert = 1:3):

volume	reagent
0.5 µl	F136 (201.2 ng/µl, 5916 bp)
4.07 µl	F138 (9.6 ng/µl, 771 bp)
12.43 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F143 + F144 (npt-csette + t35S) (vector:insert = 1:3):

volume	reagent
0.53 µl	F143 (188.8 ng/µl, 3587 bp)
6.05 µl	F144 (3.0 ng/µl, 217 bp)
10.42 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Negative control was prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed overnight at 16 °C.

Friday, June 28th

Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed, for re-transformation, 1 µl of P388 was added to 150 µl of competent cells

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

QuikChange III of AlcR (P376)

Investigator: Andi, Johanna

Aim of the experiment: QuikChange III of AlcR (P376)

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P376), (1:120 diluted)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O84 (=10 µM)
1 µl	1:10 dilution of O85 (=10 µM)
19 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
		52 °C	1 min
		68 °C	5 min
3		4 °C	hold

Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)

Investigator: Katrin

Aim of the experiment: Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)

Procedure:

reaction batch for P388(Ig-Kappa-SigP_SpyTag)

volume	reagent
20 µl	P388
4 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

reaction batch for P246(Nanoluc)

volume	reagent
20 µl	P246
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV-HF (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

reaction batch for F133(PCR of pActin)

volume	reagent
25 µl	F133
5 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	PstI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Reaction batches were incubated for 3 h at 37 °C.

4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.

Preparative geleletrophoresis was performed at 90 V for 60 min.

Lane	Digestion of F133 with XbaI & PstI	1 kbp DNA ladder	Digestion of P246 with NgoMIV,PstI	Digestion of P388 with AgeI, PstI
result	as expected (but smaller fragment visible), upper band was cut out		as expected, lower band was cut out	as expected

Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)

Investigator: Katrin

Aim of the experiment: Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)

Procedure:

Ligation batch for F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc)

volume	reagent
0.90 µl	F146 (111.2 ng/µl, 2189 bp)
3.99 µl	F147 (17.5 ng/µl, 510 bp)
12.11 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F145+F42 (pActin + pSB1C3) (batch: 30 ng vector!)

volume	reagent
23.35 µl	F145 (2.5 ng/µl, 1353 bp)
0.41 µl	F42 (73.5 ng/µl, 2086 bp)
2.7 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=27 µl	TOTAL

Negative controls were prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature.

Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc

Procedure:

one clone per plate was picked
4 ml LB-medium, 4 µl chloramphenicol

Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel

Investigator: Katrin

Aim of the experiment: Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel

Procedure:

1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellchloramphenicol plate.

0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid

Electrophoresis on 1% agarose gel

Lane	1 kbp DNA ladder	QCIII product of AlcR
Results		as expected

Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103))

Investigator: Jeff

Aim of the experiment: Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103)).

Procedure:

25 µL of 100 pmol/µl of mMCSII_fw (O98) and mMCSII_rv (O99) were pooled and mixed together/25 µL of 100 pmol/µl of GGGGSx5_TEV_fw (O102) and GGGGSx5_TEV_rv (O103) were pooled and mixed together/

Heating up to 95 °C for 5 min

Cooling at RT in a styropor box overnight

Saturday, June 29th

Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)

Procedure:

Miniprep according to QIAGEN QIAprep Kit

Transformation of E. coli XL1-blue with ligation product F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)

Investigator: Katrin

Aim of the experiment: Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)

Procedure:

reaction batch

volume	reagent
20 µl	P402
4 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF (20 U/µl)
1 µl	SpeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated for 3 h at 37 °C.

Preparative gelelectrophoresis of F148, F149 and digestion product of P402

Investigator: Jeff

Aim of the experiment: Preparative gelelectrophoresis of F148, F149 and digestion product of P402.

Procedure:

Preparative gelelectrophoresis was performed on a 1% agarose gel at 90 V for 45 min.

Lane:	1 kbp DNA ladder	F148	F149	Digestion of P402 with AgeI & SpeI
Result:		as expected	as expected	as expected (lower band was cut out)

Gelpurified products were extracted via QIAquick gel extraction kit, QIAGEN.

PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA)

Investigator: Jeff

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA).

Procedure:

Operational sequence:

Reaction batch with Q5 Mastermix for P143 (pActin amplification):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl	10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl	Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

Reaction batch with Q5 Mastermix for P344 (FluA triple mutant amplification):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O104 (FluA_tripmut_fw)
2.5 µl	10 µM Reverse Primer O105 (FluA_tripmut_rv)
1 µl	Plasmid DNA (P344)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme for P143 (pActin amplification):

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	62 °C	30 s
	72 °C	45 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

The PCR program was performed after following scheme for P344 (FluA triple mutant amplification)

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	69 °C	30 s
	72 °C	16 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Sunday, June 30th

Miniprep of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3), F143 + F144 (npt-csette + t35S) and ReTraFo of P388 (IgKappa-SigP_SpyTag)

Investigator: Andi

Aim of the experiment: Miniprep of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3), F143 + F144 (npt-csette + t35S) and ReTraFo of P388 (IgKappa-SigP_SpyTag).

Procedure:

Miniprep according to QIAGEN QIAprep Kit

Analytical digestion and gelelectrophoresis of P403 – P418

Investigator: Rosario

Aim of the experiment: Analytical digestion and gelelectrophoresis of P403 – P418.

Procedure:

23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
46 µl	CutSmart Buffer (10x)
5.75 µl	EcoRI-HF (20 U/µl)
5.75 µl	PstI-HF (20 U/µl)
345 µl	ddH₂O
=402.5 µl	TOTAL

23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
17.5 µl	Mastermix for EcoRI & PstI
2.5 µl	Plasmid DNA (P403 – P418)
=20 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2.22 µl of DNA loading buffer (10x) was added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	1000 bp DNA ladder	Digestion of P403 with EcoRI & PstI	Digestion of P404 with EcoRI & PstI	Digestion of P405 with EcoRI & PstI	Digestion of P406 with EcoRI & PstI	Digestion of P407 with EcoRI & PstI	Digestion of P408 with EcoRI & PstI	Digestion of P409 with EcoRI & PstI	Digestion of P410 with EcoRI & PstI	Digestion of P374 with EcoRI & PstI	F153	F154
Result:		corrupt	as expected	as expected	as expected	corrupt	corrupt	corrupt	corrupt	as expected (control)	no PCR product; corrupt	as expected

Lane:	1000 bp DNA ladder	Digestion of P411 with EcoRI & PstI	Digestion of P412 with EcoRI & PstI	Digestion of P246 with EcoRI & PstI (Control)	Digestion of P413 with EcoRI & PstI	Digestion of P414 with EcoRI & PstI	Digestion of P391 with EcoRI & PstI (Control)
Result:		as expected	as expected	as expected	as expected	as expected	as expected

Lane:	1000 bp DNA ladder	Digestion of P415 with EcoRI & PstI	Digestion of P416 with EcoRI & PstI	Digestion of P417 with EcoRI & PstI	Digestion of P418 with EcoRI & PstI	Digestion of P385 with EcoRI & PstI
Result:		as expected	as expected	as expected	as expected	as expected (Control)

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Ingmar

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin). Procedure:

Operational sequence:

Reaction batch with Q5 Mastermix for P143 (pActin amplification):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl	10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl	Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme for P143 (pActin amplification):

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	62 °C	30 s
	72 °C	45 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 60 min using NEB 2-Log DNA ladder.

Lane:	1 kbp DNA ladder	1:1000 P143	1:1000 P143	1:10 P143
Result:		length as expected, but low concentration, small amount of side products	length as expected, but low concentration, small amount of side products	length as expected; higher concentration but increased amount of side products, too. Fragment was labelled F155.

Preparative digestion and gelelectrophoresis of P367 (NLS+thermonuclease), P416 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6), P418 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 ), P404 (t35S_npt-casette), P395 (SpyTag)and F154 (FluA triple mutant)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P367 (NLS+thermonuclease), P416 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6), P418 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 ), P404 (t35S_npt-casette), P395 (SpyTag)and F154 (FluA triple mutant).

Procedure:

Batch for preparative digestion of P404 with EcoRI and XbaI, P395 with NgoMIV and SpeI, F154 with XbaI and AgeI, P367 with XbaI and NgoMIV, P416 with SpeI and Pst I and P418 with SpeI and PstI

volume	reagent
20 µl	Plasmid DNA
4 µl	CutSmart Buffer (10x)
1 µl	Enzyme 1 (20 U/µl)
1 µl	Enzyme 2 (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

volume	reagent
25 µl	PCR product
5 µl	CutSmart Buffer (10x)
1 µl	Enzyme 1 (20 U/µl)
1 µl	Enzyme 2 (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Plasmid	Name	Enzyme 1	Enzyme 2
P404	t35S_npt-casette	EcoRI	XbaI
P395	SpyTag	NgoMIV	SpeI
P418	PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6	SpeI	PstI
P367	NLS+thermonuclease	XbaI	NgoMIV
P416	PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6	SpeI	PstI
F154	FluA triple mutant	XbaI	AgeI

Incubation for 2.5 h at 37 °C.

4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane	Digestion of P367 with XbaI & NgoMIV	1kb DNA ladder	Digestion of P416 with SpeI & PstI	Digestion of P418 with SpeI & PstI
Results	as expected		as expected	as expected

Lane	Digestion of P404 with EcoRI & XbaI	1kb DNA ladder	Digestion of P395 with NgoMIV & SpeI	Digestion of F154 with XbaI & AgeI
Results	as expected		as expected; lower band was extracted	as expected

Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Andi

Aim of the experiment: Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

Ligation batch for F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3):

volume	reagent
2.03 µl	F123 (49.3 ng/µl, 2086 bp)
14.97 µl	F152 (488.2 ng/µl, 102 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3):

volume	reagent
0.63 µl	F156 (158.3 ng/µl, 2596 bp)
16.37 µl	F152 (488.2 ng/µl, 102 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):

volume	reagent
0.67 µl	F157 (148.7 ng/µl, 6711 bp)
1.71 µl	F131 (16.4 ng/µl, 627 bp)
14.62 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):

volume	reagent
0.76 µl	F158 (132.4 ng/µl, 6711 bp)
1.71 µl	F131 (16.4 ng/µl, 627 bp)
14.59 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3):

volume	reagent
1.78 µl	F159 (56.1 ng/µl, 3812 bp)
15.22 µl	F151 (229.4 ng/µl, 37 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F150 + F160 (SERK-SigP_Nanoluc_SpyTag):

volume	reagent
0.59 µl	F150 (169.3 ng/µl, 2660 bp)
16.41 µl	F160 (109 ng/µl, 39 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F123 + F161 (FluA triple mutant in pSB1C3):

volume	reagent
2.03 µl	F123 (49.3 ng/µl, 2086 bp)
2.58 µl	F161 (29.1 ng/µl, 522 bp)
12.39 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Week 11

Monday, July 1st

Everyday I'm swaffeling

Investigator: Le swaffleur passive

Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter)

Investigator: Ingmar

Aim of the experiment: Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter).

Procedure:

Batch for preparative digestion of F155 (pActin PCR product) with XbaI and PstI

volume	reagent
28.5 µl	F155
4 µl	CutSmart Buffer (10x)
1 µl	XbaI
1 µl	PstI
5.5 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P393 (Stress inducible Promoter)with EcoRI and SpeI"

volume	reagent
20 µl	P393
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	SpeI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Incubation for 2.5 h at 37 °C.

4.5 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Lane	1kb DNA ladder	Digestion of F155 with XbaI & PstI	Digestion of P393 with EcoRI & SpeI
Results		as expected, the upper band was cut out	as expected, the lowest band was cut out

Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit

Ligation of F162 (pActin) + F42(pSB1C3 backbone)

Investigator: Ingmar

Aim of the experiment: Ligation of F162 (pActin) + F42(pSB1C3 backbone)

Procedure:

Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)

volume	reagent
1.36 µl	F42 (100 ng)
6.64 µl	F162
91 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (NEB)
=20 µl	TOTAL

Negative controls were prepared by replacing the volume of the insert by ddH₂O.

The ligation was performed for 1 hour at room temperature; the ligation batch was transferred to 16 °C afterwads over night.

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Tuesday, July 2nd

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of F123 + F152, F157 + F131, F158 + F131, F159 + F151 and F123 + F161

Investigator: Louise

Aim of the experiment: Picking of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).

Procedure:

Picked colonies were transfered into 5 ml LB-medium with 5 µl of Chloramphenicol and incubated for at 37 °C in the 180 rpm cell-culture shaker.

Ligation of F162 (pActin) + F42(pSB1C3 backbone) and of F163 (stress inducible Promoter + RFP ) + F159 (t35S-NPT-pSB1C3)

Investigator: Ingmar

Aim of the experiment: As the ligation of the prmoter done on Monday failed, it is repeated with an decreased overall volume resulting in an increased DNA concentration: Ligation of F162 (pActin) + F42(pSB1C3 backbone). Furthermore the stress inducible Promoter cloned to RFP (F163) should be ligated into a pSB1C3 in front og t35S and NPT.

Procedure:

Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)

volume	reagent
1.36 µl	F42 (100 ng)
6.64 µl	F162
1 µl	T4 ligase buffer (10x)
1 µl	T4 ligase
=10 µl	TOTAL

The reaction was prepared twice: one time using a ligase provided by NEB and one time using a ligase providied by Thermo Scientific. Negative controls were prepared by replacing the volume of the insert by ddH₂O.

Ligation batch for ligation of F163 (stress inducible promoter + RFP) + F159(t35S-NPT-pSB1C3 backbone)

volume	reagent
1.79 µl	F159 (100 ng)
8.61 µl	F163
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase
6,6 µl	H2O
=20 µl	TOTAL

The ligation was performed for 2 hours at room temperature; the ligation batch was transferred to 16 °C afterwads over night.

Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3)

Investigator: Ingmar

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

PCR of P143 (pLuc-Actin, Amplification of pActin)

Investigator: Ingmar

Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin). The PCR product was phosphorylated afterwards to test blunt end cloning into a vector. Procedure:

Operational sequence:

Reaction batch for PCR with Q5 Mastermix for P143 (pActin amplification):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl	10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl	Plasmid DNA (P143)- 1:10 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme for P143 (pActin amplification):

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	62 °C	30 s
	72 °C	45 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Reaction batch for phosphorylation of thePCR product:

volume	reagent
15 µl	PCR product
2 µl	10 x forward buffer A
2 µl	10 mM ATP
1 µl	Polynucleotide Kinase (Thermo Scientific)
=20 µL	TOTAL

The reaction was performed for 1 h at 37 °C. Afterwards the mixture was inactivated by heat for 10 min at 75 °C.

Quickchange mutagenesis of P143 (Actin promoter)

Investigator: Ingmar

Aim of the experiment: Deliting the forbidden EcoRI restriction site in the promoter.

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid P143 template
0.5 µl	1:10 dilution of O106
19. 5 µl	ddH2O
1 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid P143 template
0.5 µl	O107
19.5 µl	ddH2O
1 µl	dNTP mix
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		67°C	6,5 min

Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied. Furthermore 1 µl Pfu Ultra II DNA polymerase was added.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag)

Investigator: Louise, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag).

Batch for preparative digestion of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):

volume	reagent
20 µl	P352
4 µl	CutSmart Buffer (10x)
1 µl	SpeI
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):

volume	reagent
20 µl	P358
4 µl	CutSmart Buffer (10x)
1 µl	SpeI
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P395 (SpyTag):

volume	reagent
20 µl	P395
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV
1 µl	SpeI
14 µl	ddH₂O
=40 µl	TOTAL

Incubation at 37 °C; 2.5 h

4.44 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches, digested with NgoMIV & SpeI (P395), after digestion and they were loaded on a 1% agarose gel.

Preparative gelelectrophoresis was performed at 90 V for 60 min.

Reaction batches which were digested with SpeI & PstI were purified with QIAquick PCR purification kit, QIAGEN.

Lane	1kb DNA ladder	Digestion of P395 with NgoMIV & SpeI
Results		as expected; lower band was cut out

Extracted via QIAquick gel extraction kit, QIAGEN.

Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES) and F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)

Investigator: Jeff

Aim of the experiment: Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).

Procedure:

Ligation batch for F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:

volume	reagent
1.22 µl	F164 (81.7 ng/µl, 3524 bp)
3.25 µl	F131 (16.4 ng/µl, 627 bp)
12.53 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:

volume	reagent
1.65 µl	F165 (60.7 ng/µl, 3224 bp)
3.56 µl	F131 (16.4 ng/µl, 627 bp)
11.79 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at RT for 1 h.

Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)

Investigator: Jeff

Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).

Procedure:

Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:

volume	reagent
0.59 µl	F150 (169.3 ng/µl, 2660 bp)
2.67 µl	F166 (3.3 ng/µl, 39 bp)
13.79 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Investigator: Ingmar, Jeff

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).

The resulting tubes were labeled as P420–P432.

Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)

Investigator: Jeff

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).

Procedure:

Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight

Wednesday, July 3rd

Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)

Investigator: Ingmar

Aim of the experiment: Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).

Procedure:

Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 5 mL of LB-medium + 5 µL chloramphenicol(1000x).

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.

Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).

The resulting tubes were labeled as P433 – P435.

Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Analytical digestion and gelelectrophoresis of P420 – P435

Investigator: Ingmar

Aim of the experiment: Analytical digestion and gelelectrophoresis of P420 – P435.

Procedure:

Lane:	1000 bp DNA ladder	Digestion of P420 with EcoRI & PstI	Digestion of P421 with EcoRI & PstI	Digestion of F123 with EcoRI & PstI	Digestion of P422 with EcoRI & PstI	Digestion of P429 with EcoRI & PstI	Digestion of P430 with EcoRI & PstI	Digestion of P431 with EcoRI & PstI	Digestion of P432 with EcoRI & PstI	1000 bp DNA ladder
Result:			as expected	as expected	as expected	corrupt	corrupt	corrupt	corrupt

Lane:	1000 bp DNA ladder	Digestion of P424 with EcoRI & PstI	Digestion of P425 with EcoRI & PstI	Digestion of P426 with EcoRI & PstI	Digestion of P427 with EcoRI & PstI	Digestion of P428 with EcoRI & PstI	Digestion of P404 with EcoRI & PstI	Digestion of P433 with EcoRI & PstI	Digestion of P434 with EcoRI & PstI	Digestion of P435 with EcoRI & PstI	Digestion of P367 with EcoRI & PstI	1000 bp DNA ladder
Result:			as expected	as expected	as expected	corrupt	corrupt	corrupt	corrupt	as expected (control)	no PCR product; corrupt	as expected

Thursday, July 4th

Quickchange mutagenesis I of P444 (Actin promoter)

Investigator: Louise

Aim of the experiment: Deleting the forbidden EcoRI restriction site in the promoter.

Procedure: PCR

Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid P444 template
1.0 µl	1:10 dilution of O106
1.0 µl	1:10 dilution of O107
18.0 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
12.5 µl	Q5 Polymerase Mastermix
1 µl	Plasmid P444 template
1.25 µl	1:10 dilution of O106
1.25 µl	1:10 dilution of O107
8.0 µl	ddH2O
1 µl	dNTP mix 50 mM

PCR cycling parameters for batch 1

Cycles	Temperature	Time
1	95 °C	30 sec
20	95°C	30 sec
	52°C	1 min
	68°C	5 min
	4°C	HOLD

PCR cycling parameters for batch 2

Cycles	Temperature	Time
1	98 °C	30 sec
20	98°C	10 sec
	55°C	30 sec
	72°C	4 min
	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

The successful amplification was verified on an agerose gel and the PCR-product was transformed (by Jeff)

Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150

Investigator: Jeff, Katrin

Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150

Procedure:

Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:

volume	reagent
0.59 µl	F150 (169.3 ng/µl, 2660 bp)
2.67 µl	F166 (3.3 ng/µl, 39 bp)
13.79 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of insert F166

Test digestion of pAct in pSB1C3 (p435 to p448)

Investigator: Jeff

App. 500 ng of DNA from 13 different clones were digeted with EcoRI and PstI
Mastermix consisting of 3 µl of each enzyme 30 µl Cutsmart buffer and 120 µl water

p444 and p447 were sent for sequencing

Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls

Investigator: Katrin

Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Preparative digestion of P434, P412, P462, P453

Investigator: Katrin

Aim of the experiment: Preparative digestion of P434, P412, P462, P453

Batch for preparative digestion of P434

volume	reagent
20 µl	P434
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P412

volume	reagent
20 µl	P412
4 µl	CutSmart Buffer (10x)
1 µl	XbaI
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P462

volume	reagent
20 µl	P462
4 µl	CutSmart Buffer (10x)
1 µl	PstI-HF
1 µl	SpeI
14 µl	ddH₂O
=40 µl	TOTAL

Batch for preparative digestion of P453

volume	reagent
20 µl	P453
4 µl	CutSmart Buffer (10x)
1 µl	PstI-HF
1 µl	SpeI
14 µl	ddH₂O
=40 µl	TOTAL

Incubation at 37 °C; 3 h, then put into freezer

Ligation of F142+F147 (IgK-SigP_Spycatcher + NanoLuc) and F141+F139 (SpyCatcher + IgK-SigP_NanoLuc)

Investigator: Louise

Aim of the experiment: Ligation of F142+F147 and F139+F141

Procedure:

Ligation batch for F142+F147:

volume	reagent
0.81 µl	F142 (123.4 ng/µl, 2489 bp)
3.51 µl	F147 (17.5 ng/µl, 510 bp)
12.68 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F141+F139

volume	reagent
1.83 µl	F141 (54.6 ng/µl, 2425 bp)
3.43 µl	F139 (21.0 ng/µl, 583 bp)
11.74 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of inserts F147 and F139

Friday, July 5th

Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Investigator: Katrin

Aim of the experiment: Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Procedure:

Ligation batch for F122+F169

volume	reagent
1.99 µl	F122 (50.3 ng/µl, 2369 bp)
4.32 µl	F169 (18.1 ng/µl, 618 bp)
10.69 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F170+F168

volume	reagent
2.01 µl	F170 (49.7 ng/µl, 4168 bp)
4.11 µl	F168 (11.0 ng/µl, 628 bp)
10.88 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of inserts F169 and F168

Transformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Investigator: Katrin

Aim of the experiment: TTransformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)

Investigator: Katrin

Aim of the experiment: Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
2 colonies were picked for F150+F166 and F139+F141, 8 colonies were picked for F142+F147 (many colonies on negatibe controls)
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.

Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B)

Investigator: Rosario

Aim of the experiment: Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B).

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2	Barcode3	Barcode4
P3	FR02233147 (O5)
P341	FR02233146 (O3)	FR02233145 (O4)	FR02233144 (O100)	FR02233143 (O101)
P453	FR02233142 (O4)
P462	FR02233141 (O4)
P300	FR02233140 (O3)	FR02233139 (O4)	FR02233138 (O110)	FR02233137 (O111)

Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site

Investigator: Jeff

Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.

Procedure:

Reaction batch 1:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid P447 template (1:100)
1.0 µl	1:10 dilution of O106 (=10 µM)
1.0 µl	1:10 dilution of O107 (=10 µM)
18.0 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

Reaction batch 2:

volume	reagent
25 µl	Q5 Polymerase Mastermix
1 µl	Plasmid P447 template (1:100)
2.5 µl	1:10 dilution of O106 (=10 µM)
2.5 µl	1:10 dilution of O107 (=10 µM)
8.0 µl	ddH₂O
1 µl	dNTP mix 50 mM

PCR cycling parameters for batch 1

Cycles	Temperature	Time
1	95 °C	30 sec
20	95°C	30 sec
	52°C	1 min
	68°C	4 min
1	4°C	HOLD

PCR cycling parameters for batch 2

Cycles	Temperature	Time
1	98 °C	30 sec
20	98°C	10 sec
	55°C	30 sec
	72°C	2 min
3	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

QuikChange of pYes (P3)

Investigator: Rosario

Aim of the experiment: QuikChange of pYes (P3)

Reaction batch

volume	reagent	Additional information
2.5 µl	10x Pfu Ultra II buffer
1 µl	Plasmid template (P3), (1:10 diluted)	need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl	1:10 dilution of O5 (=10 µM)
19 µl	ddH2O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 s
2	20	95 °C	30 s
		52 °C	1 min
		68 °C	4 min
3		4 °C	hold

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100)

Investigator: Jeff

Procedure: Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100) to check whether QuikChange might be worked.

Procedure:

4.5 µl of the QuikChange products of P447 (PfuUltraII Polymerase batch and Q5 Polymerase batch (see above)) and P3 (pTEF2 in pTUM100) were mixed together 0.5 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.

Lane:	2 log DNA ladder	QC of P3 with O44 & O45 (PfuUltraII)	QC of P447 with O106 & O107 (PfuUltraII)	QC of P447 with O106 & O107 (Q5)
Result:		as expected	no product visible	no product visible

Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100) (see above).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Saturday, July 6th

Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site

Investigator: Le swaffeleur

Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.

Procedure:

Reaction batch 1:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P447 template (1:100)
1.0 µl	1:10 dilution of O106 (=10 µM)
16.0 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl	TOTAL

Reaction batch 2:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P447 template (1:100)
1.0 µl	1:10 dilution of O107 (=10 µM)
16.0 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl	TOTAL

The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	54°C	1 min
	68°C	4 min
1	4°C	HOLD

After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles	Temperature	Time
1	98 °C	30 sec
20	98°C	10 sec
	54°C	30 sec
	72°C	2 min
3	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3)

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3).

Procedure:

9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 30 min on a 1% agarose gel.

Lane:	2 log DNA ladder	QC of P447 with O106 & O107 (PfuUltraII, without DMSO)	QC of P447 with O106 & O107 (PfuUltraII, with DMSO)
Result:		as expected	as expected

Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3)

Investigator: Jeff, Le Swaffeleur

Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.

Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

2 colonies were picked for F122+F169 and F170+F168.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Sunday, July 7th

Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)

Investigator: Jeff, Katrin

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).

The resulting tubes were labeled as P475 and P476.

Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)

Investigator: Jeff, Katrin

Aim of the experiment: Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)

16x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
17.5 µl	Mastermix for EcoRI & PstI
2.5 µl	Plasmid DNA (P464 – P476, P402)
=20 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	1000 bp DNA ladder	Digestion of P464 with EcoRI & PstI	Digestion of P465 with EcoRI & PstI	Digestion of P466 with EcoRI & PstI	Digestion of P467 with EcoRI & PstI	Digestion of P402 with EcoRI & PstI (control)	Digestion of P475 with EcoRI & PstI	Digestion of P476 with EcoRI & PstI
Result:		as expected	as expected	as expected	as expected	as expected	as expected	as expected

Lane:	1000 bp DNA ladder	Digestion of P468 with EcoRI & PstI	Digestion of P469 with EcoRI & PstI	Digestion of P470 with EcoRI & PstI	Digestion of P471 with EcoRI & PstI	Digestion of P472 with EcoRI & PstI	Digestion of P473 with EcoRI & PstI	Digestion of P474 with EcoRI & PstI
Result:		corrupt	corrupt	corrupt	corrupt	corrupt	corrupt	corrupt

PCR of P378 (Proteinphosphotase 1)

Investigator: Katrin, Jeff

Aim of the experiment: PCR of P378 (Proteinphosphotase 1).

Procedure:

Reaction batch for PCR with Q5 Mastermix for P378 (Proteinphosphotase 1):

volume	reagent
25 µl	2x Q5 Master Mix
1 µl	dNTP mix (normally not necessary!)
2.5 µl	10 µM Forward Primer O112 (PP1 for)
2.5 µl	10 µM Reverse Primer O113 (PP1 rev)
1 µl	Plasmid DNA (P378) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme for P378(PP1 amplification):

Cycling parameters:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	62 °C	30 s
	72 °C	30 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

The resulting product was labeled as F174.

Analytical gelelectrophoresis of F174

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F174.

Procedure:

9 µl of the PCR product F174 (=PCR of P378 with O112/O113) products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

Lane:	2 log DNA ladder	F174
Result:		as expected

Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag)

Investigator: Katrin, Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag).

Procedure:

Reaction batch for P422 (FluA triple mutant):

volume	reagent
20 µl	P422
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV
1 µl	SpeI
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P464 (IgKappa-SigP_NanoLuc_SpyCatcher):

volume	reagent
20 µl	P464
4 µl	CutSmart Buffer (10x)
1 µl	SpeI
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P466 (IgKappa-SigP_NanoLuc_SpyTag):

volume	reagent
20 µl	P466
4 µl	CutSmart Buffer (10x)
1 µl	SpeI
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P422 with NgoMIV & SpeI	2 log DNA ladder	Digestion of P464 with XbaI & PstI	Digestion of P466 with XbaI & PstI
Results	as expected; lower band was cut out		as expected; lower band was cut out	as expected; lower band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1)

Investigator: Katrin, Jeff

Aim of the experiment: Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1).

Procedure:

Reaction batch for P379 (IgKappa-SigP_SpyCatcher):

volume	reagent
20 µl	P379
4 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF
1 µl	PstI-HF
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for F174 (Proteinphosphotase 1):

volume	reagent
25 µl	F174
5 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF
1 µl	PstI-HF
18 µl	ddH₂O
=50 µl	TOTAL

Reaction batches were incubated at 37 °C for 4 h.

After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)

Investigator: Jeff

Aim of the experiment: Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).

Procedure:

Ligation batch for F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES

in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), vector:insert = 1:3:

volume	reagent
2.01 µl	F170 (49.7 ng/µl, 4168 bp)
3.50 µl	F179 (12.9 ng/µl, 628 bp)
11.49 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), vector:insert = 1:3:

volume	reagent
1.65 µl	F171 (60.05 ng/µl, 3868 bp)
5.80 µl	F178 (12.4 ng/µl, 928 bp)
9.55 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), vector:insert = 1:3:

volume	reagent
0.61 µl	F176 (163.9 ng/µl, 2489 bp)
3.51 µl	F147 (17.5 ng/µl, 510 bp)
12.88 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), vector:insert = 1:3:

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2117 bp)
2.90 µl	F177 (25.5 ng/µl, 522 bp)
13.18 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F123+F175 (pSB1C3 only + PP1), vector:insert = 1:3:

volume	reagent
0.92 µl	F123 (49.3 ng/µl, 2086 bp)
2.90 µl	F175 (12.3 ng/µl, 972 bp)
3.59 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 22 °C for 1 h

Negative controls were also prepared with water instead of insert.

Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).

8 colonies were picked for QuikChange products of P447 (pActin)and F139+F141 and 4 colonies were picked for ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Week 12

Monday, July 8th

Analytical digestion and gelelectrophoresis of P477-P484 (pActin)

Investigator: Rosario, Ingmar

Aim of the experiment: Analytical digestion and gelelectrophoresis of P477-P484 (pActin in pSB1C3) after QC of forbidden EcoRI Restriction site.

10x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
15 µl	Cut Smart buffer
2 µl	EcoRI
2 µl	PstI
30 µl	ddH2O
=49 µl	TOTAL

The volume of plasmid DNA added was calculated in order to have equal amounts of DNA of approximately 550 ng

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	1000 bp DNA ladder	Digestion of P477 with EcoRI & PstI	Digestion of P478 with EcoRI & PstI	Digestion of P479 with EcoRI & PstI	Digestion of P480 with EcoRI & PstI	Digestion of P481 with EcoRI & PstI (control)	Digestion of P482 with EcoRI & PstI	Digestion of P483 with EcoRI & PstI	Digestion of P484 with EcoRI & PstI
Result:		as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected

all quickchanges were successfull.

Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion

Investigator: Rosario, Ingmar

Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion.

Procedure:

Reaction batch 1:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
2,67 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O108 (=10 µM)
17.330 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl	TOTAL

Reaction batch 2:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
2.67 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O109 (=10 µM)
17.33 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl	TOTAL

The two batches were cycled seperately for the first 10 rounds.

PCR cycling parameters

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	54°C	1 min
	72°C	4.5 min
1	4°C	HOLD

After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles	Temperature	Time
1	98 °C	30 sec
20	98°C	10 sec
	54°C	30 sec
	72°C	2 min
3	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Sequencing of P477, P486, P487, P464, P466, P476, P435

Investigator: Rosario

Aim of the experiment: Sequencing of P477 (Actin promoter), P486, P487, P464, P466, P476, P435.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2
P477	FR02233136 (O3)	FR02233135 (O4)
P486	FR02233134 (O3)
P487	FR02233133 (O3)
P464	FR02233132 (O4)
P466	FR02233131 (O4)
P476	FR02233130 (O3)	FR02233129 (O4)
P435	FR02233128 (O3)

Preparative digestion and gelelectrophoresis of P187 (LMU GFP)

Investigator: Rosario

Aim of the experiment: Preparative digestion and gelelectrophoresis of P187 (LMU GFP).

Procedure:

Reaction batch for P187 :

volume	reagent
20 µl	P187
4 µl	CutSmart Buffer (10x)
1 µl	NgoMIV
1 µl	SpeI
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch was incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P187 with NgoMIV & SpeI	2 log DNA ladder
Results	as expected; lower band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP)

Investigator: Rosario

Aim of the experiment: Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP).

Procedure:

Ligation batch for F109+F180 (SERK-SigP in pSB1C3 + LMU GFP), vector:insert = 1:3:

volume	reagent
4.1 µl	F180 (726 bp)
0.9 µl	F179 (2177 bp)
12 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F92+F180 (IgKappa in pSB1C3 + LMU GFP), vector:insert = 1:3:

volume	reagent
4.15 µl	F180 (726 bp)
0.85 µl	F92 (2144 bp)
12 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 22 °C for 1 h

Negative controls were also prepared with water instead of insert.

Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1))

Investigator: Rosario

Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)).

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

2 colonies were picked for all ligation products except F176+F147 for which 4 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Tuesday, July 9th

Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag)

Investigator: Jeff, Florian

Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).

The resulting tubes were labeled as P488 to P499.

Analytical digestion and gelelectrophoresis of P488-P499 (several ligations)

Investigator: Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of P488-P499 (several ligations).

15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
30 µl	Cut Smart buffer
3.75 µl	EcoRI
3.75 µl	PstI
225 µl	ddH2O
=262,5 µl	TOTAL

17.5 µl master mix and 2.5 µl plasmid DNA were mixed.

Analytical digestion was performed at 37 °C for 1 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	1 kb DNA ladder	Digestion of P488 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI	Digestion of P489 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI	Digestion of P490 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI	Digestion of P491 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI	Digestion of P492 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI	Digestion of P493 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI
Result:		as expected (950 bp)	as expected (950 bp)	as expected (950 bp)	as expected (950 bp)	as expected (972 bp)	as expected (972 bp)

Lane:	1 kb DNA ladder	Digestion of P422 F123+F161 (FluA triple mutant + pSB1C3) with EcoRI & PstI (control)	Digestion of P494 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI	Digestion of P495 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI	Digestion of P496 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI	Digestion of P497 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI	Digestion of P498 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI	Digestion of P499 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI
Result:		as expected (522 bp)	strange	as expected (622 bp)	roughly as expected (2768 bp)	roughly as expected (2768 bp)	roughly as expected (2768 bp)	roughly as expected (2768 bp)

Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion

Investigator: Florian

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion.

Procedure:

9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

Lane:	1 kb DNA ladder	QC of P477
Result:		no product

Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion, second attempt

Investigator: Jeff

Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion.

Procedure:

Reaction batch 1:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
3 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O108 (=10 µM)
17.0 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl	TOTAL

Reaction batch 2:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
3.0 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O109 (=10 µM)
17.0 µl	ddH₂O
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl	TOTAL

The two batches were cycled seperately for the first 25 rounds.

PCR cycling parameters

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	52°C	1 min
	72°C	4.5 min
1	4°C	HOLD

After the ten cycles the two reaction batches were pooled together, 0.5 µl of polymerase and 1 µl of dNTP-mix was added and the reaction was cycled for 25 rounds with the following conditions:

PCR cycling parameters after poooling the reaction batches together:

Cycles	Temperature	Time
1	98 °C	30 sec
20	98°C	10 sec
	52°C	30 sec
	72°C	2 min
3	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Wednesday, July 10th

Preparative digestion of P495 (SERK-SigP_FluA)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P495 (SERK-SigP_FluA).

Procedure:

Reaction batch for P495 (SERK-SigP_FluA):

volume	reagent
20 µl	P495
4 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

After digestion, linearized DNA was purified with QIAquick PCR purification kit, QIAGEN.

Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1).

Procedure:

Reaction batch for P486 ((GGGGS)x5-TEV-site-linker):

volume	reagent
20 µl	P486
4 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	AgeI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P488 (IgKappa-SigP_SpyCatcher_NanoLuc):

volume	reagent
20 µl	P488
4 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P492 (Proteinphosphotase 1)):

volume	reagent
20 µl	P492
4 µl	CutSmart Buffer (10x)
2 µl	NgoMIV (10 U/µl)
2 µl	SpeI (10 U/µl)
12 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P486 with XbaI & AgeI	2 log DNA ladder	Digestion of P488 with XbaI & PstI	Digestion of P492 with NgoMIV & SpeI
Results	as expected; lower band was cut out		as expected; lower band was cut out	as expected; lower band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

Ligation batch for F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), vector:insert = 1:3:

volume	reagent
0.63 µl	F156 (158.3 ng/µl, 2596 bp)
2.88 µl	F182 (4.1 ng/µl, 102 bp)
13.49 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

volume	reagent
0.54 µl	F181 (185.7 ng/µl, 2705 bp)
6.21 µl	F135 (17.8 ng/µl, 996 bp)
10.25 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), vector:insert = 1:3:

volume	reagent
1.65 µl	F171 (60.5 ng/µl, 3859 bp)
1.22 µl	F183 (59 ng/µl, 928 bp)
14.13 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1), vector:insert = 1:3:

volume	reagent
0.92 µl	F109 (108.2 ng/µl, 2117 bp)
2.44 µl	F184 (54.9 ng/µl, 972 bp)
13.64 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Negative controls were also prepared with water instead of insert.

Thursday, July 11th

Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498

Investigator: Jeff

Aim of the experiment: Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498.

Procedure:

The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2	Barcode3
P294			FR02233072 (O100)
P296	FR02233073 (O3)	FR02233074 (O4)	FR02233075 (O100)
P342	FR02233076 (O3)	FR02233077 (O4)	FR02233078 (O100)
P488		FR02233079 (O4)
P492	FR02233080 (O3)	FR02233081 (O4)
P495	FR02233082 (O3)
P496	FR02233083 (O3)	FR02233084 (O4)
P498	FR02233085 (O3)	FR02233086 (O4)

Friday, July 12th

QuikChange of P477 (pActin) to insert missing 60 bp

Investigator: Jeff

Aim of the experiment: QuikChange of P477 (pActin) to insert missing 60 bp.

Procedure:

Reaction batch #1, with 5% DMSO and O108 as oligonucleotide:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O108 (=10 µM)
1 µl	dNTP mix 50 mM
1.25 µl	DMSO
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl	ddH₂O
=25 µl	TOTAL

Reaction batch #2, with 5% DMSO and O109 as oligonucleotide:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O109 (=10 µM)
1 µl	dNTP mix 50 mM
1.25 µl	DMSO
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl	ddH₂O
=25 µl	TOTAL

Reaction batch #3, O108 as oligonucleotide:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O108 (=10 µM)
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl	ddH₂O
=25 µl	TOTAL

Reaction batch #4, O109 as oligonucleotide:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P477 template (1:100)
1.0 µl	1:10 dilution of O109 (=10 µM)
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl	ddH₂O
=25 µl	TOTAL

Every reaction batch type was created twice; one batch of each type was used for the program with 55 °C as annealing temperature and one was used with 65 °C as annealing temperature.

The batches were cycled seperately for the first 10 rounds

PCR cycling parameters with 55 °C annealing temperature:

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	55°C	1 min
	72°C	4.5 min
1	4°C	HOLD

PCR cycling parameters with 65 °C annealing temperature:

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	65°C	1 min
	72°C	4.5 min
1	4°C	HOLD

After the ten cycles the reaction batches (#1 with #2 and #3 with #4) were pooled together and the reaction was cycled for further 20 rounds with the following conditions:

PCR cycling parameters with 55 °C annealing temperature:

Cycles	Temperature	Time
1	95 °C	30 sec
20	95°C	30 sec
	55°C	1 min
	72°C	4.5 min
1	4°C	HOLD

PCR cycling parameters with 65 °C annealing temperature:

Cycles	Temperature	Time
1	95 °C	30 sec
20	95°C	30 sec
	65°C	1 min
	72°C	4.5 min
1	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.

Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion.

Procedure:

9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 100 V for 40 min on a 1% agarose gel.

Lane:	2 log DNA ladder	QC of P477 with DMSO and annealing temperature at 55 °C	QC of P477 with DMSO and annealing temperature at 65 °C	QC of P477 with annealing temperature at 55 °C	QC of P477 with annealing temperature at 65 °C
Result:		no product	no product	no product	no product

Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).

2 colonies were picked for F156+F182, F171+F182, F109+F184 and 6 colonies were picked for ligation product F181+F135.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Saturday, July 13th

Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P508 to P519.

Analytical digestion and gelelectrophoresis of P508 – P519

Investigator: Le Swaffeleur

Aim of the experiment: Analytical digestion and gelelectrophoresis of P508 – P519.

Procedure:

15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
26 µl	Cut Smart buffer
3.25 µl	EcoRI-HF (20 U/µl)
3.25 µl	PstI-HF (20 U/µl)
195 µl	ddH2O
=227.5 µl	TOTAL

17.5 µl master mix and 2.5 µl plasmid DNA (P508 – P519) were mixed.

Analytical digestion was performed at 37 °C for 1 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2 log DNA ladder	P508, digested with EcoRI & PstI	P509, digested with EcoRI & PstI	P510, digested with EcoRI & PstI	P511, digested with EcoRI & PstI	P512, digested with EcoRI & PstI	P513, digested with EcoRI & PstI	P514, digested with EcoRI & PstI	P515, digested with EcoRI & PstI	P516, digested with EcoRI & PstI	P517, digested with EcoRI & PstI	P518, digested with EcoRI & PstI	P519, digested with EcoRI & PstI	2 log DNA ladder
Result:		as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected	as expected

Sunday, July 14th

Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct).

Procedure:

Reaction batch for P428 (MCS-t35S-npt):

volume	reagent
15 µl	P428
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	XbaI (20 U/µl)
19 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P477 (pAct):

volume	reagent
15 µl	P477
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
2 µl	SpeI (10 U/µl)
18 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 4 h.

Preparative gelelectrophoresis was performed at 90 V for 2 h in an 1% agarose gel.

Lane	Digestion of P486 with XbaI & AgeI	2 log DNA ladder	Digestion of P488 with XbaI & PstI	2 log DNA ladder
Results	as expected; intense band was cut out		as expected; lowest band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F185+F186 (pAct + MCS_t35S_npt)

Investigator: Jeff

Procedure:

Ligation batch for F185+F186 (pAct + MCS_t35S_npt), vector:insert = 1:3:

volume	reagent
4 µl	F185 (5000 bp)
4 µl	F186 (1200 bp)
1 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=10 µl	TOTAL

Ligation was performed at 22 °C for 1 h

Negative controls were also prepared with water instead of insert.

Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1).

Procedure:

Reaction batch for P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES):

volume	reagent
20 µl	P430
4 µl	CutSmart Buffer (10x)
2 µl	SpeI (20 U/µl)
1 µl	PstI-HF (20 U/µl)
13 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES):

volume	reagent
20 µl	P431
4 µl	CutSmart Buffer (10x)
2 µl	SpeI (10 U/µl)
1 µl	PstI-HF (20 U/µl)
13 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA):

volume	reagent
20 µl	P509
4 µl	CutSmart Buffer (10x)
2 µl	NgoMIV (10 U/µl)
1 µl	PstI-HF (20 U/µl)
13 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P510 (SERK-SigP_PP1)):

volume	reagent
20 µl	P510
4 µl	CutSmart Buffer (10x)
1 µl	AgeI-HF (20 U/µl)
1 µl	PstI-HF (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P430 with XbaI & AgeI	2 log DNA ladder	Digestion of P431 with XbaI & PstI	Digestion of P509 with NgoMIV & SpeI
Results	as expected		as expected	as expected; lower band was cut out

Lane	2 log DNA ladder	Digestion of P510 with AgeI & PstI
Results		as expected

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff

Aim of the experiment: Ligation of F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1).

Procedure:

Ligation batch for F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), vector:insert = 1:3:

volume	reagent
1.99 µl	F122 (50.3 ng/µl, 2369 bp)
0.98 µl	F189 (79.9 ng/µl, 618 bp)
14.03 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:

volume	reagent
0.45 µl	F190 (224.7 ng/µl, 3155 bp)
5.32 µl	F135 (17.8 ng/µl, 996 bp)
11.23 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Negative controls were also prepared with water instead of insert.

Week 13

Monday, July 15th

Transformation of the genes to be expressend in moss (retransformation) and F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)

Investigator: Jeff

Procedure for the retransformations:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

0.5 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

50 µl of this cell suspension was plated on chlorampenicol plates.

Tuesday, July 16th

Preparative digestion and gelelectrophoresis of all effectors

Investigator: Jeff

Procedure:

Mastermix:

volume	reagent
224	ddH₂O
64 µl	CutSmart Buffer (10x)
16 µl	EcoRI-HF (20 U/µl)
16 µl	PstI-HF (20 U/µl)

20 µl of DNA were mixed with 20 µl of Mastermix and were incubated at 37 °C for 4 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	2 log DNA ladder	Digestion of P20 (GFP) with EcoRI & PstI	2 log DNA ladder	Digestion of P196 (XylE) with EcoRI & PstI	2 log DNA ladder	Digestion of P245 (EreB) with EcoRI & PstI
Results	as expected	as expected	as expected	as expected	as expected	as expected

Lane	2 log DNA ladder	Digestion of P246 (nLuc) with EcoRI & PstI	2 log DNA ladder	Digestion of P330 (SERK-SigP_NLuc) with EcoRI & PstI	2 log DNA ladder	Digestion of P346 (Dehydrochlorinase) with EcoRI & PstI
Results	as expected	as expected	as expected	as expected	as expected	as expected

The digestion of P350 was separated for an additional hour because there might be contaminations of the vector that is smaller than the protein-coding BioBrick we want to express. The separated bands looked like that:

Lane	2 log DNA ladder	Digestion of P350 (EreB-Receptor) with EcoRI & PstI	2 log DNA ladder	Digestion of P354 (nLuc-Receptor) with EcoRI & PstI	2 log DNA ladder	Digestion of P402 (IgK-nLuc) with EcoRI & PstI
Results	as expected	as expected	as expected	as expected	as expected	as expected

Lane	2 log DNA ladder	Digestion of P476 (SpyCatcher-Receptor_IRES_IgK_SpyTag-nLuc) with EcoRI & PstI	2 log DNA ladder	Digestion of P496 (SpyTag-Receptor_IRES_nLuc_SpyCatcher with EcoRI & PstI	2 log DNA ladder	Digestion of P498 (SpyCatcher-Receptor_IRES_IgK_nLuc-SpyTAG) with EcoRI & PstI
Results	as expected	as expected	as expected	as expected	as expected	as expected

Lane	2 log DNA ladder	Digestion of P502 (IgK-GFP) with EcoRI & PstI	2 log DNA ladder	Digestion of P512 (SpyTAG-Receptor-IRES_SpyCatcher-nLuc) with EcoRI & PstI	2 log DNA ladder	Digestion of P516 (FluA-Recetpro) with EcoRI & PstI
Results	as expected	as expected	as expected	as expected	as expected	as expected

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Miniprep of P520 to P525: P520 (Bba_KK509000,Cauliflower Mosaic Virus 35S promoter) P521-525 (pAct(with_del)-MCS-T35S-npt

Investigator: Jeff

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P520 to P525.

Analytical digestion and gelelectrophoresis of P520 - P525

Investigator: Jeff

Procedure:

7x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:

volume	reagent
7 µl	Cut Smart buffer
1.75 µl	EcoRI-HF (20 U/µl)
1.75 µl	PstI-HF (20 U/µl)
38.5 µl	ddH2O
=49 µl	TOTAL

7 µl master mix and 3 µl plasmid DNA were mixed.

Analytical digestion was performed at 37 °C for 1.5bsp;h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2 log DNA ladder	P428, dsted with EcoRI & PstI	P522, digested with EcoRI & PstI	P522, digested with EcoRI & PstI	P523, digested with EcoRI & PstI	P524, digested with EcoRI & PstI	P525, digested with EcoRI & PstI
Result:	control	no insert ;(	no insert ;(	no insert ;(	no insert ;(	no insert ;(	no insert ;(

Wednesday, July 17th

Thursday, July 18th

Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207.

Procedure:

volume	reagent
2.5 µl	Plasmid DNA (P537 – P540)
2 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
15 µl	ddH2O
=20 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2 log DNA ladder	Digestion of P537 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI	Digestion of P538 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI	Digestion of P539 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI	Digestion of P540 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI	F206	F207
Result:		as expected (916 bp)	as expected (916 bp)	as expected (2080 bp)	as expected (2080 bp)	as expected (3484 bp)	as expected (3484 bp)

PCR to insert 60 missing base-pairs into pActin (new method)

Investigator: Jeff

Aim of the experiment: PCR to insert 60 missing base-pairs into pActin (new method)

Procedure:

Reaction batch for PCR with Q5 Mastermixes for P477 (pActin):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl	10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl	Plasmid DNA (P477) - 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl	10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl	Plasmid DNA (P477) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme:

Cycling parameters:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	72 °C	30 s
	72 °C	2 min
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

The resulting product was labeled as F206 for the 1:1000 batch and F207 for the 1:100 batch.

Friday, July 19th

Saturday, July 20th

Sunday, July 21st

Week 14

Monday, July 22nd

Arrival of the correct pActin 5 from Physcomitrella patens from Reski lab

This plasmid was named p541

Tuesday, July 23rd

Wednesday, July 24th

PCR of P541 (PppActin5)

Investigator: Jeff

Aim of the experiment: PCR of P541 (PppActin5).

Procedure:

Reaction batch for PCR with Q5 Mastermix for P541 (PppActin5):

volume	reagent
25 µl	2x Q5 Master Mix
1 µl	dNTP mix (normally not necessary!)
2.5 µl	10 µM Forward Primer O116 (PppAct_f)
2.5 µl	10 µM Reverse Primer O117 (PppAct_r)
1 µl	Plasmid DNA (P541) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL	ddH₂O Water
=50 µL	TOTAL

The content has been mixed with a pipette

The PCR program was performed after following scheme:

Cycling parameters:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	53 °C	30 s
	72 °C	30 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

The resulting product was labeled as F208.

Analytical gelelectrophoresis of F208

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F208.

Procedure:

5 µl of the PCR product F208 (=PCR of P541 with O116/O117) products were mixed with 2 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.

Lane:	2 log DNA ladder	F208
Result:		as expected

Preparative digestion of P208 (PCR of PppActin5)

Investigator: Jeff

Procedure:

Reaction batch for F208 (PCR of PppActin5):

volume	reagent
25 µl	F208
5 µl	CutSmart Buffer (10x)
1 µl	EcoRI-HF
1 µl	SpeI-HF
18 µl	ddH₂O
=45 µl	TOTAL

Reaction batch was incubated at 37 °C for 2 h.

After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.

Ligation and transformation of F209 + F41 (PppActin5 in pSB1C3)

Investigator: Jeff

Procedure:

Ligation batch for F209 (PppActin5), vector:insert = 1:3:

volume	reagent
7 µl	F209 (36.9 ng/µl, ??? bp)
1 µl	F189 (89.9 ng/µl, ??? bp)
1 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=10 µl	TOTAL

Ligation was performed at RT for 1 hour.

Negative controls were also prepared with water instead of insert.

The ligation products were transformed into E. coli XL-1 Blue.

Thursday, July 25th

Picking of E. coli XL1 blue transformed with ligation product of F208 and F41 (PppActin5 in pSB1C3)

Investigator: Jeff

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).

6 colonies were picked for ligation of F208 and F41 (PppActin5 in pSB1C3).

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Friday, July 26th

Miniprep of P542 to P547 (pSB1C2_PppActin5)

Investigator: Jeff

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P542 to P547.

Analytical digestion and gelelectrophoresis of P542 – P547

Investigator: Jeff

Procedure:

volume	reagent
3 µl	Plasmid DNA (P542 – P547)
1 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
5.5 µl	ddH2O
=10 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2 log DNA ladder	Digestion of P542 (PppActin5) with EcoRI & PstI	Digestion of P543 (PppActin5) with EcoRI & PstI	Digestion of P544 (PppActin5) with EcoRI & PstI	Digestion of P545 (PppActin5) with EcoRI & PstI	Digestion of P546 (PppActin5) with EcoRI & PstI	Digestion of P547 (PppActin5) with EcoRI & PstI
Result:		result unclear because insert and backbone have the same length!	result unclear because insert and backbone have the same length!	result unclear because insert and backbone have the same length!	result unclear because insert and backbone have the same length!	result unclear because insert and backbone have the same length!	result unclear because insert and backbone have the same length!

Unfortunately the correctness of the clones could not be seen in this experiment. Thus a second digestion was performed:

volume	reagent
3 µl	Plasmid DNA (P542 – P547)
1 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
0.25 µl	NcoI (form Fermentas
5.25 µl	ddH2O
=10 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2 log DNA ladder	Digestion of P542 (PppActin5) with EcoRI & PstI	Digestion of P543 (PppActin5) with EcoRI & PstI	Digestion of P544 (PppActin5) with EcoRI & PstI	Digestion of P545 (PppActin5) with EcoRI & PstI	Digestion of P546 (PppActin5) with EcoRI & PstI	Digestion of P547 (PppActin5) with EcoRI & PstI
Result:		Clone correct: backbone and PppActin5	Clone wrong: only backbone, no insert	Clone correct: backbone and PppActin5	Clone correct: backbone and PppActin5	Clone wrong: only backbone, no insert	Clone correct: backbone and PppActin5

Sequencing of P544 and P545

Investigator: Ingmar

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1	Barcode2
P544	FR02233095	FR02233087
P545	FR02233093	FR02233094

Saturday, July 27th

Sunday, July 28st

Week 15

Monday, July 29th

QuikChange of P544 (PppActin5)

Investigator: Jeff

Procedure:

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P544 template (1:100)
1.0 µl	1:10 dilution of O118 (=10 µM)
1.0 µl	1:10 dilution of O119 (=10 µM)
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl	ddH₂O
=25 µl	TOTAL

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P544 template (1:100)
1.0 µl	1:10 dilution of O120 (=10 µM)
1.0 µl	1:10 dilution of O121 (=10 µM)
1 µl	dNTP mix 50 mM
0.5 µl	Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl	ddH₂O
=25 µl	TOTAL

PCR cycling parameters with 55 °C annealing temperature:

Cycles	Temperature	Time
1	95 °C	30 sec
10	95°C	30 sec
	55°C	1 min
	72°C	5 min
1	4°C	HOLD

Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.

Tuesday, July 30th

Wednesday, July 31st

Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Reaction batch for P537 (SERK-SigP_SERK-TMD_(GGGGS)-TEV-site-linker_NLS_NucA):

volume	reagent
20 µl	P537
4 µl	CutSmart Buffer (10x)
2 µl	XbaI (20 U/µl)
2 µl	PstI-HF (20 U/µl)
12 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	2 log DNA ladder	Digestion of P537 with XbaI & PstI
Results		as expected; lower band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Resulting fragment was named as F210.

Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Ligation batch for F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:

volume	reagent
0.53 µl	F187 (189.9 ng/µl, 7346 bp)
0.68 µl	F210 (55.3 ng/µl, 916 bp)
15.79 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:

volume	reagent
0.73 µl	F188 (136.7 ng/µl, 7346 bp)
0.68 µl	F210 (55.3 ng/µl, 916 bp)
15.59 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Negative controls were also prepared with water instead of insert.

Thursday, August 1st

Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Friday, August 2nd

Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).

2 colonies were picked for BBa_K864100, BBa_E0020, F187+F210 and F188+F210 each.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.

Saturday, August 3rd

Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P558 to P565.

Analytical digestion and gelelectrophoresis of P558 – P565

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P558 – P565.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume	reagent
20 µl	Cut Smart buffer
2.5 µl	EcoRI-HF
2.5 µl	PstI-HF
150 µl	ddH2O
=175 µl	TOTAL

17.5 µl of the mastermix were mixed together with 2.5 µl of Plasmid DNA (P558 – P565)

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	Digestion of P558 (BBa_K864100)	Digestion of P559 (BBa_K864100)	2 log DNA ladder	Digestion of P560 (BBa_E0020)	Digestion of P561 (BBa_E0020)
Result:	as expected (723 bp)	as expected (723 bp)		as expected (723 bp)	as expected (723 bp)

Lane:	Digestion of P562 (F187+F210)	Digestion of P563 (F187+F210)	2 log DNA ladder	Digestion of P564 (F188+F210)	Digestion of P565 (F188+F210)
Result:	as expected (6191 bp)	as expected (6191 bp)		not as expected; corrupt (6191 bp)	as expected (6191 bp)

Sunday, August 4th

Week 16

Monday, August 5th

Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)

Investigator: Jeff

Aim of the experiment: Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).

Procedure:

Reaction batch for P540:

volume	reagent
20 µl	P540
4 µl	CutSmart Buffer (10x)
2 µl	EcoRI-HF (20 U/µl)
2 µl	PstI-HF (20 U/µl)
4 µl	XhoI (10 U/µl)
8 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P558, P560, P562, P565:

volume	reagent
20 µl	P558, P560, P562, P565
4 µl	CutSmart Buffer (10x)
2 µl	EcoRI-HF (20 U/µl)
2 µl	PstI-HF (20 U/µl)
12 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P540 with EcoRI & PstI & XhoI	2 log DNA ladder	Digestion of P558 with EcoRI & PstI	Digestion of P560 with EcoRI & PstI
Results	as expected; upper band was cut out		as expected; lower band was cut out	as expected; lower band was cut out

Lane	Digestion of P562 with EcoRI & PstI	2 log DNA ladder	Digestion of P565 with EcoRI & PstI
Results	as expected; upper band was cut out		as expected; upper band was cut out

DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.

Resulting fragment was named as F214 – F218.

PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes

Investigator: Jeff

Aim of the experiment: PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes. Procedure:

Reaction batch for PCR with Q5 Mastermixes for P558 (SYFP2):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O122 (SYFP2_fw)
2.5 µl	10 µM Reverse Primer O123 (SYFP2_rv)
1 µl	Plasmid DNA (P558) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

Reaction batch for PCR with Q5 Mastermixes for P560 (eCFP):

volume	reagent
25 µl	2x Q5 Master Mix
2.5 µl	10 µM Forward Primer O124 (eCFP_fw)
2.5 µl	10 µM Reverse Primer O125 (eCFP_rv)
1 µl	Plasmid DNA (P560) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL	ddH₂O Water
=50 µL	TOTAL

The content has been created on ice and mixed with a pipette

The gradient PCR program was performed after following scheme with following conditions (T_m=67 °C; ΔG=2 °C; P558 in row 1(=65.0 °C); P560 in row 12 (=69.2 °C)):

Cycling parameters:

Initial denaturation	98 °C	30 s
30 cycles	98 °C	10 s
	T_m=67 °C; ΔG=2 °C	30 s
	72 °C	22 s
Final extension	72 °C	2 min
Hold	4 °C	infinite

After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

The resulting product was labeled as F212 for the the PCR products of P558 and F213 for the PCR product of P560.

Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).

Procedure:

4.5 µl of the PCR product F212 and F213 products were mixed with 0.5 µl of DNA loading buffer (10x).

Analytical gelelectrophoresis was performed at 90 V for 60 min on an 1% agarose gel.

Lane:	F212	2 log DNA ladder	F213
Result:	as expected		as expected

Tuesday, August 6th

Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv)

Investigator: Jeff

Aim of the experiment: Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv).

Procedure:

25 µL of O128 (100 µM) and 25 µL of O129 (100 µM) were pooled together.

Heating up to 95 °C for 5 min.

Cooling at RT in a styropor box overnight.

Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).

Procedure:

Reaction batch for F212:

volume	reagent
25 µl	F212
5 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	AgeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Reaction batch for F213:

volume	reagent
25 µl	F213
5 µl	CutSmart Buffer (10x)
1 µl	XbaI (20 U/µl)
1 µl	AgeI-HF (20 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Reaction batches were incubated at 37 °C for 2 h.

Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.

Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP)

Investigator: Jeff

Aim of the experiment: Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP).

Procedure:

Ligation batch for F123+F219 (pSB1C3 + SYFP2), vector:insert = 1:3:

volume	reagent
1 µl	F123 (49.3 ng/µl, 2086 bp)
0.4 µl	F219 (114.9 ng/µl, 723 bp)
15.6 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F123+F220 (pSB1C3 + eCFP), vector:insert = 1:3:

volume	reagent
1 µl	F123 (49.3 ng/µl, 2086 bp)
1.2 µl	F220 (41.7 ng/µl, 723 bp)
14.8 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Analytical digestion and gelelectrophoresis of further clones

Investigator: Jeff

Procedure: The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.

Analytical digestion was performed at 37 °C for 2 h.

After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8
Result:		wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result

Preparative digestion of P428 (mMCS-T35S-npt) and P554 (PppAct5-QCII)

Investigator: Jeff

Procedure:

Reaction batch for P428:

volume	reagent
20 µl	P428
5 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	XbaI (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Reaction batch for P554:

volume	reagent
20 µl	F213
5 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
2 µl	SpeI (10 U/µl)
2 µl	NcoI (10 U/µl)
18 µl	ddH₂O
=50 µl	TOTAL

Reaction batches were incubated at 37 °C for 2 h.

Lane	2 log DNA ladder	Digestion of P428 with EcoRI & XbaI	Digestion of P554 with EcoRI & SpeI
Results		as expected; highest band was cut out	as expected; highest band was cut out

The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.

Ligation of F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt)

Investigator: Jeff

Procedure:

Ligation batch for F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt), vector:insert = 1:3:

volume	reagent
5 µl	F221 (42 ng/µl, 3700 bp)
11 µl	F222 (37 ng/µl, 2000 bp)
2 µl	T4 ligase buffer (10x)
2 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at room temperature overnight.

Transformation of E. coli XL1-Blue with ligation F221+F222

Investigator: Jeff

Procedure:

After 1 h of ligation 10 µl were used for transformation.

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Wednesday, August 7th

Transformation of E. coli XL1-Blue with ligation F221+F222

Investigator: Jeff

Procedure:

After 1 h of ligation 10 µl were used for transformation.

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed after 1 h

Investigator: Jeff

Procedure:

PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

12 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Thursday, August 8th

Analytical digestion and gelelectrophoresis of further clones

Investigator: Jeff

Procedure: The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.

Analytical digestion was performed at 37 °C for 2 h.

After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8	clone 9	clone 10
Result:		wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result

All these clones were discarded.
Additionally the fragments for the ligation were controlled.

Lane:	2log DNA ladder	F185	F211	F221	F222
Result:		correct	correct	correct	correct

Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed over night

Investigator: Jeff

Procedure:

PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

40 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Friday, August 9th

Miniprep of E. coli Xl1-Blue transformed with ligation of PppAct5 into mMCS-T35S-npt

Investigator: Jeff

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P574 to P613.

Analytical digestion and gelelectrophoresis of P574 – P613

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P574 – P613.

Procedure:

Mastermix for analytical digestion with EcoRI

volume	reagent
45 µl	Cut Smart buffer
8 µl	EcoRI-HF
400 µl	ddH2O
=453 µl	TOTAL

9 µl of the mastermix were mixed together with 1 µl of Plasmid DNA

Analytical digestion was performed at 37 °C for 2 h.

After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8	clone 9	clone 10
Result:		wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	good clone => only faint additional band

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8	clone 9	clone 10
Result:		wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	best clone => no additional band (best clone)	wrong result	wrong result

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8	clone 9	clone 10
Result:		wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	wrong result	correct size, but additional bands	correct size, but additional bands

Lane:	2log DNA ladder	clone 1	clone 2	clone 3	clone 4	clone 5	clone 6	clone 7	clone 8	clone 9	clone 10
Result:		wrong result	wrong result	correct size, but additional bands	wrong result	wrong result	correct size, but additional bands	wrong result	wrong result	wrong result	wrong result

Saturday, August 10th

Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P614 to P617.

Analytical digestion and gelelectrophoresis of P614 – P617

Investigator: Jeff

Aim of the experiment: Analytical digestion and gelelectrophoresis of P614 – P617.

Procedure:

Mastermix for analytical digestion with EcoRI and PstI

volume	reagent
2.5 µl	Plasmid DNA (P614 – P617)
2 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25nbsp;µl	PstI-HF
15 µl	ddH2O
=20 µl	TOTAL

Analytical digestion was performed at 37 °C for 1 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 120 V for 30 min.

Lane:	2 log DNA ladder	Digestion of P614 (SYFP2 RFC25)	Digestion of P615 (SYFP2 RFC25)	Digestion of P616 (eCFP RFC25)	Digestion of P617 (eCFP RFC25)
Result:		as expected (723 bp)	as expected (723 bp)	as expected (723 bp)	as expected (723 bp)

Sunday, August 11th

Preparative digestion of P615 (SYFP2 RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P615 (SYFP2 RFC25)

Procedure:

Preparative digestion and gelelectrophoresis of P617 (eCFP RFC25)

Investigator: Jeff

Aim of the experiment: Preparative digestion of P617 (eCFP RFC25)

Procedure:

Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2)

Investigator: Jeff

Aim of the experiment: Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2).

Procedure:

Ligation batch for F224+F223 (SYFP2 + Gly-TEV-site-Gly), vector:insert = 1:3:

volume	reagent
0.66 µl	F224 (152.1 ng/µl, 2086 bp)
0.34 µl	F223 (2506.9 ng/µl, 35 bp)
16 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at 16 °C overnight.

Miniprep of Retrafos of P516, P330, P245, P496, P346, P196, P20, P476, P554, P498, P246, P354, P428, P512, P402, P197, P350, P502

Investigator: Louise, Christopher

Aim of the experiment: Miniprep of Retrafos of P516, P330, P245, P496, P346, P196, P20, P476, P554, P498, P246, P354, P428, P512, P402, P197, P350, P502.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The new plasmids were numbered P618 - P635 (see Inventory)

Week 17

Monday, August 12th

Sequencing of P509, P512, P516, P537, P540, P562, P565, P583, P591

Investigator: Louise

Aim of the experiment: Sequencing of P509, P512, P516, P537, P540, P562, P565, P583, P591

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode1 (fw)	Barcode2 (rev)
P509	FR02233103
P632	FR02233102	FR02233101
P633	FR02233100	FR02233099
P537	FR02233098	FR02233097
P540	FR02233096	FR02233104
P562	FR02233105	FR02233106
P565	FR02233107	FR02233108
P583	FR02233109	FR02233110 (Primer O131)
P591	FR02233111	FR02233088 (Primer O131)

Transformation of E. coli XL1-Blue with P337,P540, P583 and P591

Investigator: Johanna

Aim of the experiment: Retransformation of E. coli XL1-Blue with P337,P540, P583 and P591

Procedure:

1,5 µl of dd H2O were added to the empty tubes

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Transformation of E. coli XL1-Blue with ligation product F224+F223 and negative control of F224

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F224+F223 and negative control of F224

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Tuesday, August 13th

Gelextraction and Ligation of digested P583 and P591

Investigator: Louise

Aim of the experiment: Gelextraction and Ligation of digested P583 and P591

Procedure:

After preparative digestion of P583 and P591 with SpfI and MfeI the gel extraction was performed after manufacturer`s protocol (QIAquick Gel Extraction Kit, QIAGEN)

the extracted fragments were labelled F226 (digested P583) and F227 (digested P591)

ligation was performed without extra inserts with just 100 ng of cut plasmid to test if it was cut as expected

Ligation batch for self-ligation of F226

volume	reagent
0.6 µl	F226 (164.1 ng/µl)
16.4 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for self-ligation of F227

volume	reagent
0.4 µl	F227 (243.4 ng/µl)
16.6 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_J45014

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_245014

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Picking of Retrafos of P337, P540, P583 and P591 and of Ligation F224 + F223

Investigator: Jeff

Aim of the experiment: Picking of Retrafos of P337 (SERK-SigP_XylE), P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt) and of Ligation F224 + F223 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)

Procedure:

Retrafos 1 clone each, Ligation 4 clones due to approx. 50 % positive neg. ctrl

4 ml LB medium w/ 4 µl chloramphenicol

Incubation overnight at 37 °C in the 180 rpm

Wednesday, August 14th

Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)

Investigator: Louise

Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)

Procedure:

BBa_J45014 (banana odour): 4 Colonies were picked from chloramphenicol plates.

Each colony was transferred into a tube containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

These tubes were transferred into a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of P640-P643 and P615 with EcoRI and PstI and P637, P639 with EcoRI and MfeI (old and new)

Investigator: Louise, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P640-P643 () with EcoRI and PstI and P637, P639 with EcoRI and MfeI (old and new)

Procedure:

batch for digestion of P640-P643

volume	reagent
8.5 µl	Plasmid DNA
2 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
9 µl	ddH2O
=20 µl	TOTAL

batch for digestion of P615

volume	reagent
2.5 µl	Plasmid DNA
2 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	PstI-HF
15 µl	ddH2O
=20 µl	TOTAL

batch for digestion of P637 and P639

volume	reagent
8.5 µl	Plasmid DNA
2 µl	Cut Smart buffer
0.25 µl	EcoRI-HF
0.25 µl	MfeI (old or new)
9 µl	ddH2O
=20 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane:	DNA ladder	Digestion of P615 (SYFP2 RFC25-reference)	Digestion of P637 (with old MfeI)	Digestion of P637 (with new MfeI)	Digestion of P639 (with old MfeI)	Digestion of P639 (with new MfeI)	Digestion of P640	Digestion of P641	Digestion of P642	Digestion of P643
Result:		as expected	corrupt MfeI	as expected	corrupt MfeI	as expected	as expected	as expected	as expected	as expected

Sequencing of P614, P617, P637, P639 and P641

Investigator: Flo, Johanna

Aim of the experiment: Sequencing of P614, P617, P637, P639 and P641

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode	Primer
P614	FR02233112	03
P617	FR02233113	03
P641	FR02233114	03
P637	FR02233119	061
P639	FR02233115	061

Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223

Investigator: Johanna

Aim of the experiment: Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The new plasmids were numbered as followed:

Plasmid	Miniprep-Plasmid
P337	P636
P583	P637
P540	P638
P591	P639
F224+223 (version 1)	P640
F224+223 (version 2)	P641
F224+223 (version 3)	P642
F224+223 (version 4)	P643

Miniprep of Retrafos of banana odour, P583 and P591

Investigator: Florian

Aim of the experiment: Miniprep of Retrafos of banana odour, P583 and P591.

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The new plasmids were numbered as followed:

Plasmid	Miniprep-Plasmid
Banana 1	P644
Banana 2	P645
Banana 3	P646
Banana 4	P647
P583	P648
P591	P649

Preparative digestion of P644 (banana odour), P648 & P649 (PppAct5-mMCS-T35S-npt)

Investigator: Florian

Procedure:

Reaction batch for P644:

volume	reagent
20 µl	P644
5 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	PstI (20 U/µl)
23 µl	ddH₂O
=50 µl	TOTAL

Reaction batch for P648:

volume	reagent
40 µl	P648
10 µl	CutSmart Buffer (10x)
2 µl	MfeI (20 U/µl)
0 µl was empty :'(	SbfI (20 U/µl)
46 µl	ddH₂O
=50 µl	TOTAL

Reaction batch for P649:

volume	reagent
40 µl	P649
10 µl	CutSmart Buffer (10x)
2 µl	MfeI (20 U/µl)
2 µl	SbfI (20 U/µl)
46 µl	ddH₂O
=50 µl	TOTAL

Reaction batches were incubated at 37 °C for 3 h.

Lane	2 log DNA ladder	Digestion of P428 with EcoRI & XbaI	Digestion of P554 with EcoRI & SpeI
Results		as expected; highest band was cut out	as expected; highest band was cut out

The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.

Thursday, August 15th

Preparative digestion of P414 and P640 + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi

Procedure:

Reaction batch for P414:

volume	reagent
20 µl	P414
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
1 µl	PstI (20 U/µl)
14 µl	ddH₂O
=40 µl	TOTAL

Reaction batch for P640:

volume	reagent
20 µl	P640
4 µl	CutSmart Buffer (10x)
1 µl	EcoRI (20 U/µl)
2 µl	NgoMIV (20 U/µl)
13 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.

Lane	Digestion of P414 with EcoRI & PstI	1kB DNA ladder	Digestion of P640 with EcoRI & NgoMIV
Results	as expected; upper band was cut out		as expected; lower band was cut out

Gelextraction was performed after preparative Gelelectrophoresis
The resulting fragments were named F231 (Laccase) + F232

Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218

Investigator: Louise, Florian

Aim of the experiment: Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218

Procedure:

Ligation batch for F230+F191 (plasmid with mini MCS + GFP cytoplasmatic), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
18.7 µl	F191 (2 ng/µl, 723 bp)
0.0 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F192 (plasmid with mini MCS + catecholdioxygenase cytoplasmatic), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
5.94 µl	F192 (8 ng/µl, 918 bp)
10.65 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F193 (plasmid with mini MCS + EreB cytoplasmatic), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
1.85 µl	F193 (35 ng/µl, 1254 bp)
14.74 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F194 (plasmid with mini MCS + Nanoluciferase cytoplasmatic), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
8.80 µl	F194 (3 ng/µl, 510 bp)
7.79 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F195 (plasmid with mini MCS + nanoluciferase secretory_SERK), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
3.99 µl	F195 (8 ng/µl, 616 bp)
12.60 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F196 (plasmid with mini MCS + gutathiontransferase cytoplasmatic), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
1.17 µl	F196 (28 ng/µl, 633 bp)
15.42 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F197 (plasmid with mini MCS + EreB membrane associated), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
11.12 µl	F197 (11 ng/µl, 2362 bp)
5.47 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F198 (plasmid with mini MCS + Nanoluc membrane associated), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
6.44 µl	F198 (13 ng/µl, 1618 bp)
10.15 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F199 (plasmid with mini MCS + nanoluc secretory_Ig Kappa), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
1.16 µl	F199 (26 ng/µl, 583 bp)
15.43 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F200 (plasmid with mini MCS + Nanoluc+IRES+Spytag_Nanoluc), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
3.00 µl	F200 (47 ng/µl, 2725 bp)
13.59 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F201 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spycatcher), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
3.81 µl	F201 (37 ng/µl, 2725 bp)
12.78 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F202 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spytag), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
2.66 µl	F202 (53 ng/µl, 2725 bp)
13.93 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F203 (plasmid with mini MCS + IgKappa_GFP secretory), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
1.09 µl	F203 (38 ng/µl, 800 bp)
15.50 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F204 (plasmid with mini MCS + Nanoluc+IRES+Spycatcher_Nanoluc), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
2.17 µl	F204 (65 ng/µl, 2725 bp)
14.42 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F205 (plasmid with mini MCS + FluA membrane associated), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
2.11 µl	F205 (40 ng/µl, 1630 bp)
14.48 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F214 (plasmid with mini MCS + PP1 membrane associated), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
0.95 µl	F214 (113 ng/µl, 2080 bp)
15.64 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
2.76 µl	F217 (116 ng/µl, 6191 bp)
13.83 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

volume	reagent
1.15 µl	F230 (243.7 ng/µl, 5796 bp)
0.86 µl	F217 (116 ng/µl, 6191 bp)
14.99 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F218 (plasmid with mini MCS + PIF6 safety), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
2.50 µl	F218 (128 ng/µl, 6191 bp)
14.09 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F218 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1

volume	reagent
1.15 µl	F230 (243.7 ng/µl, 5796 bp)
0.78 µl	F218 (116 ng/µl, 6191 bp)
15.07 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at roomtemperature, 1 h

Ligation of F230 with F231

Investigator: Louise, Christopher

Aim of the experiment: Ligation of F230 with F231

Procedure:

Ligation batch for F230+F231 (plasmid with mini MCS + Laccase membrane-associated), vector:insert = 1:3

volume	reagent
0.41 µl	F230 (243.7 ng/µl, 5796 bp)
14.33 µl	F231 (9.5 ng/µl, ~2630 bp)
2.26 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation was performed at room temperature, 1 h

Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230

Investigator: Louise, Christopher

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Friday, August 16th

Sequencing of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)

Investigator: Rosario

Aim of the experiment: Sequencing of P640.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode	Primer
P640	FR02233116	03

Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV

Investigators: Louise, Jeff

Aim of the experiment: Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV

Procedure:

volume	reagent
33 µl	P640
4 µl	CutSmart Buffer (10x)
2 µl	NgoMIV (10 U/µl)
1 µl	EcoRI Hf (20 U/µl)
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 3 h.

Lane	2 log DNA ladder	Digestion of P640 with EcoRI & NgoMIV
Results		as expected; band was cut out

The band was extracted from the gel via QIAGEN Gel Extraction Kit and termed F233. The concentration was 31.1 ng/µl.

Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3)

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3).

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Ligation of F233+F225 (creation of eCFP_Gly-TEV-site-Gly_SYFP2)

Investigator: Jeff

Aim of the experiment: Creation of eCFP_Gly-TEV-site-Gly_SYFP2

Procedure:

Ligation batch, vector:insert = 1:3

volume	reagent
3.22 µl	F233 (31.1 ng/µl, 2834 bp)
1.93 µl	F225 (39.6 ng/µl, 723 bp)
11.85 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

neg. ctrl. w/ water instead of insert

Ligation was performed at room temperature, 1 h

Miniprep of E. coli XL1-Blue transformed with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F217+F230, F230+F218, F218+F230, F230+F228

Investigator: Jeff, Rosario, Louise, Christopher

Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F217+F230, F230+F218, F218+F230, F230+F228

Procedure:

Ligation products were labelled B to V (in the same order as in Aim of the experiment, above)

Two clones for every ligation product except for H, L and O

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).

The resulting tubes were labeled as P650 to P688.

Analytical digestion and gelelectrophoresis of P650-P688 with XbaI and PstI-HF

Investigator: Jeff, Rosario, Louise, Christopher

Aim of the experiment: Analytical digestion and gelelectrophoresis of P650-P688 with XbaI and PstI-HF

Procedure:

batch for digestion of P650-P688

volume	reagent
5 µl	Plasmid DNA
1 µl	Cut Smart buffer
0.2 µl	XbaI
0.2 µl	PstI-HF
3.6 µl	ddH2O
=10 µl	TOTAL

Analytical digestion was performed at 37 °C for 1 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Saturday, August 17th

Picking of of E. coli XL1 blue transformed with P449, P541, F230+F231, P667(K1), P668 (K2), P669 (L1), P670 (M1), P671 (M2), P674 (O1)

Investigator: Andi, Johanna

Aim of the experiment: Picking of of E. coli XL1 blue transformed with P449, P541, F230+F231, P667(K1), P668 (K2), P669 (L1), P670 (M1), P671 (M2), P674 (O1)

Procedure:

F230+F231: 8 Colonies were picked from chloramphenicol plates.

P449: 4 Colonies were picked from chloramphenicol plates.

P541: 4 Colonies were picked from ampicilin plates.

K-Plate: 4 Colonies were picked from chloramphenicol plates.

L-Plate: 4 Colonies were picked from chloramphenicol plates.

M-Plate: 4 Colonies were picked from chloramphenicol plates.

O-Plate: 4Colonies were picked from chloramphenicol plates.

Each colony was transferred into a tube containing a relation of 1:1000 of LB-medium : antibiotic

These tubes were transferred into a cell culture shaker at 37 °C and were incubated 16 h

Preparative digestion of P625, P516, P644 + Preparative Gelelectrophoresis + Gelextraction

Investigator: Andi, Johanna

Procedure:

Reaction batch for PXXX:

volume	reagent
20 µl	PXXX
4 µl	CutSmart Buffer (10x)
2 µl	EcoRI (20 U/µl)
2 µl	PstI (20 U/µl)
12 µl	ddH₂O
=40 µl	TOTAL

Reaction batches were incubated at 37 °C for 2.5 h.

Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Gelextraction was performed after preparative Gelelectrophoresis
The resulting fragments were named F234 (digestion of P516), F235 (digestion of P625), F236 (digestion of P644).

Ligation of F230+ F234, F230+ F235, F230+F236

Investigator: Johanna, Andi

Aim of the experiment: Ligation of F230+ F234, F230+ F235, F230+F236

Procedure:

Ligation batch for F230+F234, vector:insert = 1:3

volume	reagent
6.3 µl	F234(228 ng/µl, 1600 bp)
0.4 µl	F230 (100 ng/µl, bp)
10.3 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F235, vector:insert = 1:3

volume	reagent
9.0 µl	F235(214 ng/µl, 2300 bp)
0.4 µl	F230 (100 ng/µl, bp)
7.6 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

Ligation batch for F230+F236, vector:insert = 1:3

volume	reagent
16.6 µl	F236(330 ng/µl, 1500 bp)
0.4 µl	F230 (100 ng/µl, bp)
0.0 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

neg. ctrl. w/ water instead of insert

Ligation was performed at room temperature, 1 h

Miniprep of F230+F231 (version 1-4), P541 (version 1-2), K3, K4, L2, L3, M3, M4, O2, O3, P3-P10 and P449 (version 1-2)

Investigator: Johanna, Andi

Aim of the experiment: Miniprep of F230+F231 (version 1-4), P541 (version 1-2), K3, K4, L2, L3, M3, M4, O2, O3, P3-P10 and P449 (version 1-2)

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The new plasmids were numbered as followed:

Plasmid	Miniprep-Plasmid
F230+F231 (version 1)	P689
F230+F231 (version 2)	P690
F230+F231 (version 3)	P691
F230+231 (version 4)	P692
P541 (version 1)	P693
P541 (version 2)	P694
K3	P695
K4	P696
L2	P697
L3	P698
M3	P699
M4	P700
O2	P701
O3	P702
P3	P703
P4	P704
P5	P705
P6	P706
P7	P707
P8	P708
P9	P709
P10	P710
P449 (version 1)	P711
P449 (version 2)	P712

Analytical digestion and gelelectrophoresis of P689-P712 with XbaI and PstI

Investigator: Andi, Johanna

Aim of the experiment: Analytical digestion and gelelectrophoresis of P689-P712 with XbaI and PstI

Procedure:

batch for digestion of P689-712

volume	reagent
5 µl	Plasmid DNA
1 µl	Cut Smart buffer
0.2 µl	XbaI-HF
0.2 µl	PstI-HF
3.6 µl	ddH2O
=10 µl	TOTAL

Analytical digestion was performed at 37 °C for 1.5 h.

After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.

Analytical gelelectrophoresis was performed at 90 V for 1 h.

Lane	1kB DNA ladder	Digestion of P689	Digestion of P690	Digestion of P691	Digestion of P692	Digestion of P693	Digestion of P694	Digestion of P695	Digestion of P696	Digestion of P697	Digestion of P698
Results

Lane	1kB DNA ladder	Digestion of P699	Digestion of P700	Digestion of P701	Digestion of P702	Digestion of P703	Digestion of P704	Digestion of P705	Digestion of P706	Digestion of P707	Digestion of P708
Results

Lane	1kB DNA ladder	Digestion of P709	Digestion of P710	Digestion of P711	Digestion of P712
Results

Transformation of E. coli XL1-Blue with ligation product F230+F234, F230+F235, F230+F236

Investigator: Johanna, Andi

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F234, F230+F235, F230+F236.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Week 18

Monday, August 19th

Midiprep of Safety_PIF3, PP1 membrane bound, Safety_PIF6, Safety_PIF3, Positve control mit PppActin5, EreB cyt., NanoLuc cyt., XylE cyt., NanoLuc sek. (IgKappa), NanoLuc sek. (SERK), GFP cyt., GFP sek. (IgKappa), FluA membrane bound, stress-inducible promoter, GST, NanoLuc membrane bound

Investigator: Andreas, Rosario

Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Procedure:

Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)

Gelelectrophoresis of digested Midi-Preps F243 - F260

Investigator: Ingmar

Aim of the experiment:

Procedure:

Concentrations of digestions were measured (blanked against EB-buffer), see Inventory List

5 µl of DNA loading buffer (10x) mixed with 1 µl Midiprep-DNA, on 1% agarose gel.

90 V 1 h.

Lane	1kB DNA ladder	F234	F235	F236	F237	F238	F239	F240	F241	F242	F243	F244
Results

[[File:|500px]]

Lane	1kB DNA ladder
Results

[[File:|500px]]

Lane	1kB DNA ladder
Results

Tuesday, August 20th

Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261

Investigator: Andi, Rosario, Johanna

Aim of the experiment: Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261.

Procedure:

Reaction batch for P643:

volume	reagent
20 µl	P643
5 µl	CutSmart Buffer (10x)
2 µl	EcoRI (20 U/µl)
2 µl	NgoMIV (20 U/µl)
12 µl	ddH₂O
=40 µl	TOTAL

Result of the Gelelectrophoresis:

Reaction batch was incubated at 37 °C for 2 h.

Digested product was purified with QIAquick PCR Purification Kit, QIAGEN.

Gelelectrophoresis was performed for 1 h and 90V.

Gelextraction was performed after manufacturers protocol and the resulting Fragment was called F261.

Sequencing of P643

Investigator: Andi

Aim of the experiment: Sequencing of P643.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Label	Barcode	Primer
P643	FR0223317	03

Ligation of F261+F225

Investigator: Rosario

Aim of the experiment: Ligation of F261+F225

Procedure:

Ligation batch for F261+F225, vector:insert = 1:3

volume	reagent
1,5 µl	F225
1,2 µl	F261
14,3 µl	ddH₂O
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
=20 µl	TOTAL

neg. ctrl. w/ water instead of insert

Ligation was performed at room temperature, 1 h

Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA (Ligation) and 1 µl of DNA (Retrafo) were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

Transformation of Physcomitrella patens with

Investigator: Ingmar, Jeff

Aim of the experiment: Transformation of Physcomitrella patens with

Procedure:

Extraction of moss from bioreactor culture by filtering through protoplat sieve

Incubation of moss with xxx ml Driselase to digest cell wall (2h at roomtemperature, protected from light)

Centrifugation at xxx rpm for xxx min with slow acceleration rates

tbc

Wednesday, August 21th

Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643

Investigator: Rosario

Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643.

Procedure:

F225+F261: 3 Colonies were picked from chloramphenicol plates.

P643: 1 Colony was picked from a chloramphenicol plate.

Each colony was transferred into a tube containing a relation of 1000:1 of LB-medium : antibiotic

These tubes were transferred into a cell culture shaker at 37 °C and were incubated 12 h

Transformation of E. coli XL1-Blue with ligation product F225+F261

Investigator: Rosario

Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.

@@ Line 29,281: / Line 29,281: @@
 * The pellet was resuspended in 100&nbsp;µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
+</div>
+<div class="general">
+<div class="safety_mechanism">
+<div class="effectors">
+=== Transformation of ''Physcomitrella patens'' with ===
+'''Investigator:''' Ingmar, Jeff
+'''Aim of the experiment:''' Transformation of ''Physcomitrella patens'' with
+'''Procedure:'''
+* Extraction of moss from bioreactor culture by filtering through protoplat sieve
+* Incubation of moss with xxx&nbsp;ml Driselase to digest cell wall (2h at roomtemperature, protected from light)
+*
+* Centrifugation at xxx&nbsp;rpm for xxx&nbsp;min with slow acceleration rates
+tbc
+*
+</div>
+</div>
 </div>

Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

Revision as of 10:03, 22 August 2013

Labjournal

Week 1

Monday, April 22nd

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Sequencing of RFP-Generator (RFC25, pSB1C3)

Tuesday, April 23rd

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Wednesday, April 24th

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)

Week 2

Monday, April 29th

Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Tuesday, April 30th

Preparative digestion and gelelectrophoresis of mINF1 and Leptin with HindIII and EheI

Transformation of E. coli XL1 blue with PSH21 (P17)

Thursday, May 2nd

Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system

Friday, May 3rd

Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry

Plating of received E. coli containing biobricks

Sunday, May 5th

Picking of the plated E. coli containing biobricks and the parts received from LMU

Week 3

Monday, May 6th

Miniprep of the prepared E. coli containing biobricks and the parts from LMU

Sequencing of FluA, SV40, CaMV35S, xylE, LMU mKate2, LMU GFP, LMU mCherry, DREB1C, RD29A

Preparation of ordered primers

Tuesday, May 7th

PCR, purification and analytical geleletrophoresis of EreA (P15) and EreB (P16)

Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV

Wednesday, May 8th

Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI

Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3)

Midiprep of cloning vector RFC25 RFP generating device

Thursday, May 9th

Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB)

Friday, May 10th

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Picking of transformed Plasmids F8 + F6

PCR of TEV (P14, pRK793)

Digestion of P61 with S1 Nuclease

Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)

Preparation of ordered primers

Saturday, May 11th

Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA)

Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Preparative digestion of PCR product of EreB (F2) with XbaI & AgeI

Purification and ligation of digested PCR product EreB (F14)

Transformation of ligation products of F10 + F11 into E.coli Xl1-Blue

Transformation of Quickchange Product P62 into E.coli Xl1-Blue

Quickchange mutagenesis of pTUM104

Sunday, May 12th

Analytical gelelectrophoresis of F15 (digested TEV PCR product)

Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25)

Transformation of E. coli XL1 blue with ligation products F8+F14 (EreB in pSB1C3, RFC25)

Transformation of E. coli XL1 blue with P23

Picking of transformed Plasmid F10+F11 (IRES from polio virus, blunt ligated with pSB1C3 for sequencing)

Week 4

Monday, May 13th

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F10+F11

Analyzing sequencing results

Quickchange mutagenesis SDMI of p27

Quickchange mutagenesis SDMI of p38

Quickchange mutagenesis SDMI of P63

Transformation of E. coli XL1 blue with P65

Transformation of E. coli XL1 blue with P66

Transformation of E. coli XL1 blue with P67

Tuesday, May 14th

Analytical digestion of P15 and P19 to P52

Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV)