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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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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: Leonie

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, Leonie, 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: Leonie

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,Leonie,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, Leonie

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: Leonie,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, Leonie

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, Leonie

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, Leonie, 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: Leonie, 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,Leonie,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, Leonie

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, Leonie

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, Leonie

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, Leonie

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, Leonie, 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, Leonie

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, Leonie, 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, Leonie, 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: Leonie,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: Leonie, 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, Leonie

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, Leonie, 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, Leonie, 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, Leonie

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, Leonie, 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, Leonie, 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, Leonie, 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: Leonie, 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, Leonie, 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: Leonie, 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: Leonie, 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: Leonie, 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: Leonie, 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, Leonie, 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, Leonie

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, Leonie

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, Leonie

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: Leonie, 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: Leonie

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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie, 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, Leonie

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, Leonie

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: Leonie

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: Leonie

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, Leonie

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, Leonie, 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, Leonie, 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, Leonie

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, Leonie

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: Leonie, 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: Leonie, 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: Leonie, 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: Leonie, 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, Leonie

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, Leonie

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, Leonie

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: Leonie, 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, Leonie

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, Leonie

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, Leonie

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, Leonie, 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, Leonie

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: Leonie

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, Leonie

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, Leonie

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: Leonie, 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, Leonie, 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: Leonie

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

1kb dna ladder	P361	P362	P363	P364	P365	P366
	xxx bp	xxx bp	xxx bp	xxx bp	xxx bp	xxx bp

Dpn1 digestion and transformation of QCII of pActin (insertion) + analytical Gel

Investigator: Jeff, Leonie

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, Leonie

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: Leonie

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: Chinese 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: Chinese, Italiener

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)

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Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

Revision as of 12:57, 27 June 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)