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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 29nd

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)

Investigator: Andi

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

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 ligation products P28, P117, P118, P119, P120, P121, P122, P123.

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 P122
	to be announced	to be announced	to be announced	to be announced	to be announced	to be announced	to be announced	to be announced	to be announced

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 25st

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	P45 digested with XbaI&AgeI (=F28)	P45 digested with XbaI&AgeI (=F28)	P39 digested with AgeI&PstI (=F29)	P39 digested with AgeI&PstI (=F29)	P20 digested with NgoMIV&PstI (=F30)	P20 digested with NgoMIV&PstI (=F30)
	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 psB1C3 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 psB1C3(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 psB1C3(P10) with XbaI & PstI.

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 psB1C3(P10) with EcoRI and SpeI.

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.

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.

@@ Line 7,322: / Line 7,322: @@
 * Gelpurified F31 and F32 were labelled as F43 and F44.
+</div>
+<div class = "general">
+<div class = "safetey_mechanism">
+===  Transformation of ''E.&nbsp;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.&nbsp;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.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
+* 10&nbsp;µl of DNA were added to 200&nbsp;µl of competent cells and gently mixed.
+* 30&nbsp;min incubation on ice
+* 5&nbsp;min. heat shock at 37&nbsp;°C
+* Adding of 1&nbsp;ml LB-medium to each tube.
+* Incubation for 45&nbsp;min at 37&nbsp;°C in the 180&nbsp;rpm cell-culture shaker.
+* 100&nbsp;µl of the cell suspension was plated on one chloramphenicol plate.
+* The rest were centrifuged for 1&nbsp;min at 13000&nbsp;rpm and the supernatant was dicarded.
+* The pellets were resuspended in 100&nbsp;µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.
+</div>
 </div>

Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

Revision as of 17:52, 30 May 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 29nd

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)