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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 of P14 with öljafljafs

Investigator: Katrin, Jeff

Digestion of P61 with S1 Nuclease

Investigator: Katrin

Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)

Investigator: Leonie

Saturday, May 11th

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.

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

Investigator: Louise

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

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

Procedure: PCR Purification of EreB PCR (F2)

To purify the digested Fragment(F14) the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be X 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)
9,28 µl	F2 (X ng/µl, 1257 bp)
2 µl	T4 ligase buffer (10x)
1 µl	T4 ligase (1 U/µl)
X 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

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.

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

Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

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 of P14 with öljafljafs

Digestion of P61 with S1 Nuclease

Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)

Saturday, May 11th

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