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June

Wednesday, June 13th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Hybridisation of the primers O1 with O2 and O3 with O4

Primer preparation:

centrifugation
dilution in the denoted quantity in bidest. water (concentration = 100 pmol/µl)
centrifugation

Hybridisation:

volume	reagent
34 µl	ddH2O
5 µl	PNK-buffer
2.5 µl	Primer O1
2.5 µl	Primer O2
1 µl	PNK (10mM)

volume	reagent
34 µl	ddH2O
5 µl	PNK-buffer
2.5 µl	Primer O3
2.5 µl	Primer O4
1 µl	PNK (10mM)

30 min 37 °C
10 min 80 °C
put the Thermo Block with the mixture in a styrofoam box and let it cool down over night

Thursday, June 14th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Digestion of pYES2 with HindIII and XbaI

volume	reagent
12 µl	Miniprep (pYES2 SH 1.7.3 with a concentration of 87.8 ng/µl)
5 µl	NEB2
5 µl	10x BSA
2 µl	XbaI (10 U/µl)
2 µl	HinIII (10U/µl)
24 µl	ddH2O

Incubation: 37 °C, 1.75 h

DNA preparative gel electrophoresis

gel: 1% with LMP-agarose
band 1: 10 µl DNA ladder (1kb)
band 2: 50 µl probe + 5 µl loading dye
70 V, 90 min

Gelextration

cut the bands (5.7-5.8 kb) and split it in two eppis
- m1=165.3 mg
- m2=211.1 mg
QIAquick Gel Extractrion Kit
- eppi1: 495.9 µl QG-buffer
- eppi2: 633.3 µl QG-buffer
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation

the product was named P5

Transformation of plasmids from Prof. Schwab in E.coli XL-1 Blue

Investigator: Lara, Andrea

Aim of the experiment: Preparation of the plasmids for transformation

Overnight cultures of cells with limonenesynthase-plasmid from Prof. Schwab

resuspend 50 µl / 200 µl of competent cells with 50 ml LB medium
add 0,1 ml Ampicillin (100 µg/ml) and 0,28 ml Chloramphenicol (170 µ/ml) for strain 108
add 0,07 ml Kanamycin (50 µg/ml) and 0,28 ml Chloramphenicol (170 µ/ml) for strain 106
incubate at 37 °C

Friday, June 15th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Analytical DNA gel electrophoresis

gel: 1.2 %
band 1: 10 µl DNA ladder (1kb)
band 2: 3 µl pYES2 digested + 7 µl TAE-buffer + 1 µl loading dye
band 3: 3 µl O5 + 7 µl TAE-buffer + 1 µl loading dye
band 4: 3 µl O6 + 7 µl TAE-buffer + 1 µl loading dye
band 5: 10 µl DNA ladder (100 bp)

Ligation of Plasmid P5 (pYES2 digested) with the hybridized primers O5 and O6

volume	reagent
4 µl	pYES2 digested (P5)
1 µl	O5
1 µl	O6
2 µl	T4-ligase buffer (10x)
0,5 µl	T4 DNA-ligase
11.5 µl	ddH2O

Negative control

volume	reagent
4 µl	pYES2 digested (P5)
2 µl	T4-ligase buffer (10x)
0,5 µl	T4 DNA-ligase
13.5 µl	ddH2O

water bath 16 °C
after 3 h a probe for the transformation was taken
the rest was ligated over the weekend

Transformation of E. coli with ligated products (P6)

competent cells: SHXL1 Blue (by Simon)
Transformation with
- ligation product (P6)
- negative control

results:

P6 (100 µl): 1 clone
P6 (concentrated): 30 clones
negative control (100 µl): 0 clones
negative control (concentrated): 6 clones

Transformation of plasmids from Prof. Schwab into E.coli XL-1 Blue

Investigator: Andrea

Aim of the experiment: Preparation of the plasmids for transformation

Determination of the concentration with Nano Drop

Sample	concentration [ng/µl]
P3	1353
P4	no result

the strain 106 culture was not grown satisfying and were incubated for 2 more days

Miniprep of pGex-4T-1 of strain 108 from Prof. Schwab

see QIAprep Spin Miniprep Kit
stored as P3 (-20 °C)

Sunday, June 17th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Picking clones for Miniprep

10 clones of transformed E.coli with P6 were picked
medium: 5ml LB with Amp

Monday, June 18th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Miniprep of transformed E.coli with P6

QIAprepS Spin Miniprep Kit
- step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
the 10 Minipreps were named: P7 - P16

Determination of the concentration with Nano Drop

Sample	concentration [ng/µl]	260/280
P7	157.6	2.32
P8	207.3	1.63
P9	171.2	2.02
P10	183.1	1.57
P11	160.4	2.2
P12	179.9	1.75
P13	179.2	2.07
P14	188.3	1.6
P15	166.7	2.05
P16	174.6	2.08

Controll digestion with HindIII XbaI and NgoMIV

Samples P7-P16: 2.5 µl
Negative controll pYES SH 1.7.3: 2.5 µl

Master Mix HindIII and XbaI: 17,5 µl for a 20 µl preparation

volume	reagent
3 µl	HindIII
3 µl	XbaI
24 µl	NEB2
24 µl	10x BSA
156 µl	ddH2O

Master Mix NgoMIV: 17,5 µl for a 20 µl preparation

volume	reagent
6 µl	NgoMIV
24 µl	NEB4
180 µl	ddH2O

Incubation: 37 °C, 1.5 h

Analytical gel electrophoresis of P7-P16

gel: 1.5 %
gel 1:
- band 1: 10 µl DNA ladder (1 kb)
- band 2: 3 µl pYES2 SH 1.7.3 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye
- band 3 - 12: 3 µl P7-P16 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye

gel 2:
- band 1: 10 µl DNA ladder (1 kb)
- band 2: 3 µl pYES2 SH 1.7.3 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye
- band 3 - 12: 3 µl P7-P16 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye

Tuesday, June 19th

Transformation of BBa_I742111 (Limonenesynthase) into E.coli XL-1 Blue

Investigator: Andrea

Aim of the experiment: Transformation

For each Biobrick 100 µL cells were used and pooled together with 2 µL of plasmid DNA in a ERG on ice.

Incubation on ice for 30 min.

5 min heat shock at 37 °C.

Each ERG now is transferred in a new ERG prefilled with 1 mL of LB-medium and incubated in a cell-culture shaker at 37 °C for 45 min.

100 µL of these cell suspension were plated on antibiotic selection plates (Ampicillin for LexA and Kanamycin for heme oxygenase).

The rest of the cell suspension is centrifuged at 13000 rpm for 60 s and the supernatant is discarded.

The pellet is resuspended with 100 µL for each ERG and is plated on another antibiotic selection plate

These 4 plates were put at 37 °C overnight

Wednesday, June 20th

Exchange of the Multiple Cloning Site of pYES2

Investigator: Saskia, Daniela

Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2

Sequencing of P13 and P14: pYES2 with new MCS

sequencing primer:
- 1.6 µM forward primer O9
- DNA P13 and P14

Transformation of BBa_I742111 (Limonenesynthase) into E.coli XL-1 Blue

Investigator: Daniela

Aim of the experiment: Transformation

Picking of Clones

6 clones were picked
Incubation at 37 °C in LB + Amp

Thursday, June 21st

Transformation of BBa_I742111 and plasmids from Prof. Schwab into E.coli XL-1 Blue

Investigator: Lara, Andrea

Aim of the experiment: Controll of Transformation

Controll digestion

Sample P3

volume	reagent
14 µl	Plasmid-DNA
0,25 µl	NcoI
2 µl	Buffer Tango (Fermentas)
0,25 µl	HindIII
2 µl	Buffer Red (Fermentas)
1,5 µl	ddH2O

Sample P4

volume	reagent
6 µl	Plasmid-DNA
0,25 µl	EcoRI
2 µl	Buffer EcoRI (Fermentas)
0,25 µl	NotI
2 µl	Buffer Orange (Fermentas)
9,5 µl	ddH2O

Sample Biobrick-clones

volume	reagent
5 µl	Plasmid-DNA
0,25 µl	EcoRI
2 µl	Buffer EcoRI (Fermentas)
0,25 µl	PstI
2 µl	Buffer Orange (Fermentas)
10,5 µl	ddH2O

Analytic Gelelectrophoresis

Friday, June 22nd

Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS

Investigator: Ingmar, Volker

Aim of the experiment: Plasmid amplification

Operation Sequence:

melting of 100 µl Ca-competent E.coli XL1-Blue cells
addition of 1 µl of the Plasmid pKS2µHyg-PAL-4Cl-CHS
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

Saturday, June 23rd

Quick Change mutagenis to remove NgoMIV from pYES2

Investigator: Ingmar, Volker

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR
Reaction batch

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P7 pYes2_RFC25 MCS 1.1 template
0.5 µl	1:10 dilution of O38 (10 pmol/µL)
0.5 µl	1:10 dilution of O39 (10 pmol/µL)
17 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		68°C	6 min

The vector resulting from the PCR-product was named pYes2_RFC25 MCS 1.2.
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
addition of 1 µl of the Plasmid P7 pYes2_RFC25 MCS 1.2
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, June 24th

Miniprep of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS

Investigator: Ingmar, Volker

Aim of the experiment: Plasmid purification

Operation Sequence:

A single clone of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS was picked an transferred to 5 ml LB Amp on saturday evening. Incubation overnight at 37°C 180 rpm.
Using a Quiagen kit a miniprep of the overnight culture was done.

Quick Change mutagenis to remove NgoMIV from pYES2

Investigator: Ingmar, Volker

Aim of the experiment: Removal of a NgoMIV restriction site in the backbone of pYes2.

Operational sequence:

A single clone of E. coli pYes2_RFC25 MCS 1.2 was picked an transferred to 5 ml LB Amp. Incubation overnight at 37°C 180 rpm.

Transformation of 2 Biobricks into E. coli XL1-Blue

Investigator: Jeffery Truong

Aim of the experiment: Transformation of Biobricks into E. coli for plasmid propagation for PCR with new RFC pre- and suffix primer in order to do protein fusions.

2 Biobricks from the distribution kit were used: First, LexA (BBa_K105005, Plate 3 Well 9E) in the pSB1A2 plasmid with ampicillin resistance and second, the heme oxygenase (BBa_I15008, Plate 2 Well 13J) in the pSB2K3 plasmid with kanamycin resistance.

10 µL of autoclaved H2O were added to each well on the distribution kit. The well immediately turned red which means that one does it right.

The now resuspended DNA liquids were transferred into a new ERG on ice.

CaCL2 competent E. coli XL1-Blue cells from the stock were gently defrezed on ice.

For each Biobrick 100 µL cells were used and pooled together with 2 µL of plasmid DNA in a ERG on ice.

Incubation on ice for 30 min.

5 min heat shock at 37 °C.

Each ERG now is transferred in a new ERG prefilled with 1 mL of LB-medium and incubated in a cell-culture shaker at 37 °C for 45 min.

100 µL of these cell suspension were plated on antibiotic selection plates (Ampicillin for LexA and Kanamycin for heme oxygenase).

The rest of the cell suspension is centrifuged at 13000 rpm for 60 s and the supernatant is discarded.

The pellet is resuspended with 100 µL for each ERG and is plated on another antibiotic selection plate

These 4 plates were put at 37 °C overnight

Monday, June 25th

Miniprep of E. coli XL1-Blue with pYes2_RFC25 MCS 1.2

Investigator: Alois, Martin

Aim of the experiment: proof of successful removal of NgoMIV in the backbone of pYes2

Operation Sequence:

Mini prep of pYes2 1.2. The resulting purified DNA is P33.
Control digest of pYes2_RFC25 MCS 1.2 and p13 (+ analytical gel electrophoresis: 90 V, 1 h:

*  15 µl ddH20
*   2 µl NEBuffer4
* 0,5 µl NgoMIV
* 2,5 µl pYes2 1.2/p13
* 37°C, 1 h.

Quick Change mutagenis to remove SpeI from pYES2_RFC25 MCS 1.2

Investigator: Ingmar, Volker

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR
Reaction batch

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P33 template
0.5 µl	1:10 dilution of O44 (10 pmol/µL)
0.5 µl	1:10 dilution of O45 ((10 pmol/µL)
17 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		68°C	6 min

The procedure was furthermore applied to P13 and P14.
The vector resulting from the PCR-product was named pYes2_RFC25 MCS 1.3.
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

This operation sequence was applied to the PCR prducts of P33, P13 and P14 respectively.
melting of 100 µl Ca-competent E.coli XL1-Blue cells
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

Picking of E. coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Investigator: Jeffery Truong

Aim of the experiment: Picking colonies from transformed E. coli XL1-Blue, 4x picked for each Biobrick.

pSB1A2 plasmid with BBa_K105005 (LexA): Colonies were on both ampicillin selection plates, the one with diluted cell suspension and the one with concentrated E. coli cell suspension. Typical E. coli colony morphology. Picking was performed on the plate with diluted cell suspension.

pSB2K3 plasmid BBa_I15008 (heme oxygenase): Colonies were only on the kanamycin selection plate with concentrated cell suspension. The one with diluted susepension was empty. Typical but very small E. coli colonies. Picking was performed from the first plate.

Picked pipette tips was transferred into a special cell-culture tubes with air-permeable, but sterile cover. In each tube 4 mL of LB-medium + ampicillin (???)(for pSB1A2) or kanamycin (35 mg/mL) (for pSB2K3).

4 colonies for each Biobrick was picked; total: 8 tubes overnight culture.

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

Tuesday, June 26th

Quick Change mutagenis to remove SpeI from pYes2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Removal of a SpeI restriction site in the backbone of pYes2.

Operational sequence:

For each transformation of the PCR-products of P14 and P33 a single clone was picked an transferred to 6 ml LB Amp. Incubation overday at 37°C 180 rpm. The transfomation with the PCR product of P13 was not successfull. Therfore no clone could be picked.
Using a Quiagen kit a miniprep of the overnight culture was done.
The resulting purified DNA was aliquoted in new tubes labeled as follows:

PCR product of P33(transformation done by Ingmar): P29

PCR product of P33(transformation done by Saskia&Jara): P30

PCR product of P14(transformation done by Ingmar): P31

PCR product of P14(transformation done by Saskia&Jara): P32

Afterwards a control digestion of P29-P32 was done.

Reaction batch

Plasmid	P29	P30	P31	P32
NEB4 buffer	2 µl	2 µl	2 µl	2 µl
DNA	2,5 µl	2,5 µl	5 µl	5 µl
SpeI-HF	0.25 µl	0.25 µl	0.25 µl	0.25 µl
NgoMIV	0.25 µl	0.25 µl
ddH2O	15 µl	15 µl	12.75 µl	12.75 µl
Sum	20 µl	20 µl	20 µl	20 µl

Incubation at 37 °C for 1h.
Verification of control digest by agarose gel electrophoresis:

20 µl of each digest product was mixed with 2 µl DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V.

Verification of the PCR products P30, P31 and P33

Investigator: Saskia, Jara

Aim of the experiment: Verification of the PCR produts P30, P31 and P33

Nano Drop

Sample	concentration [ng/µl]	260/280
P33	1072.6	1.01
P30	1588	1.28
P31	926.2	0.82

Analytical gel electrophoresis

gel: 1 %
band 1: 10 µl DNA ladder (1kb)
band 2: P30
band 3: P33
band 4: P31

Control of the competent cells and transformation with P20

Investigator: Saskia, Jara

Aim of the experiment: Control of the competent cells and transformation with P20 Transformation

competent cells: by Simon and Ingmar
plasmid: P20

result:

successful transformation: red colonies

PCR of PAL, 4CL, CHS, OMT (Coumaryl-CoA)

Investigator: Daniela, Mary

Determination of concentration of plasmids (Nanodrop): c(pKS2µHyg-PAL-4CL-CHS) = 500 ng/µl c (pOMT) = 20 ng/µl

PCR:

Name of tube	Enzyme	consensus (+)/ consensless (-)	used Oligos
CHS -	CHS	-	O13 and O24
CHS +	CHS	+	O23 and O24
PAL -	PAL	-	O15 and O16
PAL +	PAL	+	O22 and O16
OMT -	OMT	-	O17 and O26
OMT +	OMT	+	O25 and O26
4CL -	4CL	-	O19 and O20
4CL +	4CL	+	O21 and O20

Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
4 µl	dNTP's (each 2.5 mM)
0.5 µl	Pfu Ultra II (2.5 U/µL)
5 µl	1:10 dilution of used forward primers (10µM)
5 µl	1:10 dilution of used reversed primers (10µM)
1 µl	DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL or pOMT 20 ng/µL)
29.5 µL	bidest. sterile Water

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	5 min (and adding Pfu Ultra after 3 min)
2	30	95°C	30 sec
		46°C	2.5 min
		72°C	1.5 min
3		72°C	5 min

PCR purification

Purification was done using QIAquick PCR Purification Kit (250)

Analytical Gel Electrophoresis:

-> going on with CHS, 4CL and OMT; the PCR of PAL will be repeated

Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Investigator: Jeffery Truong, Georg Schützinger

Aim of the experiment: Plasmid isolation from the picked transformed overnight E. coli cells with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase).

The LB-medium with antibiotics of every tube was opaque which means that the picked cells were successfully inoculated.

Centrifugation step at 5000 rpm for 10 min at 16 °C.

Every single step now was performed on ice.

Miniprep (Qiagen Qiaprep spin) after manufacturer's protocoll.

Analytical restriction master mix was prepared after following scheme (Using XbaI and PstI):
- 4.4 µL XbaI
- 4.4 µL PstI
- 17.6 µL Tango-buffer (10x)
- 132 µL ELGA H2O

17.5 µL from the master mix was poooled together with 2.5 µL of plasmid DNA. That corresponds to 2.5 µL of plasmid DNA, 0.25 µL XbaI, 0.25 µL PstI, 2 µL Tango-buffer (10x), 15 µL ELGA H2O.

Incubation at 37 °C for 120 min on a ERG heating unit.

BUT: error was performed during preparing the digested plasmid DNA for analytical gelelectrophoresis in the dilution step. 1:10 dilution of analyctical probe with DNA loading buffer:
- 3.3 µL sample (contains already 1x loading buffer!)
- 0.7 µL loading buffer (?x)
- 6 µL TAE-buffer

Should have done: 3 µL sample + 1 µL loading buffer (10x) + 6 µL TAE-buffer (1x)

DNA-ladder preperation: 10 µL ladder stock solution + 10 µL DNA loading buffer + 80 µL TAE-buffer. 10 µL of this solution was pipetted in one gel pocket of the prepared 1% agarose gel including ehtiudiumbromid.

20 µL of each samples were also pipetted into the gel pockets.

The gel pockets were pipetted after following scheme:

Heme oxygenase (colony 1)	Heme oxygenase (colony 2)	Heme oxygenase (colony 3)	Heme oxygenase (colony 4)	DNA-ladder	LexA (colony 1)	LexA (colony 2)	LexA (colony 3)	LexA (colony 4)

Gel electrophoresis at 90 V

After 20 min the resolution was still poor; 20 min longer.

Analytical Gel okay, but samples interchanged

Friday, June 29th

Preparative digest of PCR-products of 4CL, CHS and OMT

Investigator: Katrin, Mary

each digestion will dure 2.5h at 37°C

CHS: digestion with Xba1 and HF-Age1 (both NEB)

volume	reagent
25µl	PCR-product
5µl	Buffer NEB4
0.5µl	BSA
1µl	Xba1 (NEB; 20u/µl)
1µl	HF-Age1 (NEB; 20u/µl)
17.5µl	bidest. sterile H2O

OMT: digestion with Xba1 and HF-Age1 (both NEB)

volume	reagent
25µl	PCR-product
5µl	Buffer NEB4
0.5µl	BSA
1µl	Xba1 (NEB; 20u/µl)
1µl	HF-Age1 (NEB; 20u/µl)
17.5µl	bidest. sterile H2O

4CL: digestion with Xba1 and Pst1 (both Fermentas)

volume	reagent
25µl	PCR-product
5µl	Buffer Tango
2µl	Xba1 (Fermentas; 10u/µl)
3µl	Pst1 (Fermentas; 10u/µl)
15µl	bidest. sterile H2O

Preparative Gelelectrophoresis of PCR-products of 4CL, CHS

Investigator: Katrin, Mary

picture follows!

Gelextraction of 4CL+, 4CL-, CHS+, CHS- (bands are as expected; +=with consensus-sequence, -=without consensus-sequence)

DNA-purification with Kit from Quiagen

Quick Change mutagenesis to remove PstI in URA3 from pYES2_RFC25 MCS 1.2

Investigator: Ingmar

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR
Reaction batch

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P29 template
0.5 µl	1:10 dilution of O40 (10 pmol/µL)
0.5 µl	1:10 dilution of O41 ((10 pmol/µL)
17 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		67°C	6.5 min

The procedure was furthermore applied to P31.
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

This operation sequence was applied to the PCR prducts of P29 and P31 respectively.
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

Saturday, June 30th

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.

Operational sequence:

For each transformation of the PCR-products of P29 and P30 a single clone was picked an transferred to 6 ml LB Amp. Incubation overnight at 37°C 180 rpm. The transfomation with the PCR product of P31, P32 and P33 was not successfull. Therfore no clone could be picked from these plates and four instead of one clone was picked from the plates containing the transformations of P29.

July

Sunday, July 1st

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.

Operational sequence:

Using a Quiagen kit a miniprep of the overnight culture was done.
The resulting purified DNA was aliquoted in new tubes labeled as follows:

1st PCR product of P29: P34

2nd PCR product of P29: P35

3rd PCR product of P29: P36

4th PCR product of P29: P37

PCR product of P30: P38

Afterwards a control digestion of P34-P38 was done.

Reaction batch

Plasmid	P34	P35	P36	P37	P38
Fermentas 10x R buffer	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl
DNA	2 µl	2 µl	2 µl	2 µl	5 µl
PstI	0.25 µl	0.25 µl	0.25 µl	0.25 µl	0.25 µl
ddH2O	17.25 µl	17.25 µl	17.25 µl	17.25 µl	14.25 µl
Sum	20 µl	20 µl	20 µl	20 µl	20 µl

Incubation at 37 °C for 1h.
Verification of control digest by agarose gel electrophoresis:

20 µl of each digest product was mixed with 4 µl 6x DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V.

All digest products show the expected two bonds at 3526 bp and at 2332 bp. The Miniprep product P35 was chosen to be used for the further Quickchange Mutagenesis.

Quick Change mutagenesis to remove PstI in the 2µ ori from pYES2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.

PCR
Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P35 template
0.5 µl	1:10 dilution of O42 (10 pmol/µL)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

Reaction batch 2

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P35 template
0.5 µl	1:10 dilution of O43 (10 pmol/µL)
16.5 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo 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

Monday, July 2nd

Repetition of PCR of PAL

Investigator: Mary

Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
4 µl	dNTP's (each 2.5 mM)
0.5 µl	Pfu Ultra II (2.5 U/µL)
5 µl	1:10 dilution of used forward primers (10µM)
5 µl	1:10 dilution of used reversed primers (10µM)
1 µl	DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL)
29.5 µL	bidest. sterile Water

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	2 min (and adding Pfu Ultra after 2 min)
2	30	95°C	30 sec
		55°C	1 min
		72°C	2.5 min
3		72°C	5 min

PCR of LexA with primers including the RFC25 pre- and suffix

Investigator: Jeffery Truong, Georg Schützinger

Aim of the experiment: The Biobrick BBa_K105005 (LexA) has a RFC10 pre- and suffix, but we need RFC25 pre- and suffix for protein fusion, so we have to do a PCR with primer containing the RFC25 pre- and suffix.

The received forward and reverse primer TUM12-LexA-fw and TUM12-LexA-rv are resuspended in 204 µL and 221 µL ELGA water as described in the data sheet to get a final primer concentration of 100 pmol/µL=100 µM. For the PCR reaction mixture we took 0.5 µL of these resuspended primer and add 4.5 µL of ELGA water to get a final primer concentration of 10 µM.

Clone 3 of BBa_K105005 (LexA) has beed choosen for the PCR (ERG No. P23).

PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer
1 µl	10 µM Reverse Primer
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (BBa_K105005) from P23 (Clone 3)
35.75 µL	ELGA Water
=50 µL	TOTAL

The PCR program was performed after following scheme:

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

Tuesday, July 3rd

Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix

Investigator: Jeffery Truong

Aim of the experiment: Analytical gelelectrophoresis of cleaned up PCR product from LexA (BBa_K105005) with primer containing the RFC25 pre- and suffix (TUM12-LexA-fw and TUM12-LexA-rv).

The clean-up of the PCR product LexA (BBa_K105005) with primer containing the RFC25 pre- and suffix (TUM12-LexA-fw and TUM12-LexA-rv) was performed with the QIAquick PCR Purification Kit from Qiagen after manufacturer's protocoll.

5 µL of the purificated PCR product was taken to perform a analytical gelelectrophoresis to verify the success of the PCR.

1% agarose gel containing ethidium bromide was used for the analytical gelelectrophoresis.

The analytical gelelectrophoresis was performed for 60 min at 90 V.

Scheme of the gel:

100 bp ruler	PCR product of BBa_105005	1000 bp ruler

LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase

Investigator: Lara Kuntz

Aim of the experiment: To get colonies of BL21 strains containing lavendula LS for amplification and subsequent plasmid extraction.

Schwab strain #106 was plated on a chloramphenicol containing LB plate. #108 was plated on a amp+chlp containing LB plate. The plates were incubated at 37°C over night.

Quick Change mutagenis to remove PstI in the 2µ Ori from pYes2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.

Operational sequence:

From the transformation of the PCR-product of P35 two single clones were picked an transferred to 6 ml LB Amp. Incubation overnight at 37°C 180 rpm.

PCR of BBa_I742111 (Limonenesynthase) Clone 3 (Trafo 19.06.12)

Investigator: Andrea

PCR used forward primer with consensus sequence; PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer O27
1 µl	10 µM Reverse Primer O30
0.25 µl	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µl)
1 µl	Plasmid DNA (BBa_I742111) Clone 3
35.75 µl	dd water
50 µL	TOTAL

PCR used forward primer without consensus sequence; PCR reaction mixture

volume	reagent
10 µl	5x OneTaq Standard Reaction Buffer
1 µl	10 mM dNTPs
1 µl	10 µM Forward Primer O28
1 µl	10 µM Reverse Primer O30
0.25 µl	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µl)
1 µl	Plasmid DNA (BBa_I742111) Clone 3
35.75 µl	dd water
50 µL	TOTAL

The PCR program was performed after following scheme:

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	47 °C	30 s
	68 °C	1,75 min
Final extension	68 °C	5 min
Hold	4 °C	1 h

Analytical Gelelectrophoresis

5 µl DNA + 1 µl loading buffer

Wednesday, July 4th

Quick Change mutagenis to remove PstI in the 2µ Ori from pYes2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.

Operational sequence:

Using a Quiagen kit a miniprep of the overnight culture was done.
The resulting purified DNA was aliquoted in new tubes labeled as follows:

1st Transformation of P35: P39

2nd Transformation of P35: P40

Afterwards a control digestion of P39 and P40 was done.

Reaction batch

Plasmid	P39	P40
Fermentas 10x R buffer	0.5 µl	0.5 µl
DNA	5 µl	5 µl
PstI	0.25 µl	0.25 µl
ddH2O	14.25 µl	14.25 µl
Sum	20 µl	20 µl

Incubation at 37 °C for 1h.
Verification of control digest by agarose gel electrophoresis:

20 µl of each digest product was mixed with 4 µl 6x DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V.

All digest products show the expected bond at 5858 bp. The Miniprep product P40 was chosen to be used for the further Quickchange Mutagenesis.

Quick Change mutagenis to insert Ala in front of the Strep - tag II in pYES2_RFC25 MCS

Investigator: Ingmar

Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector; operating purfication possibility via Strep-tag II.

PCR
Reaction batch

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P40
1 µl	1:10 dilution of O54 (10 pmol/µL)
1 µl	1:10 dilution of O55 ((10 pmol/µL)
16 µl	ddH2O
0.5 µl	dNTP mix
0.5 µl	Pfu Turbo DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	16	95°C	30 sec
		55°C	1 min
		68°C	6 min

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
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

Analytical Gelelektrophoresis of PCR-Products of PAL

Investigator: Mary

Aim of the experiment: purification and testing if PCR was successful

Purification of PCR-Products with purification kit from quiagen analytical gelelectrophoresis of PAL+, PAL-; expected band at 2,1 kb

piture follows

-> PCR was not successful

Schwab expression stains #106 & #108 which contain lavendula limonene synthase

Investigator: Andrea

Aim of the experiment: Getting clones of cells with plasmids containing the gene for limonenesynthase

Picking of Clones

2 clones of every stain were picked
Incubation at 37 °C in LB + Amp (#108) / LB + Kan (#106)

Preparative Gelelectrophoresis of PCR-products of OMT

Investigator: Mary, Katrin

Aim of the experiment: Purification of the previously digested DNA, test if digestion was successful

picture follows!

Gelextraction of OMT- and OMT+ (bands are as expected; +=with consensus-sequence, -=without consensus-sequence)

DNA-purification with Kit from Quiagen

Thursday, July 5th

Preparation of plasmids containing lavendula LS

Investigator: Lara

Aim: Purify Schwab plasmids containing lavendula limonene synthase

Experiment was conducted using Qiagen Plasmid Miniprep Kit.

P39: Plasmid from Schwab strain #106 (1); 35 ng/µl

P40: Plasmid from Schwab strain #106 (2); 72 ng/µl

P41: Plasmid from Schwab strain #108 (1); 240 ng/µl

P42: Plasmid from Schwab strain #108 (2); 120 ng/µl

Restriction digest of Schwab plasmids

Investigator: Lara

Aim: To check whether extracted plasmids from Schwab expression strains #106 & #108 are OK.

Digest of plasmid from strain #106 was conducted with the following protocoll:

500 ng Plasmid DNA

0,25 µl Nco1

0,25 µl Hind 3

2 µl Buffer Tango

dd H2O to a total volume of 20 µl.

Digest of plasmid from strain #108 was conducted with the following protocoll:

500 ng Plasmid DNA

0,25 µl EcoR1

0,25 µl Not1

2 µl Buffer Orange

dd H2O to a total volume of 20 µl.

(All enzymes and buffers were from Fermentas).

Analytical gel electrophoresis of digested Schwab plasmids

Investigator: Lara Aim: Check plasmids for insert.

Plasmids were digested, 2 µl loading dye (10x) was added to each sample. Gel was loaded in the following order:

1. 6 µl gene ruler 1 kb, 2.-6. 11 µl of: P39, P40, P41, P42, Coumaryl-Plasmid (Kathrin)

Analytical Gelelektrophoresis of plasmid pKS2µHyg-PAL-4Cl-CHS

Investigator: Katrin

Aim of the experiment: testing if PAL is part of the plasmid that was sent to us (troubleshooting because PCR of PAL was not successful)

digestion took 1 h at 37°C

digestion with ApaI (Fermentas)

volume	reagent
9.3 µl	plasmid pKS2µHyg-PAL-4Cl-CHS (miniprep)
2 µl	Buffer B
0.5 µl	ApaI (Fermentas; 10u/µl)
8.2 µl	bidest. sterile H2O

analytical gelelectrophoresis: expected band at 3,14 kb (Gal-PAL-XK)

picture follows

-> experiment was successful, PAL is part of the plasmid pKS2µHyg-PAL-4Cl-CHS

Team:TU Munich/Notebook/Labjournal

From 2012.igem.org

Contents

June

Wednesday, June 13th

Exchange of the Multiple Cloning Site of pYES2

Thursday, June 14th

Exchange of the Multiple Cloning Site of pYES2

Transformation of plasmids from Prof. Schwab in E.coli XL-1 Blue

Friday, June 15th

Exchange of the Multiple Cloning Site of pYES2

Transformation of plasmids from Prof. Schwab into E.coli XL-1 Blue

Sunday, June 17th

Exchange of the Multiple Cloning Site of pYES2

Monday, June 18th

Exchange of the Multiple Cloning Site of pYES2

Tuesday, June 19th

Transformation of BBa_I742111 (Limonenesynthase) into E.coli XL-1 Blue

Wednesday, June 20th

Exchange of the Multiple Cloning Site of pYES2

Transformation of BBa_I742111 (Limonenesynthase) into E.coli XL-1 Blue

Thursday, June 21st

Transformation of BBa_I742111 and plasmids from Prof. Schwab into E.coli XL-1 Blue

Friday, June 22nd

Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS

Saturday, June 23rd

Quick Change mutagenis to remove NgoMIV from pYES2

Sunday, June 24th

Miniprep of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS

Quick Change mutagenis to remove NgoMIV from pYES2

Transformation of 2 Biobricks into E. coli XL1-Blue

Monday, June 25th

Miniprep of E. coli XL1-Blue with pYes2_RFC25 MCS 1.2

Quick Change mutagenis to remove SpeI from pYES2_RFC25 MCS 1.2

Picking of E. coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Tuesday, June 26th

Quick Change mutagenis to remove SpeI from pYes2_RFC25 MCS

Verification of the PCR products P30, P31 and P33

Control of the competent cells and transformation with P20

PCR of PAL, 4CL, CHS, OMT (Coumaryl-CoA)

Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase)

Friday, June 29th

Preparative digest of PCR-products of 4CL, CHS and OMT

Preparative Gelelectrophoresis of PCR-products of 4CL, CHS

Quick Change mutagenesis to remove PstI in URA3 from pYES2_RFC25 MCS 1.2

Saturday, June 30th

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

July

Sunday, July 1st

Quick Change mutagenis to remove PstI in the URA 3 gene from pYes2_RFC25 MCS

Quick Change mutagenesis to remove PstI in the 2µ ori from pYES2_RFC25 MCS

Monday, July 2nd

Repetition of PCR of PAL

PCR of LexA with primers including the RFC25 pre- and suffix

Tuesday, July 3rd

Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix

LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase

Quick Change mutagenis to remove PstI in the 2µ Ori from pYes2_RFC25 MCS

PCR of BBa_I742111 (Limonenesynthase) Clone 3 (Trafo 19.06.12)

Wednesday, July 4th

Quick Change mutagenis to remove PstI in the 2µ Ori from pYes2_RFC25 MCS

Quick Change mutagenis to insert Ala in front of the Strep - tag II in pYES2_RFC25 MCS

Analytical Gelelektrophoresis of PCR-Products of PAL

Schwab expression stains #106 & #108 which contain lavendula limonene synthase

Preparative Gelelectrophoresis of PCR-products of OMT

Thursday, July 5th

Preparation of plasmids containing lavendula LS

Restriction digest of Schwab plasmids

Analytical gel electrophoresis of digested Schwab plasmids

Analytical Gelelektrophoresis of plasmid pKS2µHyg-PAL-4Cl-CHS