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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) and 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

Incubation on ice for 30 min

5 min heat shock at 37 °C

cells were 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)

cell suspension was centrifuged at 13000 rpm for 60 s for resuspending the pellet with 100 µl LB and plating also

incubation 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

The Multiple Cloning Site was exchanged successfully!!!

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 Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	15	95°C	30 sec
		55°C	1 min
		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 Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

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

Wednesday, June 27th

Repetition of analytic gel of 21st June

Investigator: Andrea

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

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 Ultra II 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 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

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: P43

2nd Transformation of P35: P44

Afterwards a control digestion of P43 and P44 was done.

Reaction batch

Plasmid	P43	P44
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 P44
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

Analytical Gel Electrophoresis: File:TUM12 20120704 PAL-PCR v2.tiff

-> PCR was not successful, no band at 2,1 kb

-> next steps: new Design of Primer and repetition of PCR with new primers

Picking of clones of Schwab expression stains #106 & #108

Investigator: Andrea

Aim of the experiment: Getting clones of cells with plasmids containing the gene for limonenesynthase and amplification of these clones for further plasmid preparation.

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 (Katrin)

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)

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

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.

Operational sequence:

From the transformation of the PCR-product of P44 two single clones were picked an transferred to 6 ml LB Amp. Incubation overday at 37°C 180 rpm.
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 picked clone of P44: P45

2nd picked clone of P44: P46

Friday, July 6th

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

Afterwards a control digestion of P45 and P46 was done.

Reaction batch

Plasmid	P45	P46
Fermentas 10x O buffer	2 µl	2 µl
DNA	3 µl	3 µl
PstI	0.25 µl	0.25 µl
ddH2O	14.75 µl	14.75 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 bonds at 5858 bp. The Miniprep product P45 was chosen to check the insertion of Ala in front of the strep-tag II via DNA sequencing.

The results of the sequencing are shown below:

The sequencing results show that the insertion of Alanin in front of the Strep-tag II was not successful.

PCR of Schwab plasmid DNA to amplify gene for lavendula LS

Instructor: Lara

Aim: PCR of Schwab plasmids with primers which were designed to amplify the lavendula LS gene and to add RFC25 restriction sites.

3 different primer combinations were used:

1. O33/O37

2. O34/O37

3. O35/O37

Each primer combination was used for plasmid DNA amplification of P40 and P41.

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 (Final: 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
	50 °C	30 s
	68 °C	1,75 min
Final extension	68 °C	5 min
Hold	4 °C	1 h

Analytic Gel of PCR of Schwab plasmid DNA

Instructor: Andrea

Saturday, July 7th

Quick Change mutagenesis 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.

As the sequencing of the first attempt to introduce Ala in front of the Strep-tag II did not show a successfull insertion of Ala, the quickchange mutagense was performed once again with a modified setup. Presumably a formation of primer dimers was responsable for the experiment's results. Therefore the second PCR was operated in two steps as shown below.

PCR
Reaction batch 1

volume	reagent
2.5 µl	10x Pfu Ultra II buffer
4 µl	Plasmid P44 template
0.5 µl	1:10 dilution of O54 (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 P44 template
0.5 µl	1:10 dilution of O55 (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

Transformation of E.coli XL1 blue with ADH1 promoter, ADH1 terminator and TEF2 promoter

Investigator: Georg

Monday, July 9th

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder (part 1/4)

Investigator: Jeff, Alois, Martin

Aim of the experiment: Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. Saccharomyces cerevisiae does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried Spirulina platensis powder.

Operational sequence:

50 g of Spirulina platensis powder was (from concept-vitalprodukte.de) resuspended in 1.5 l of H2O (30 mg/l) in a beaker covered with aluminium foil.

Stirring for 10 min at RT.

Green Spirulina suspension was divided in 6 centrifuge bottles, covered in aluminium foil.

Centrifation at 10500 rpm at 4 °C (SLA-3000 rotor, Thermo Scientific) for 1 h.

Supernatant was discarded

25 ml of MeOH added to each bottle and was heavily shaked to resuspend the pellet for the next cleaning step with MeOH.

Each bottle with the resuspended pellet were filled with MeOH to a final volume of 250 ml and shaked again to fully resuspend the pellet.

Centrifugation at 10500 rpm at 4 °C (SLA-3000 rotor, Thermo Scientific) for 10 min.

Supernatant was discarded.

The last 4 steps were repeated until the supernatant of the washed pellet was colorless or cyanblue but not green anymore (Regulary, it takes 3 or 4 times). Pellet should be cyanblue now.

The pellet of the 6 centrifuge tubes was collected in a sole centrifugation tube, covered in aluminium foil tube, by scratching it out with a small spoon.

The remaining rest of the pellet which cannot be scratched out were resuspended in a small amount of MeOH and were transformed from tube to tube with a interim shaking step.

This suspension was transferred into the tube with the scratched-out pellet.

Centrifugation at 10500 rpm at 4 °C (SLA-3000 rotor, Thermo Scientific) for 10 min.

Supernatant was discarded.

Finally washed pellet was stored, wrapped in foil overnight for the methanolysis next day.

Tuesday, July 10th

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder (part 2/4)

Investigator: Jeff, Alois, Martin

Aim of the experiment: Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. Saccharomyces cerevisiae does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried Spirulina platensis powder.

Operational sequence:

Washed pellet from the day before was resuspended in 500 ml MeOH.

Heat suspension in a 500 ml flask in a water bath at 70 – 75 ºC with a condensing coil cooled with water for 5 – 8 hrs.

After this, the suspension was transferred into a new centrifuge tube and centrifuged at 10500 rpm at 4 °C (SLA-3000 rotor, Thermo Scientific) for 20 min.

The supernatant was decanted trough a filter paper into new centrifugation tube and stored, wrapped in a aluminium foil, at -20 °C.

The pellets also was stored, wrapped in a aluminium foil, at -20 °C.

Plating of received E. coli containing biobricks

Investigator: Jeff

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: BBa_K207001 in pSB1A2, BBa_K243006 in BBa_K157000, BBa_K300001 in K300007 (part for other subproject), BBa_K268000 in pSB6A0 (part for other subproject), BBa_K365005 RFC25 in pSB1C3, BBa_K365000 in pSB1C3, BBa_K207000 in pSB3K3, BBa_K165031 in pSB1AK3

Wednesday, July 11th

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder (part 3/4)

Investigator: Jeff, Alois, Martin

Aim of the experiment: Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. Saccharomyces cerevisiae does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried Spirulina platensis powder.

Operational sequence:

The pellet after the first methanolysis of the day before was undergone a second methanolysis step to ensure high efficiacy of PCB extraction. Operational sequence was performed like the first methanolysis including the centrifugation and filtering step. The pellet after the second methanolysis should be more colorless and the filtered supernatant was freezed, like the one from the first methanolysis, at -20 °C, wrapped in aluminium foil

Transformation of E. coli XL1-Blue with pGADT7 and pGBKT7 plasmid for Yeast-two-hybrid (Y2H)

Investigator: Jeff

Aim of the experiment: pGADT7 and pGBKT7 are plasmids containing the transcriptional activation domain or the DNA binding domain of the transcription activator Gal4. pGADT7 contains the transcriptional activation domain of gal4; we want to clone the the first 100 residues of Pif3 (BBa_K365000) into this plasmid. As a result we have a fusion contruct of Gal4 and Pif3, which is nescassary for the light-switchable promoter system. pGBKT7 is for backup, if the ordered biobricks are not working.

Operational sequence:

E. coli XL1-Blue are transformed with pGADT7 and pGBKT7 seperately after standard protocol of our lab.

Introducing new Saccharomyces cerevisiae strain (Y190 strain) from Schwab lab for Y2H

Investigator: Jeff

Aim of the experiment: The Y190 Saccharomyces cerevisiae is a special strain for Yeast-two-hybrid. This strain carries a Gal4 and Gal80 deletion to higher the signal/noise-ratio of protein-protein interactions. Reporter for protein-protein interactions are HIS3, lacZ and MEL1 and are encoded in the genomic DNA. Transformation markers are trp1, leu2 and cyhR2 and are encoded on the transformation plasmids.

Operational sequence:

The freshly plated Y190 cells are transferred with a inoculation loop from the original plate on a new YPD agar plate.

After 2 days the the plate was put in 4 °C.

Repetition of PCR of PAL

Investigator: Katrin, Daniela

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
		52°C	1 min
		72°C	2.5 min
3		72°C	5 min

PCR purification

Purification was done using QIAquick PCR Purification Kit (250)

Analytical gelelektrophoresis of PCR-Products of PAL

-> PCR was not successful, no band at 2,1 kb (picture follows)

Preparative Digest of pYES_iGEM

Investigator: Katrin, Daniela

Digestion of P50 with Xbal and NgOMIV

volume	reagent
10 µl	P50 (concentration: 264.5 ng/µl)
2.5 µl	NEB4
2.5 µl	10x BSA
1 µl	XbaI (20 U/µl)
2 µl	NgOMIV (10U/µl)
7 µl	ddH2O

Incubation: 37 °C, 3 h

Digestion of P50 with Xbal and PstI

volume	reagent
10 µl	P50 (concentration: 264.5 ng/µl)
5 µl	Tango buffer
2 µl	XbaI
3 µl	PstI
7.5 µl	ddH2O

Incubation: 37 °C, 3 h

DNA preparative gel electrophoresis

gel: 1% with LMP-agarose
band 1: P50 digested with XbaI and PstI
band 2: P50 digested with XbaI and NgOMIV
70 V, 90 min

Gelextration

cut the bands
QIAquick Gel Extractrion Kit was used
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation

Ligation of digested P50 with digested PCR-products of PCR 15-20 (4CL, CHS and OMT)

Investigator: Katrin, Daniela

Concentration (Nano Drop:
4CL+ (PCR 15) = 40.3 ng/µl
4CL- (PCR 16) = 37.3 ng/µl
CHS+ (PCR 17) = 46.1 ng/µl
CHS- (PCR 18) = 51.2 ng/µl
OMT+ (PCR 19) = 22.3 ng/µl
OMT- (PCR 20) = 16.7 ng/µl

P50 digested with XbaI and PstI = 23.9 ng/µl (the digested Plasmid has the number P71)
P50 digested with XbaI and NgOMIV = 28.4 ng/µl (the digested Plasmid has the number P72)

required volumes were calculated using Lab Tools

4CL+ (PCR 15) with P50 digested with XbaI and PstI

volume	reagent
5.27 µl	P50 digested
2.73 µl	4CL+ (PCR 15)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

4CL- (PCR 16) with P50 digested with XbaI and PstI

volume	reagent
5.13 µl	P50 digested
2.87 µl	4CL- (PCR 16)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

CHS+ (PCR 17) with P50 digested with XbaI and NgOMIV

volume	reagent
5.84 µl	P50 digested
2.16 µl	CHS+ (PCR 17)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

CHS- (PCR 18) with P50 digested with XbaI and NgOMIV

volume	reagent
6 µl	P50 digested
2 µl	CHS- (PCR 18)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT+ (PCR 19) with P50 digested with XbaI and NgOMIV

volume	reagent
4.45 µl	P50 digested
3.55 µl	OMT+ (PCR 19)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT- (PCR 20) with P50 digested with XbaI and NgOMIV

volume	reagent
3.87 µl	P50 digested
4,13 µl	OMT- (PCR 20)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

Negative control

volume	reagent
5 µl	P50 digested with XbaI and PstI or NgOMIV
3 µl	ddH2O
1 µl	T4 DNA-ligase
1 µl	T4-ligase buffer (10x)

water bath 16 °C

Preparation of YPD medium

Investigator: A lot of Alois, Martin

Manual for YPD production Yeast Extract Peptone Dextrose Medium (1 liter)

1% yeast extract
2% peptone
2% dextrose (D-glucose)

1. Dissolve the following in 1000 ml of water:

10 g yeast extract
20 g peptone
20 g dextrose (see note below if making plates)

2. Autoclave for 20 minutes on liquid cycle.

3. Store medium at room temperature. The shelf life is approximately one to two months.

Thursday, July 12th

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder (part 4/4)

Investigator: Martin, Jeff

Aim of the experiment: Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. Saccharomyces cerevisiae does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried Spirulina platensis powder.

Operational sequence:

The supernatants (~1l in total) of the two previous experiments were pooled and put into a rotary evaporater in order to acquire a concentrate of approximately 100ml.
The settings of the rotary evaporater: ~160 millibars, waterbath temperature of 25-30°C
Furthermore, there were measures to be taken to protect the solution from direct sunlight: blinds down, aluminum foil wrapped around the waterbath

Afterwards we transfered the Methanol solution into a separating funnel and added another 100ml of aqua dest..
By means of adding chloroform we created two phases. The lower (chloroform) phase with the solved Phycocyanobilin was separated and put into another rotary evaporater flask. This step was repeated three times in order to get all the Phycocanobilin into the next step

Then the chloroform was completely removed in the rotary evaporater (same settings as before), the pure (?) Phycocyanobilin in the flask was solved in 60 ml DMSO, transfered into a new flask and frozen at -20 °C.

Picking of transformated (pGADT7 and pGBKT7) E. coli cells on antibiotic selection plates

Investigator: Jeff

Aim of the experiment: pGADT7 and pGBKT7 are plasmids containing the transcriptional activation domain or the DNA binding domain of the transcription activator Gal4. pGADT7 contains the transcriptional activation domain of gal4; we want to clone the the first 100 residues of Pif3 (BBa_K365000) into this plasmid. As a result we have a fusion contruct of Gal4 and Pif3, which is nescassary for the light-switchable promoter system. pGBKT7 is for backup, if the ordered biobricks are not working.

Operational sequence:

A E. coli colony was picked for each plasmid and was transferred in a tube containing 4 ml of LB-medium containing antibiotics (Amp for pGADT7 and Kan for pGBKT7).

Overnight culture at 37 °C in a cell-culture shaker.

Miniprep of transformated E. coli from overnight culture (8 plasmids containing biobricks)

Investigator: Andrea

Aim of the experiment: Miniprep of transformated E. coli from overnight culture to get the plasmids with biobricks

NanoDrop Measure

Plasmid	Concentration
P73	41.6 ng/µl
P74	85.2 ng/µl
P75	72.4 ng/µl
P76	254.3 ng/µl
P77	74.4 ng/µl
P78	50.0 ng/µl
P79	46.2 ng/µl
P80	116.7 ng/µl
P81	77.1 ng/µl
P82	65.5 ng/µl

Analytical digestion and gelelectrophoresis of biobricks (8 plasmids containing biobricks)

Investigator: Jeff

Aim of the experiment: Checking whether the biobricks are part of the plasmid-backbone.

Operational sequence:

Reaction batch for each plasmid:

Reagent	Volume in µl
Tango Buffer 10x	4 µl
XbaI (Fermentas)	0.25 µl
PstI (Fermentas)	0.5 µl
Plasmid DNA	2.5 µl
ddH2O	12.75 µl
TOTAL	20 µl

Incubation at 37 °C for 1 h 45 min.

Analytical gelelectrophoresis at 90 V for 1 h.

Order of gel-pockets:

1 kbp ladder	P73	P74	P75	P76	P79	P80	P81	P82	100 bp ladder
	BAD	OK	OK	OK	OK	OK	BAD	OK

P73 and P81, both parts from Havard university are bad. To exclude errors, one should pick another colony for each plasmid and do again the analytical digestion and gelelectrophoresis after the miniprep.

Transformation of Ligationproducts of pYES2 + OMT, 4Cl and CHS in E.coli

Investigator: Mary

adding 5µl ligation product in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (Withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
plate on agar with Ampicillin over night

Repetition of PCR of PAL

Investigator: Daniela

Aim of the experiment: Repetition of PCR of PAL (so far not successful) with the use of different polymerase and try of 3 temperatures

only with PAL+, 3 different temperatures (see cycling parameters) and one batch at 52.5 °C with DMSO

Reaction batch

volume	reagent
10 µl	5x Herculase II reaction buffer
0,5 µl	dNTP Mix (each dNTP 2.5 mM)
1 µl	Herculase II fusion DNA Polymerase
1,25 µl	1:10 dilution of used forward primers (O22)
1,25 µl	1:10 dilution of used reversed primers (O59)
1 µl	1:10 dilution of DNA (P19=pKS2µHyg-PAL-4CL-CHS 50 ng/µL) -> 5 ng/µL
35 µL	ddH2O

Reaction batch with DMSO

volume	reagent
10 µl	5x Herculase II reaction buffer
0,5 µl	dNTP Mix (each dNTP 2.5 mM)
1 µl	Herculase II fusion DNA Polymerase
1,25 µl	1:10 dilution of used forward primers (O22)
1,25 µl	1:10 dilution of used reversed primers (O59)
1 µl	1:10 dilution of DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL) -> 5 ng/µL
1,5 µl	DMSO (3% des Ansatzes)
33.5 µL	ddH2O

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	2 min
2	30	95°C	30 sec
		52.5°C	30 sec
		72°C	4 min
3		72°C	3 min

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	2 min
2	30	95°C	30 sec
		45°C	30 sec
		72°C	4 min
3		72°C	3 min

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	2 min
2	30	95°C	30 sec
		56.2°C	30 sec
		72°C	4 min
3		72°C	3 min

Analytical gel electrophoresis

no bands at all (picture follows)
possibly due to low concentration of P19 (5 ng/µl)

Ligation of P93 (pSB1C3 digested with XbaI and AgeI) with digested PCR-products of PCR 17-20 (CHS and OMT)

Investigator: Daniela

Concentration (Nano Drop:
CHS+ (PCR 17) = 46.1 ng/µl
CHS- (PCR 18) = 51.2 ng/µl
OMT+ (PCR 19) = 22.3 ng/µl
OMT- (PCR 20) = 16.7 ng/µl

P93 (digested with XbaI and AgeI) = 4.8 ng/µl

CHS+ (PCR17) with P93 digested with XbaI and AgeI

volume	reagent
7.04 µl	P93
0.96 µl	CHS+ (PCR 17)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

CHS- (PCR 18) with P93 digested with XbaI and AgeI

volume	reagent
7.13 µl	P93
0.87 µl	CHS- (PCR18)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT+ (PCR19) with P93 digested with XbaI and AgeI

volume	reagent
6.19 µl	P93
1.81 µl	OMT+ (PCR19)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT- (PCR20) with P50 digested with XbaI and AgeI

volume	reagent
5.75 µl	P93
2.25 µl	OMT- (PCR20)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

Negative control

volume	reagent
5 µl	P93
3 µl	ddH2O
1 µl	T4 DNA-ligase
1 µl	T4-ligase buffer (10x)

water bath 16 °C
products were named P94-P97

Preparative digest of PCR1-PCR7 and P50

Investigator: Andrea

Digestion of P50 with XbaI and NgoMIV

volume	reagent
20 µl	P50
4 µl	NEB Buffer
0.4 µl	BSA
1 µl	XbaI (10 U/µl)
2 µl	NgoMIV (10 U/µl)
27.4 µl	ddH2O

Digestion of PCR1-PCR7 with XbaI and AgeI

volume	reagent
25 µl	PCR-Product
5 µl	NEB Buffer
0.5 µl	BSA
1 µl	XbaI (10 U/µl)
1 µl	AgeI (10 U/µl)
32.5 µl	ddH2O

Incubation: 37 °C, 3 h

Preperative gel electrophoresis

Investigator: Andrea

Aim of the experiment: Analytical gel electrophoresis of products from restriction digest of plasmids PCR1 - PCR7 and PCR9

Ligation of digested PCR1-PCR7 (digested with XbaI and AgeI) with pSB1C3 (digested with XbaI and AgeI) and with pYESnew (digested with XbaI and NgoMIV)

Investigator: Andrea

Concentration (Nano Drop:
LIMS Citrus (PCR 1) = 13.4 ng/µl
LIMS Citrus (PCR 2) = 10.3 ng/µl
LIMS Lavendula (PCR 3) = 2.2 ng/µl
LIMS Lavendula (PCR 3) = 9.9 ng/µl
LIMS Lavendula (PCR 3) = 9.6 ng/µl
LIMS Lavendula (PCR 3) = 9.8 ng/µl
LIMS Lavendula (PCR 3) = 12.3 ng/µl

P50 (digested with XbaI and NgoMIV) = 10.3 ng/µl

P93 (digested with XbaI and AgeI) = 4 ng/µl

PCR1 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.89µl	P50
3.11 µl	PCR 1
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR2 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.38 µl	P50
3.62 µl	PCR2
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR3 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
1.64 µl	P50
6.36 µl	PCR3
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR4 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.3 µl	P50
3.7 µl	PCR4
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR5 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.26 µl	P50
3.74 µl	PCR5
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR6 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.26 µl	P50
3.74 µl	PCR6
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR7 with P50 digested with XbaI and AgeI/NgoMIV

volume	reagent
7.83 µl	P50
3.27 µl	PCR7
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

Negative control

volume	reagent
5 µl	P50
3 µl	ddH2O
1 µl	T4 DNA-ligase
1 µl	T4-ligase buffer (10x)

PCR1 with P93 digested with XbaI and AgeI

volume	reagent
4.89 µl	P93
3.11 µl	PCR1
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR2 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.38 µl	P93
3.62 µl	PCR2
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR3 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
1.52 µl	P93
6.48 µl	PCR3
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR4 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.3 µl	P93
3.7 µl	PCR4
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR5 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.26 µl	P93
3.74 µl	PCR5
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR6 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.26 µl	P93
3.74 µl	PCR6
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

PCR7 with P93 digested with XbaI and AgeI/NgoMIV

volume	reagent
4.72 µl	P93
3.28 µl	PCR7
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

water bath 16 °C
products were named P100-P115

Friday, July 13th

PCR of P73, P80 and P81 to add RFC25 pre- and suffix

Investigator: Jeff, Saskia

Aim of the experiment: Introducing RFC25 pre- and suffix into parts of P73, P80 and P81.

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 (For P73: O46 (TUM12-PhyBGal4bd-fw); for P80: O31 (TUM12-LSPS-fw); for P81: O50 (TUM12-Phyb-fw))
1 µl	10 µM Reverse Primer (For P73: O47 (TUM12-PhyBGal4bd-rv); for P80: O32 (TUM12-LSPS-rv ); for P81: O51 (TUM12-Phyb-rv))
0.25 µL	OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl	Plasmid DNA (P80 or P73 or P81)
35.75 µL	ELGA Water
=50 µL	TOTAL

The gradient PCR program was performed after following scheme with following conditions (Tm=58 ΔG=5 °C; P73 in row 9(=61.1 °C); P80 in row 1 (=53.0 °C); P81 in row 7(=58.4 °C):

Initial denaturation	94 °C	30 s
30 cycles	94 °C	30 s
	Tm=58 °C; ΔG=5 °C	150 s
	68 °C	60 s
Final extension	68 °C	5 min
Hold	4 °C	overnight

Miniprep, analcytical digestion and gelelectrophoresis of pGADT7 and pGBKT7

Investigator: Jeff, Saskia

Aim of the experiment:Miniprep, analcytical digestion and gelelectrophoresis of pGADT7 (tube P98, EcoRI and BamHI)and pGBKT7 (tube P99, BamHI and PstI)

Operational sequence:

Operated after standard protocol of the lab for analytical digestion and gelelectrophoresis.

Gel OK, like expected

Preperative digestion and gelelectrophoresis of P79 and O56???

Investigator: Jeff, Saskia, Georg

Aim of the experiment: Preperativ gelelectrophoresis and gel of digested PCR product of LexA (NgoMIV+PstI) and pSB1C3 containing the RFC25 compatible 20aa linker with RFC25 pre- and suffix (AgeI and PstI).

Operational sequence:

Operated after standard protocol of the lab for preperative digestion and gelelectrophoresis.

Gel OK, like expected.

Preparative digest of P19

Investigator:Daniela, Ingmar

Digestion of P19 with ApaI

volume	reagent
20 µl	P19 (concentration: 53.8 ng/µl)
4 µl	Buffer B
2 µl	ApaI (10 U/µl)
14 µl	ddH2O

Incubation: 37 °C, 3 h

DNA preparative gel electrophoresis

gel: 1% with LMP-agarose
band 1: P50 digested with XbaI and PstI
band 2: P50 digested with XbaI and NgOMIV
70 V, 90 min

Gelextration

cut the bands
QIAquick Gel Extractrion Kit was used
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation

Gradient PCR of PAL to optimize the primer annealing temperature

Investigator: Ingmar

Aim of the experiment: As all PCR experiments to amplify the PAL gene contained in P19 failed, we decided to run a gradient PCR to optimize the primer annealing temperature.

PCR
Reaction batch

volume	reagent
1 µl	10x Pfu Ultra II buffer
0.5 µl	Plasmid P19 template
0.5 µl	1:10 dilution of O15 (10 pmol/µL)
0.5 µl	1:10 dilution of O59 (10 pmol/µL)
0.25 µl	dNTP mix
7 µl	ddH2O
0.25 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	29	95°C	30 sec
		gadient 33-47.5 °C	1 min
		68°C	3 min
3	1	68°C	5 min
4	1	4°C	infinity

Verification of the PCR by agarose gel electrophoresis:

10 µl of each PCR tube was mixed with 2 µl 6x DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V in a 1% agarose gel.

A bond at the expected length of 2160 bp appears at 47 °C. Therefore a PCR using this primer annealing temperature will done on sunday.

Transformation of P94 - P97 (ligationproducts of pSB1C3 with CHS and OMT respectively) in E.coli

Investigator: Daniela

adding 5µl ligation product (P94-P97) in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (Withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
plate on agar with Ampicillin over night

wrong antibiotica was used!!! Repetition follows!!! Nevertheless, some colonies could be observed.

Ligation of LIMS in pYESnew and pSB1C3

Investigator:Andrea

Transformation

adding 5µl ligation product (PCR1-PCR7) in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min, 180 rpm
plate on agar with Ampicillin (pYESnew) or Chloramphenicol (pSB1C3) over night

Sunday, July 15th

PCR purification of PCR products of P73, P80 and P81 and analytical gelelectrophoresis

Investigator: Jeff

Aim of the experiment: PCR was performed to introduce restriction sites to the gene of interest. MfeI and BamHI for P73 and RFC25 pre- and suffix for P80 and P81. The aim is to contruct fusion proteins with the aid of these restriction sites.

Operational sequence:

PCR cleaning with Qiagen PCR purification kit after manufacturer's protocol.

Analytical gelelectrophoresis (1% agarose) at 90 for 60&min.

PCR of PAL consless using the optimized primer annealing temperature of 47 °C

Investigator: Ingmar

Aim of the experiment: Amplification of the PAL gene contained in P19.

PCR
Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
2.5 µl	Plasmid P19 template
2.5 µl	1:10 dilution of O15 (10 pmol/µL)
2.5 µl	1:10 dilution of O59 (10 pmol/µL)
1.25 µl	dNTP mix
35 µl	ddH2O
1.25 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	29	95°C	30 sec
		47 °C	1 min
		68°C	3 min
3	1	68°C	5 min
4	1	4°C	infinity

PCR successful - Band at about 2,1 kb :

Monday, July 16th

Preperative digestion and gelelectrophoresis of P98 and PCR26

Investigator: Jeff, Georg

Aim of the experiment: Construction of Gal4AD-Pif3, a part of the light-switchable promoter system.

Operational sequence:

Preperative digestion after manufacturer's (Fermentas) advise for double-digestion for EcoRI+BamHI and MfeI(MunI)+BamHI; EcoRI+BamHI: Buffer 2X Tango™, 2-fold excess of BamHI; MfeI(MunI)+BamHI: Buffer G.

Preperative gelelectrohphoresis after laboratory's standard protocol. (70 V, 90 min)

Plating of received E. coli containing biobrick BBa_K165055

Investigator: Jeff

Aim of the experiment: The received biobrick BBa_K165055 (LexA binding sites + mCYC + Kozak + YFPx2 + ADH1 terminator) 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 was BBa_K165055 in BBa_J63009 plasmid (AmpR, low copy plasmid)

Preparatory culture of S. cerevisiae

Investigator: Alois, Martin

Aim of the experiment: In order to have sufficient viable cells to inoculate an experimental culture in which we want to compare the growth rate of s. cerevisiae with a strain of brewing yeast (34/70 s. pastorianus weihenstephan) we aim to establish a preparatory over night culture.

Operational sequence: A sample of -80°C stored s. cerevisiae (glycerol stock) is used to inoculate 20ml of YPD-medium. This is shaken overnight at 30°C.

Miniprep of plasmids containing lavendula limonene synthase/citrus limonene synthase

Investigator: Lara

Aim of the experiment: Extract plasmids (pYESnew and pBS1C3) that contain limonene synthase/citrus limonene synthase.

Miniprep with Qiagen Kit; Plasmids p116-p122. Restriction digest with Xba1 and Pst1.

Restriction digest of p116-p122

Investigator: Lara

Aim of the experiment: Analytical restriction digest of plasmids p116, p117, p118, p119, p120, p121, p122.

Restriction digest with Xba1 and Pst1.

2 µl DNA

0,25 µl Pst1-HF

0,25 µl Xba1

2 µl Puffer 4

15,3 µl ddH2O

20 µl total

Analytical gel electrophoresis

Investigator: Lara

Aim of the experiment: Analytical gel electrophoresis of products from restriction digest of plasmids p116, p117, p118, p119, p120, p121, p122.

p116: PCR1/pYES

p117: PCR2/pYES

p118: PCR4/pYES

p119: PCR4/pSB1C3

p120: PCR5/pYES

p121: PCR6/pYES

p122: PCR7/pYES

Midiprep of pSB1C3 (RFC25-compatible)

Investigator:Mary

Aim of the experiment: Get a lot of pSB1C3 for further experiments

Midipreparation of the pellet from over night cultures (E.coli, Transformed with pSB1C3 - RFC25; name in registry: K365005)

was done with the Midiprep Kit from Qiagen

Result was named as P123 and had the concentration: 469,5 ng/µl (100µl at all)

PCR-Products of PAL (consless): digestion, extraction and ligation

investigator: Daniela, Mary

Aim of the experiment: digestion of PCR-Products to gain the correct cutting sites for ligation in pYES2 and pSB1C3 afterwards

Purification of PCR-Products with PCR-Purification Kit

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

digestion took 3h at 37°C

extraction from preparative gel:

PCR of PAL+cons using the optimized primer annealing temperature of 47 °C

Investigator: Daniela

Aim of the experiment: Amplification of the PAL cons gene contained in P19.

PCR
Reaction batch

volume	reagent
5 µl	10x Pfu Ultra II buffer
2.5 µl	Plasmid P19 template
2.5 µl	1:10 dilution of O22 (10 pmol/µL)
2.5 µl	1:10 dilution of O59 (10 pmol/µL)
1.25 µl	dNTP mix
35 µl	ddH2O
1.25 µl	Pfu Ultra II DNA polymerase (2.5 U / µl)

PCR cycling parameters

Segment	Cycles	Temperature	Time
1	1	95 °C	30 sec
2	29	95°C	30 sec
		47 °C	1 min
		68°C	3 min
3	1	68°C	5 min
4	1	4°C	infinity

PCR was succesful, band at 2,1kb:

Transformation of P94 - P97 (ligationproducts of pSB1C3 with CHS and OMT respectively) in E.coli

Investigator: Daniela

adding 5µl ligation product (P94-P97) in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 45 min, 180 rpm
plate on agar with chloramphenicol over night

-> were succesfull: some colonies were grown

Tuesday, July 17th

Picking of transformed E. coli containing biobrick BBa_K165055

Preparative digest of P123 (pSB1C3 RFC25)

Investigator: Mary, Daniela

Digestion of P123 with Xbal/PstI and XbaI/AgeI

volume	reagent
23.1 µl	ddH2O
4 µl	NEB4
0.4 µl	10x BSA
1 µl	XbaI (20 U/µl)
1.5 µl	PstI (10U/µl)
10 µl	P123

volume	reagent
23.35 µl	ddH2O
4 µl	NEB4
0.4 µl	10x BSA
1 µl	XbaI (20 U/µl)
1.25 µl	AgeI (10U/µl)
10 µl	P123

Incubation: 37 °C, 3 h

DNA preparative gel electrophoresis

gel: 1% with LMP-agarose
band 1: P123 digested with XbaI and PstI
band 2: P123 digested with XbaI and AgeI
70 V, 90 min

Gelextration

cut the bands
QIAquick Gel Extractrion Kit was used
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation

Products were names as follows:

P123 digested with XbaI and PstI: P132
P123 digested with XbaI and AgeI: P133

PCR-Products of PAL (with consensussequence): digestion and extraction

investigator: Daniela, Mary

Aim of the experiment: digestion of PCR-Products to gain the correct cutting sites for ligation in pYES2 and pSB1C3 afterwards

Purification of PCR-Products with PCR-Purification Kit

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

digestion took 3h at 37°C

extraction from preparative gel:

Picking Clones of CHS and OMT in pSB1C3-RFC25

investigator: Daniela, Mary

Aim of the experiment: preculture over night for the miniprep next day

Repetition of ligation of PCR 15-20 (4CL, CHS and OMT) with pYES (P71 and P72 respectively)

Investigator: Mary, Daniela

Concentration (Nano Drop:
4CL+ (PCR 15) = 40.3 ng/µl
4CL- (PCR 16) = 37.3 ng/µl
CHS+ (PCR 17) = 46.1 ng/µl
CHS- (PCR 18) = 51.2 ng/µl
OMT+ (PCR 19) = 22.3 ng/µl
OMT- (PCR 20) = 16.7 ng/µl

P50 digested with XbaI and PstI = 23.9 ng/µl (the digested Plasmid has the number P71)
P50 digested with XbaI and NgOMIV = 28.4 ng/µl (the digested Plasmid has the number P72)

required volumes were calculated using Lab Tools

4CL+ (PCR 15) with P71

volume	reagent
5.27 µl	P71
2.73 µl	4CL+ (PCR 15)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

4CL- (PCR 16) with P71

volume	reagent
5.13 µl	P71
2.87 µl	4CL- (PCR 16)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

CHS+ (PCR 17) with P72

volume	reagent
5.84 µl	P72
2.16 µl	CHS+ (PCR 17)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

CHS- (PCR 18) with P72

volume	reagent
6 µl	P72
2 µl	CHS- (PCR 18)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT+ (PCR 19) with P72

volume	reagent
4.45 µl	P72
3.55 µl	OMT+ (PCR 19)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

OMT- (PCR 20) with P72

volume	reagent
3.87 µl	P72
4,13 µl	OMT- (PCR 20)
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

Negative control

volume	reagent
5 µl	P71 or P72
3 µl	ddH2O
1 µl	T4 DNA-ligase
1 µl	T4-ligase buffer (10x)

water bath 16 °C

Transformation of ligationproducts of 4CL, OMT and CHS respectively in pYES in E.coli

Investigator: Mary,Daniela

adding 5µl ligation product in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 45 min, 180 rpm
plate on agar with ampicillin over night

results:

CHS+ in pYES (P72) 100 µl: 7 clones
CHS- in pYES (P72) 100 µl: 9 clones
CHS+ in pYES (P72) Pellet: 111 clones
CHS- in pYES (P72) Pellet: 77 clones

4CL+ in pYES (P71) 100 µl: 9 clones
4CL- in pYES (P71) 100 µl: 3 clones
4CL+ in pYES (P71) Pellet: 47 clones
4CL- in pYES (P71) Pellet: 45 clones

OMT+ in pYES (P72) 100 µl: 7 clones
OMT- in pYES (P72) 100 µl: 5 clones
OMT+ in pYES (P72) Pellet: 30 clones
OMT- in pYES (P72) Pellet: 53 clones

negative control P71 100 µl: 14
negative control P71 Pellet: 71
negative control P72 100 µl: 7
negative control P72 Pellet: 64

Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70 (part 1/3)

investigator: Maddin, Aloisius

Aim of the experiment: Discrimination of growing ability in wort medium

4 different worts:

21%, without hop
21%, without hop, autoclaved
16%, with hop
16%, with hop, autoclaved

were diluted with ELGA to a solution of 12%. These 4 different worts were inoculated with 5ml of preparatory culture of S. cerevisiae and 1ml of brewing yeast strain 34/70 (provided by the Forschungsbrauerei, Weihenstephan). Start OD was measured at 550nm (for table see next experiment). Those 8 flasks were incubated over night at room temperature and 130 rpm.

Wednesday, July 18th

Ligation of plasmid pSB1C3 containing 20aaLinker with LexA and plasmid containing Gal4AD with Pif3 for fusion protein construction

Miniprep of overnight culture from picked transformed E. coli containing biobrick BBa_K165055

Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70 (part 2/3)

Investigators: Martin, Alois

Aim of the experiment: Discrimination of growing ability in wort medium.

After 12h of cultivating the optical density (OD) at 550nm was determined:

Assay	Yeast	Autoclaved	Hop	OD 0h	OD 12h	delta OD
A1	S. cerevisae	No	No	1.2	2.2	+ 1.0
A2	S. cerevisae	Yes	No	2.0	2.6	+ 0.6
A3	S. cerevisae	No	Yes	1.8	2.4	+ 0.6
A4	S. cerevisae	Yes	Yes	2.4	2.7	+ 0.3
B1	34/70	No	No	1.6	2.5	+ 0.9
B2	34/70	Yes	No	3.0	2.9	- 0.1
B3	34/70	No	Yes	2.2	2.5	+ 0.3
B4	34/70	Yes	Yes	2.7	2.8	+ 0.1

After 12h of cultivating the S. cerevisiae is growing quite well. There is a tendency that the brewing yeast strain 34/70 is not capable to grow sufficiently in autoclaved (e.g. proteinfree) medium. Since the 34/70 was stored at 4°C over 5 days, directly used to inoculate the medium (no preparatory culture) and we only used 1ml to inoculate with (compared to 5ml of the preparatory culture of S. cerevisiae - because of obvious differences in opacity of the inoculation media) there is not yet a conclusion to be drawn. We decided to incubate for another 24h at least.

new Miniprep of P50 pYES2

Investigators: Andrea

NanoDrop concentration: 303.6 ng/µl (260/280: 1.89)

Transformation of P50 (pYes) in E.coli

Investigator: Katrin

Aim of the experiment: Get more P50(pYes) for further experiments

adding 2-3 µl of plasmid P50 in 100µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
plate on agar with Ampicillin over night

Ligation of 4CL and PAL in pSB1C3-RFC25 and ligation of PAL in pYES

Investigator: Katrin, Daniela

Concentration (Nano Drop:
4CL+ (PCR 15) = 40.3 ng/µl
4CL- (PCR 16) = 37.3 ng/µl
PAL+ (PCR 32) = 25 ng/µl
PAL- (PCR 33) = 16.8 ng/µl

P132 (pSB1C3-RFC25 digested with Xbal and Pstl) = 33.5 ng/µl
P133 (pSB1C3-RFC25 digested with Xbal and AgeI) = 41.8 ng/µl

required volumes were calculated using Lab Tools

4CL+ (PCR 15) with P132 (pSB1C3-RFC25)

volume	reagent
2.64 µl	P132
5.36 µl	4CL+ (PCR 15)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

4CL- (PCR 16) with P132 (pSB1C3-RFC25)

volume	reagent
2.51 µl	P132
5.49 µl	4CL- (PCR 16)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

PAL+ (PCR 32) with P133 (pSB1C3-RFC25)

volume	reagent
1.29 µl	P133
6.71 µl	PAL+ (PCR 32)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

PAL- (PCR 33) with P133 (pSB1C3-RFC25)

volume	reagent
0.91 µl	P133
7.09 µl	PAL- (PCR 33)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

PAL+ (PCR 32) with P72 (pYES)

volume	reagent
3.55 µl	P72
4.45 µl	PAL+ (PCR 32)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

PAL- (PCR 33) with P72 (pYES)

volume	reagent
2.79 µl	P72
5.21 µl	PAL- (PCR 33)
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)
9 µl	ddH2O

Negative control:

volume	reagent
5 µl	P72, P132 or P133
12 µl	ddH2O
1 µl	T4 DNA Ligase
2 µl	T4-ligase buffer (10x)

water bath 16 °C

Miniprep of CHS and OMT in pSB1C3-RFC25

Investigator: Daniela

Aim of the experiment: Extract plasmids (pSB1C3-RFC25) that contain CHS and OMT

QIAprepS Spin Miniprep Kit

step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)

the Minipreps were named as follows:

P135: CHS+ in pSB1C3-RFC25 (c = 150.9 ng/µl)
P136: CHS- in pSB1C3-RFC25 (c = 204 ng/µl)
P137: OMT+ in pSB1C3-RFC25 (c = 179.3 ng/µl)
P138: OMT- in pSB1C3-RFC25 (c = 146.7 ng/µl)

Control digest of CHS and OMT in pSB1C3-RFC25

Investigator: Katrin, Daniela

Aim of the experiment: Test whether the ligation of CHS and OMT in pSB1C3 was successful

volume	reagent
2.5 µl	P135 / P136 / P137 / P138
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
14.8 µl	ddH2O

pSB1C3: 2070 bp
CHS: 1173 bp
OMT: 1059 bp

It seems as if the ligation was successful. However it is strange that the band of OMT is lower that the one of CHS. we had the wrong information about the length of OMT -> the right length of OMT is 1059 bp, so everything is fine :)

Picking clones of 4CL, CHS and OMT in pYES

investigator: Katrin, Daniela

Aim of the experiment: preculture over night for the miniprep next day

Thursday, July 19th

Transformation of P124+PCR29 and P126+PCR31

Investigator: Daniela

Aim of the experiment: Transformation of the ligation of P124+PCR29 and P126+PCR31 overnight, to see if ligation has been successful.

Operational sequence:

P124+PCR29 ->Chlp
negative control: NK1 ->Chlp
P126+PCR31 ->Amp
negative control: NK2 ->Amp

adding 5µl ligation product in 100 µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min for ampicillin and 45 min for chloramphenicol, 180 rpm
plate on agar with ampicillin or chloramphenicol over night

EDIT from 20.07.2012: Unfortunately, no colonies for P124+PCR29 can be identified but colonies on from the concentrated pellet of P126+PCR31 ligation, BUT more but very small colonies on the control plate from the pellet. For further identification the colonies of the ligation has been picked on 21.07.2012 for miniprep and analytical digestion and gelelectrophoresis.

Restriction digest of P79

Investigator: Roman

Aim of the experiment: Double digest of p79 with Xba1 and Age1 to prepair it for ligation with an N- terminal nuclear localization signal sequence

40 µl composition

18 µl plasmid DNA
1 µl Age1
1 µl Xba1
4 µl NEBuffer 4 (10x)
15,6 µl ddH2O
0,4 µl BSA (100x)

The mixture was incubated over night at 37 °C

Oligohybridization of single-stranded DNA for creating SV40 nuclear localization signal for fusion proteins for translocating them into the nucleus

Investigator: Jeff

Aim of the experiment: Oligohybridization of single-stranded DNA (TUM12-SV40-fw and TUM12-SV40-rv)for creating SV40 nuclear localization signal for fusion proteins for translocating them into the nucleus

Operational sequence:

25 µL of 100 pM of TUM12-SV40-fw and 25 µL of 100 pM TUM12-SV40-rv in one ERG

Heating up to 95 °C for 30 min

Cooling at RT in a styropor box overnight.

Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70 (part 3/3)

Investigators: Andrea, (Martin)

Aim of the experiment: Discrimination of growing ability in wort medium.

After 40h of cultivating the optical density (OD) at 550nm was determined:

Assay	Yeast	Autoclaved	Hop	OD 0h	OD 12h	OD 40h	delta OD
A1	S. cerevisae	No	No	1.2	2.2	2.5	+ 1.3
A2	S. cerevisae	Yes	No	2.0	2.6	2.7	+ 0.7
A3	S. cerevisae	No	Yes	1.8	2.4	2.6	+ 0.8
A4	S. cerevisae	Yes	Yes	2.4	2.7	2.8	+ 0.4
B1	34/70	No	No	1.6	2.5	2.8	+ 1.2
B2	34/70	Yes	No	3.0	2.9	3.0	0
B3	34/70	No	Yes	2.2	2.5	2.8	+ 0.6
B4	34/70	Yes	Yes	2.7	2.8	2.9	+ 0.2

After 40h of cultivating the S. cerevisiae is growing comparably to the brewing yeast strain, though we seem to have entered a phase of substrate limitation. Autoclaved media lack solved protein and is thus for neither of the yeasts a satisfying medium; added hop also represses their growth (S. cerevisiae shows a higher susceptability - which seems logic, since the brewing yeast is selected to survive and prosper in beer brewing environment).

The next step would be to brew with our "laboratory strain" S. cerevisiae.

Preparative digest of PCR1 and PCR2 and P50 and P123

Investigator: Andrea

Digestion of PCR1 and PCR2 with XbaI and AgeI

volume	reagent
10 µl	PCR-Product
2 µl	NEB Buffer
0.2 µl	BSA
1 µl	XbaI (10 U/µl)
1 µl	AgeI (10 U/µl)
7 µl	ddH2O

Digestion of P50 with XbaI and NgoMIV

volume	reagent
10 µl	P50
4 µl	NEB Buffer
0.4 µl	BSA
1 µl	XbaI (10 U/µl)
2 µl	NgoMIV (10 U/µl)
22.6 µl	ddH2O

Digestion of P123 with XbaI and AgeI

volume	reagent
20 µl	P123
4 µl	NEB Buffer
0.4 µl	BSA
1 µl	XbaI (10 U/µl)
1 µl	AgeI (10 U/µl)
14 µl	ddH2O

Incubation: 37 °C, 3 h

Preparative gel

Investigator: Andrea

40 µl pSB1C3 + 4 µl loading dye
40 µl pYES2 + 4 µl loading dye
20 µl PCR1 + 2 µl loading dye

Ligation of digested PCR1 (digested with XbaI and AgeI) with pSB1C3 (digested with XbaI and AgeI)

Investigator: Andrea

Concentration (Nano Drop:
LIMS Citrus (PCR 1) = 18.6 ng/µl

P50 (digested with XbaI and NgoMIV) = 24.9 ng/µl

P123 (digested with XbaI and AgeI) = 44.5 ng/µl

PCR1 with P123 digested with XbaI and AgeI/NgoMIV

volume	reagent
1.19µl	P123
6.81 µl	PCR 1
1 µl	T4 DNA Ligase
1 µl	T4-ligase buffer (10x)

water bath 16 °C over night

Miniprep of 4CL, CHS and OMT in pYES (P71 and P72)

Investigator: Daniela

Aim of the experiment: Extract plasmids (pYES) that contain 4CL, CHS and OMT

The day before 2 clones were picked for each enzyme.

QIAprepS Spin Miniprep Kit

step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)

Concentration:

4CL+ in pYES clone 1: c = 172.5 ng/µl
4CL- in pYES clone 1: c = 192.1 ng/µl
CHS+ in pYES clone 1: c = 136.6 ng/µl
CHS- in pYES clone 1: c = 85.7 ng/µl
OMT+ in pYES clone 1: c = 116.0 ng/µl
OMT- in pYES clone 1: c = 129.7 ng/µl

4CL+ in pYES clone 2: c = 160.0 ng/µl
4CL- in pYES clone 2: c = 144.5 ng/µl
CHS+ in pYES clone 2: c = 128.5 ng/µl
CHS- in pYES clone 2: c = 116.2 ng/µl
OMT+ in pYES clone 2: c = 142.8 ng/µl
OMT- in pYES clone 2: c = 134.5 ng/µl

Control digest of 4CL, CHS and OMT in pYES

Investigator: Daniela

Aim of the experiment: Test whether the ligation of 4CL, CHS and OMT in pYES was successful

volume	reagent
2.5 µl	4CL+ in pYES clone 1 / 4CL- in pYES clone 1
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
14.8 µl	ddH2O

volume	reagent
3 µl	4CL+ in pYES clone 2
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
14.3 µl	ddH2O

volume	reagent
3.5 µl	4CL- in pYES clone 2
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
13.8 µl	ddH2O

volume	reagent
3.5 µl	CHS+ in pYES clone 1 / OMT+ in pYES clone 1 / OMT- in pYES clone 1 / CHS+ in pYES clone 2 / OMT+ in pYES clone 2 / OMT- in pYES clone 2
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
13.8 µl	ddH2O

volume	reagent
4 µl	CHS- in pYES clone 2
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI(20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
13.3 µl	ddH2O

volume	reagent
5 µl	CHS- in pYES clone 1
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI(20U/µl)
2 µl	NEB4 (10x)
0.2 µl	BSA (100x)
12.3 µl	ddH2O

expected bands:

pYES: about 5800bp
4CL: 1685bp
CHS: 1173bp
OMT: 1222bp

Transformation of ligationsproducts of 4CL and PAL in pSB1C3-RFC25 and of PAL in pYES in E.coli (see July, 18th)

Investigator: Daniela

4CL+ (PCR15) in pSB1C3-RFC25 (P132) ->Chlp
4CL- (PCR16) in pSB1C3-RFC25 (P132) ->Chlp
PAL+ (PCR32) in pSB1C3-RFC25 (P133) ->Chlp
PAL- (PCR33) in pSB1C3-RFC25 (P133) ->Chlp
PAL+ (PCR32) in pYES(P72) ->Amp
PAL- (PCR33) in pYES(P72) ->Amp
negative control: P72 ->Amp
negative control: P132 ->Chlp
negative control: P133 ->Chlp

adding 5µl ligation product in 100 µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min for ampicillin and 45 min for chloramphenicol, 180 rpm
plate on agar with ampicillin or chloramphenicol over night

Friday, July 20th

Gel purification of Xba1/ Age1 digested P79

Investigator: Roman

Aim of the Experiment: Aim of the experiment is the purification of the double digested vector p79 (pSB1C3_RFC25_Linker), in which the N- terminal SS is to be cloned.

Operational sequence:

The DNA was cut out of the gel (2 pieces) and weight ==> 200 mg each piece. Afterwards the plasmid DNA was extracted as described in the Quiagen Gel Purification protocol. Elution was performed in two steps (à 25 µl) with elution buffer (heated up to 40°). To improve the yeald of plasmid DNA during the elution, the column was incubated at RT for about 4 minutes before centrifugation

Miniprep of transformed E. coli XL1-Blue with pYES2 new from P50 tube (4x), BBa_K365005 (4x), BBa_K207000 (1x)and BBa_K207001 (1x)

Investigator: Jeff

Aim of the study:: Miniprep

Operational sequence:

Miniprep has been performed after manufacturer's protocol. QIAGEN - QIAprep Spin Miniprep Kit.

Analytical digestion and gelelectrophoresis of P152, P155, P156, P157, P158, P159, P160, P161

Investigator: Jeff

Aim of the study: Analytical digestion and gelelectrophoresis of P152, P155, P156, P157, P158, P159, P160, P161, to prove the insert and backbone size.

Operational sequence:

Analytical digestion and gelelectrophoresis were performed after lab's standard protocol.

Digestion with XbaI and PstI-HF in Buffer 4.

Picking from the overnight transformation of the ligated P126+PCR31

Investigator: Jeff

Aim of the study: Picking from the overnight transformation of the ligated P126+PCR31, to see whether ligation is successful or not.

Operational sequence:

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

PCR of P23 (LexA, BBa_K105005) to indroduce RFC25 pre- and suffix because last ligation was not successful

Investigator: Jeff

Aim of the experiment: Last ligation with LexA was not successful and the tube with the PCR product was empty from the ligation, so one has to do another PCR for another ligation.

Operational sequence:

Clone 3 (tube P23) of BBa_K105005 (LexA) has beed choosen for the PCR.

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

Freezed at -20 °C. Stil has to be purified with PCR purification kit on the next day

Control digest of pYES (P153 and P154)

Investigator: Mary, Ingmar, Daniela

Aim of the experiment: Test whether the vector pYES is right.

volume	reagent
1.5 µl	P153
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
16.25 µl	ddH2O

volume	reagent
1.5 µl	P153
0.25 µl	Xbal (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
14.25 µl	ddH2O

volume	reagent
1.5 µl	P153
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
14 µl	ddH2O

volume	reagent
1.5 µl	P153
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
14 µl	ddH2O

volume	reagent
1.5 µl	P153
0.25 µl	NgoMIV (10U/µl)
2 µl	NEB4 (10x)
16.25 µl	ddH2O

volume	reagent
1.5 µl	P153
0.25 µl	NgoMIV (10U/µl)
0.25 µl	PstI (20U/µl)
0.25 µl	SpeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
13.75 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
14.95 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	Xbal (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
12.95 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
12.7 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
12.7 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	NgoMIV (10U/µl)
2 µl	NEB4 (10x)
14.95 µl	ddH2O

volume	reagent
2.8 µl	P154
0.25 µl	NgoMIV (10U/µl)
0.25 µl	PstI (20U/µl)
0.25 µl	SpeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
12.45 µl	ddH2O

P153

P154

The vector seems to be correct. However NgoMIV did not digest the plasmid properly.

Saturday, July 21st

Miniprep of transformed E. coli XL1-Blue with ligation product of P126+PCR31

Investigator: Jeff

Aim of the study: Miniprep

Operational sequence:

Miniprep has been performed after manufacturer's protocol. QIAGEN - QIAprep Spin Miniprep Kit.

Analytical digestion and gelelectrophoresis of the minipreps of transformed E. coli XL1-Blue with ligation product of P126+PCR31

Investigator: Jeff

Aim of the experiment: To see whether ligation ist successful.

Reaction batch for each plasmid:

Reagent	Volume in µl
Tango Buffer 10x	4 µl
NdeI (Fermentas)	0.5 µl
BamHI (Fermentas)	0.5 µl
Plasmid DNA	2.5 µl
ddH2O	12.5 µl
TOTAL	20 µl

Incubation at 37 °C for 1 h 30 min.

Analytical gelelectrophoresis at 90 V for 1 h.

Order of gel-pockets:

100 bp ladder	P172	P173	P174	1000 bp ladder
	corrupt	corrupt	corrupt

Ligation was not successful!

PCR purification of PCR products from P23

Investigator: Jeff

Aim of the experiment Purification of PCR products from P23.

Operational sequence:

The 4 tubes with PCR products of P23 were purificated with the PCR purification kit from Qiagen after manufacturer'S protocol.

Sunday, July 22nd

Preparative digest of pYes2 RFC 25 and ligation with PCR products PCR 15 - 20 and 32+33

Investigator: Ingmar

Aim of the experiment: Intsert the genes of PAL, 4CL, CHS and OMT in pYEs2 RFC 25 in order to test their expression in yeast.

Preparative digest of P 154 with XbaI and NgoMIV and of P 153 with XbaI and PstI

volume	reagent
7 µl	ddH2O
2.5 µl	NEB 4 buffer
2.5 µl	Tango buffer
4 µl	XbaI (10 U/µl)
4 µl	NgoMIV (10 U/µl)
30 µl	P 153

volume	reagent
10 µl	ddH2O
4 µl	Tango buffer
2 µl	XbaI (10 U/µl)
4 µl	PstI (10 U/µl)
20 µl	P 154

Incubation: 37 °C, 3 h

DNA preparative gel electrophoresis

gel: 1% with LMP-agarose
Each digest product was mixed with an adequate volume of DNA loading buffer and loaded into the gel
The separation process lasted 90 min at 70 V.

Gel picture of control digest with PstI

All digest products show the expected bonds at 5849 bp (digest with XbaI and NgoMIV) and 5785 bp (digest with XbaI and PstI) respectively.

Gelextration

cut the bands
QIAquick Gel Extractrion Kit was used
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation

Products were named as follows:

P154 digested with XbaI and NgoMIV: P175
P153 digested with XbaI and PstI: P176

Ligation

PCR product	Volume	Vector	Volume	Volume T4 DNA Ligase	Volume T4 DNA Ligase buffer	Volume ddH20	New Plasimd Number
PCR15	5.25	P 176	2.75	1 µl	2 µl	9 µl	P 177
PCR16	5.25	P 176	2.75	1 µl	2 µl	9 µl	P 178
PCR 17	7.2	P 175	0.8	1 µl	2 µl	9 µl	P 179
PCR 18	7.2	P 175	0.8	1 µl	2 µl	9 µl	P 180
PCR 19	7.1	P 175	0.9	1 µl	2 µl	9 µl	P 181
PCR 20	7.1	P 175	0.9	1 µl	2 µl	9 µl	P 182
PCR 32	7.25	P 175	0.75	1 µl	2 µl	9 µl	P 183
PCR 33	7.5	P 175	0.5	1 µl	2 µl	9 µl	P 184

Monday, July 23nd

Miniprep of PAL, 4CL in pSB1C3 and PAL in pYES

Investigator: Mary

Aim of the experiment: Extract plasmids (pYES and pSB1C3-RFC25) that contain 4CL and PAL´

The day before one clone were picked for each enzyme from plates from 19th July 2012.

QIAprepS Spin Miniprep Kit

step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)

Concentration:

4CL+ (PCR15) in pSB1C3-RF25 (P132): c = 340 ng/µl
4CL- (PCR16) in pSB1C3-RF25 (P132): c = 385 ng/µl
PAL+ (PCR32) in pSB1C3-RF25 (P133): c = 395/µl
PAL- (PCR33) in pSB1C3-RF25 (P133): c = 255 ng/µl

PAL+ (PCR32) in pYES (P72): c = 190 ng/µl
PAL- (PCR33) in pYES (P72): c = 238 ng/µl

Control digest of 4CL, PAL in pSB1C3-RFC25 and PAL in pYES

Investigator: Mar

Aim of the experiment: Test whether the ligation of 4CL, PAL in pSB1C3-RFC25 and PAL in pYES was successful

Plasmids are taken from miniprep from 23.07.2012

volume	reagent
2.5 µl	4CL+ in pSB1C3-RFC25 / 4CL- in pSB1C3-RFC25
0.25 µl	Xbal (20U/µl)
0.25 µl	PstI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
13 µl	ddH2O

volume	reagent
2.5 µl	PAL+ in pSB1C3-RFC25 / PAL- in pSB1C3-RFC25
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
13 µl	ddH2O

volume	reagent
2.5 µl	PAL+ in pSB1C3-RFC25 / PAL- in pSB1C3-RFC25
0.25 µl	Xbal (20U/µl)
0.25 µl	AgeI (20U/µl)
2 µl	NEB4 (10x)
2 µl	BSA (10x)
13 µl	ddH2O

expected bands:

pYES: about 5800bp
pSB1C3-RFC25: ?
4CL: 1685bp
PAL: 2151bp

Ligation of PAL+/- in pYES was not succesful Ligation of PAL+/- in pSB1C3-RFC25 was succesful - but what is the second bond at 4CL?

APT Solubilisation and Transformation

Investigator: Mary

Aim of the experiment: solubilisation of the synthetic gene of APT and transformation of sent plasmid (including APT) in E.coli to copy it if necessary

short zentrifugation of sent product and adding 50µl bidest. H2O to it (5µg in 50µl = concentration of 0.1 µg/µl) the dissolved product was named as G1 (as Geneproduct number 1) and stored at -20°C

2µl of solved product used for transformation in Ecoli (Kit of Quiagen) and Amp-resistance (said GeneArt) Plating cells on Agar with Amp, 37°C over night

Preparative digest of APT

Investigator: Mary

Aim of the experiment: Extraction of the sequence of APT out of the sent plasmid.

volume	reagent
10µl	ddH2O
4 µl	NEB 4 buffer
4 µl	BSA (10x)
1 µl	XbaI (20 U/µl)
1 µl	AgeI (20 U/µl)
20 µl	solubilised APT-product (see Solubilisation APT)

Incubation: 37°C, 2.5h

Bond at 1244bp as expected:

The bond was cutted out of the gel and stored at -20°C (P189)

Transformation of ligationsproducts of 4CL, CHS, OMT and PAL in pYES in E.Coli (Ligation see 22th of July)

Investigator: Mary

Aim of the experiment: ligation of enzymes in pYES to transform it into yeast if this was successful.

4CL+ (PCR15) in pYES (P176) -> new name: P177
4CL- (PCR16) in pYES (P176) -> new name: P178
CHS+ (PCR17) in pYES (P175) -> new name: P179
CHS- (PCR18) in pYES (P175) -> new name: P180
OMT+ (PCR19) in pYES (P175) -> new name: P181
OMT- (PCR20) in pYES (P175) -> new name: P182
PAL+ (PCR32) in pYES (P175) -> new name: P183
PAL- (PCR33) in pYES (P175) -> new name: P184

adding 5µl ligation product in 100 µl competent XL blue E.coli cells
incubation 30 min on ice
5 min at 37°C
adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min 180 rpm
plate on agar with ampicillin and incubate over night at 37°C

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)

Wednesday, June 27th

Repetition of analytic gel of 21st June

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

Picking of clones of Schwab expression stains #106 & #108

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

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

Friday, July 6th

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

PCR of Schwab plasmid DNA to amplify gene for lavendula LS

Analytic Gel of PCR of Schwab plasmid DNA

Saturday, July 7th

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

Transformation of E.coli XL1 blue with ADH1 promoter, ADH1 terminator and TEF2 promoter