Team:TU Munich/Notebook/Labjournal

From 2012.igem.org

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'''Aim of the experiment:''' Analytical gel electrophoresis of products from restriction digest of plasmids PCR1 - PCR7 and PCR9 and P77, P78
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Revision as of 16:27, 18 July 2012

Display: Vector Design Limonene Coumaryl Thaumatin Caffeine Constituve promoter Light-switchable promoter
You can also click on individual experiments to show/hide them


Contents

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

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.

Gel picture of control digest with NgoMIV and SpeI

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: TUM12 20120629 PCR von p2µHyp-PAL-CHS-4CL und pOMT.jpg

-> 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

Analytical gel after digestion with XbaI and PstI

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)

preparative gel of 4CL

preparative gel of CHS


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.

Gel picture of control digest with PstI

  • 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
  • Analytical gelelectrophoresis of PCR product of BBa_K105005 with primer TUM12-LexA-fw and TUM12-LexA-rv

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.

Gel picture of control digest with PstI

  • 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)

TUM12 VerdauSchwabPlasmide0507.png

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)

Analytical gelelectrophoresis of PCR product of PAL

-> 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.

Gel picture of control digest with PstI

  • 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:

Sequencing results of P45 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

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.

Green dried Spirulina platensis powder Cyanblue Pellet after 4x of MeOH washing step Cyanblue supernatant of the last washing step with MeOH. It indicates that all the green chromophores are already washed out, which means that only the protein-bound phycocyanobilin is still arrested in he pellet Resuspended Pellet in MeOH to pool all the pellet Analytical sample of the dried pellet before the methanolysis for later analysis

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.

Analytical gelelectrophoresis with XbaI and PstI

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

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
33.27 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

Analytical gel electrophoresis

Investigator: Andrea

Aim of the experiment: Analytical gel electrophoresis of products from restriction digest of plasmids PCR1 - PCR7 and PCR9 and P77, P78

File:C:\Users\Andrea\Dropbox\iGEM Teamordner\Gelfotos\Limonen\Bild1.png

Friday, July 13th

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

Investigator: Jeff, Saskia

Miniprep, analcytical digestion and gelelectrophoresis of pGADT7 and pGBKT7

Investigator: Jeff, Saskia

Aim of the experiment:

Operational sequence:

Analytical digestion and gelelectrophoresis of pGADT7 (EcoRI and BamHI)and pGBKT7 (??? and ???).

Preperative digestion and gelelectrophoresis of P80 and O56

Investigator: Jeff, Saskia, Georg

Preperativ gelelectrophoresis and gel of digested PCR product of LexA and pSB1C3 containing the RFC25 compatible 20aa linker with RFC25 pre- and suffix. Digestion with AgeI and PstI.

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.

Gel picture of gradient PCR of P19

  • 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 4CL and CHS 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!!!

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

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 of P73, P80 and P81. As expected the only working PCR is P80 because the miniprep of P73 and P81 from Havard university are already corrupt!

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 :

Analytical gelelectrophoresis of PCR product of PAL

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:

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.

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.

TUM12 LS gelelectrophoresis1607.png


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)

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:

TUM 12 PAL consless (digested with Xba Age).png

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:

TUM12 120717 PCR PALconsens.jpg

Transformation of P94 - P97 (ligationproducts of pSB1C3 with OMT and CHS 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

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:

TUM12 120717 PAL+ prepGel.jpg

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 P??- P??(ligationproducts of pYES with OMT and CHS respectively) in E.coli

  • adding 5µl ligation product (P??- P??) 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

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

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
Retrieved from "http://2012.igem.org/Team:TU_Munich/Notebook/Labjournal"