Team:Frankfurt/Notebook
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Contents |
Labwork
May and June 2012
- Arrangements for labwork
preparation of competent cells (E.coli, S.cerevisiae), agarose plates (LB, YEPD, SCD-ura,…), medium for E.coli and S.cerevisiae
- Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)
- Primer design
July 2012
- Plasmid isolation of p426, p423, pUD8e, pUD22e from E.coli
- Isolation of chromosomal DNA of CEN.PK2-1C
- Trials to get the genes, promoters and terminators via PCR
August 2012
- PCR of the genes, promoters and terminators
all genes (without KO and KAH), promoters and terminators could be amplified
Templates | Amplified DNA Fragments |
---|---|
synthesized sequence of HMG-CoA | HMG-CoA |
synthesized sequence of GGPPS | GGPPS |
synthesized sequence of Cps/Ks | CPS/KS |
chromosomal DNA of CEN.PK2-1C | ERG20 |
![](/wiki/images/3/39/Gel2_1.png)
As a control it is also the circular p426 and p423 shown. The linear p426 and p423 are about 6300 bp long. pSB1C3-linear means the restriction of the plasmid with EcoRI and PstI in this case (it is 2050 bp long). As PCR templates for HMG-CoA, GGPPS and Cps/Ks were used the synthesized DNA fragments and for ERG20 chromosomal DNA of CEN.PK2-1C.
- Linearization of p426 and p423 with SpeI and XhoI
- Biobrick production of the genes HMG-CoA, ERG20, CPS/KS
- restriction of 3 µg of the genes with EcoRI and PstI
- ligation of biobrick genes with linear pSB1C3
- transformation of the ligation in E.coli
- plasmid isolation of E.coli clones
- control restriction of biobrick plasmids with EcoRI and SpeI
- restriction of 3 µg of the genes with EcoRI and PstI
- Formation of the mevalonate overexpression plasmid via gap repair
- first and second yeast transformation with equimolar quantities of DNA fragments for mevalonate overexpression (p426 with 7 inserts): only very small colonies could grow after the first and second transformation
- using pure GGPPS (purification of a preparative gel) for the third yeast transformation: normal size of the colonies
- inoculation of several clones of the third yeast transformation
- plasmid preparation of the clones
- transformation of the plasmids in E.coli
- Amplifying pSB1C3 for biobrick production
- trials to amplify pSB1C3, whose blunt ends were ligated and transformed in E.coli
- pSB1C3 should be linearized by EcoRI and PstI : did not work (two fragments instead of one)
- preparative gel of the linear fragment: very low concentration of linear pSB1C3 (was not sufficient for ligation)
- GC analysis
- GC analysis of the wild type CEN.PK2-1C (standard GGOH): as expected no GGOH could be observed
- Assembly of the KO and the KAH fragments
- amplification of the fragments via PCR (there are four fragments of each gene with an overhang to the fragment beside of 30 bp)
- Gibson assembly of the fragments of KO and KAH: did not work
September 2012
- Formation of the mevalonate overexpression plasmid via gap repair
- plasmid isolation (p426 with 7 inserts) from E.coli
- control restriction of the plasmids with EcoRI and SpeI: one clone out of 10 got the right sizes
- Ergosterol experiment
- idea: maybe the clones of the first and the second yeast transformation grow better after ergosterol addition (0,02 g/l)): wildtype with and without ergosterol and one of the clones with and without ergosterol (no significant difference in growth could be observed)
- GGPPS-PCR
- PCR of GGPPS with shorter synthesis time in order to get only the correct fragment
- Amplification of pSB1C3 for biobrick production
- transformation of pSB1C3-RFP in E.coli
- isolation of the plasmid from E.coli
- linearization with EcoRI and PstI: it worked (only the correct fragment was observed)
- PCR of biobrick-promoters and -terminators
- PCR of biobrick promoters and terminators that were used to build the mevalonate overexpression plasmid
- Assembly of the KO and the KAH fragments
- trials to assembly the KO and the KAH fragments via PCR (the overhangs were used as primer): KO worked, KAH not
- amplification of KO
- Biobrick production of the genes GGPPS, KO and the promoters and terminators
- restriction of 3 µg of the DNA fragments with EcoRI and PstI
- ligation of the promoters, terminators, KO and the GGPPS with pSB1C3
- transformation of the ligation in E.coli
- plasmid isolation of E.coli clones
- control restriction of biobrick plasmids with EcoRI and SpeI
- Midi-preparation of plasmids for sequencing
- biobrick plasmids
- mevalonate overexpression plasmid
- GC
- GC analysis of the wild type CEN.PK2-1C with p426 and the wild type with the mevalonate overexpression plasmid in SCD-ura
- standard GGOH
- Formation of the plasmid for steviol synthesis
- yeast transformation with equimolar quantities of DNA fragments for steviol synthesis (p423 with 7 inserts)
- problem: could not assemble KAH, assembled KAH1/2 and KAH3/4 were used (KAH1/2 and KAH3/4 only got a 30 bp overhang instead of a 45 bp overhang): gap repair did not work
Methods and Protocols
Plasmid Preparation
Plasmid Preparation of E.coli (Mini Preparation)
1.transfer 1,5 ml of an overnight culture in a reaction tube
2.centrifuge at 8000 rpm at RT for 5 min
3.resuspend the pellet in 100 µl solution 1
4.addition of 200 µl solution 2
5.mix till the solution is clear
6.incubate at RT for 5 min
7.addition of 150 µl cold solution 3, mix till protein clumps are build
8.incubate 10 min on ice, centrifuge 15 min with 10000 rpm
9.transfer the supernatant in a clean reaction tube
10.fill the reaction tube with 96 % EtOH, mix and let it precipitate at -20 °C for 10 min
11.centrifuge 10 min at 10000 rpm
12.wash the pellet with 70 % EtOH
13.dry the pellet and resuspend it with 30 µl water or TE buffer
Solution 1
- 50 mM glucose
- 10 mM EDTA
- 25 mM Tris-HCl pH8
Solution 2
- 0,2 M NaOH
- 1 % SDS
Solution 3
- 3 M KaAc pH 5,5
Plasmid Preparation of Saccharomyces cerevisia
1. overnight culture in 5 ml
2. centrifuge 2 ml of the cells for 1-2 min
3. wash the cells with water
4. resuspend the pellet in 400 µl buffer 1 with RNase
5. addition of 400 µl buffer 2 and mix carefully
6. addition of 2/3 volume of glass beads
7. cell destruction: vibrax the cellsin a 2 ml reaction tube for 5 min at 4°C
8. transfer 500 µl of the supernatant in a clean reaction tube
9. addition of 250 µl buffer 3, mix, incubate it for 10 min on ice
10. centrifuge at 10000 rpm, 15 min
11. transfer the supernatant in a clean reaction tube, fill with isopropanol and mix
12. centrifuge at 13000 rpm, 30 min
13. wash the pellet with cold 70 % EtOH and let it dry
14. resuspend the DNA in 30 µl water or TE buffer
P1
- 50 mM Tris/HCl pH 8
- 10 mM EDTA
- 100 µg/ml RNase A
P2
- 0,2 M NaOH
- 1 % SDS
P3
- 3 M KAc
Transformation
Yeast Transformation
1. Inoculate the synthetic complete medium (SC) with the strain
2. Incubate with shaking overnight at 30°C
3. Harvest the cells at a OD600 0,5-0,6 by centrifugation (3000x g, 5 min, RT) 4. Wash the cells with 0,5 vol of sterile water (resuspend by shaking, centrifugate with 3000x g, 5 min, RT) 5. Resuspend the cells in 0,01 vol of sterile water, transfer the suspension to a reaction tube and pellet the cells (3000x g, 5 min, RT) 6. Resuspend the pellet in 0,01 vol of sterile filtrated FCC (frozen competent cell) solution 7. Aliquot 50 µl of the solution into the reaction tubes 8. Store the cells at -80°C for at least one night (up to one year)
9. Mastermix for the FCC transformation mixture:
Substance volume [µl] PEG 3350 (50% (w/v)) 260 LiAcetat 1.0 M 36 Single-stranded carrier DNA (10 mg/ml) 10 Total volume 306
10. Prepare DNA aliquots: Solute enough DNA (e.g. 100 ng plasmid) in 54 µl of water 11. Unfreeze the cells in a 37°C block for 15-30 sec 12. Centrifuge the solution at 13000x g for 2 minutes 13. Remove the supernatant 14. Add 306 µl of FCC transformation mixture to the cells 15. Add 54 µl of the DNA to the solution and vortex shortly 16. When all the reaction tubes are prepared, vortex the samples well until all pellets are completely resoluted 17. Incubate the samples for 40 minutes at 42°C in a heating block 18. Centrifuge the cells at 13000x g for 30 sec and pour off the supernatant 19. Resuspend the cells in sterile water by vortexing 20. Spread onto the appropriate medium 21. Let the cells grow at 30°C
E.coli Transformation
PCR
Culture Medium
Full Medium (YEPD) for Yeast | |||
---|---|---|---|
Yeast Extract | 1 % (weight/volume) | ||
Pepton | 2 % (w/v) | ||
Glucose | 2 % (w/v) |
Synthetic Complete Medium (SC) for Yeast | |||
---|---|---|---|
Yeast Nitrogen Base | 0.17 % (w/v) | ||
Ammoniumsulfate | 0.5 % (w/v) | ||
Glucose | 2 % (w/v) | ||
Amino Acid Mix footnoteaödlfkj/footnote | 50 ml/l | ||
Histidin** | 0.25 mM | ||
Tryptophan** | 0.19 mM | ||
Leucin** | 0.35 mM | ||
Uracil** | 0.44 mM |
pH has to be regulated with KOH to pH=6.3
- contains no His, Leu, Trp and Uracil
** addition of this components depents on the respective selection medium
SOC-Medium for Regeneration of transformed Escherichia coli`s after Electroporation | |||
---|---|---|---|
Trypton | 2 % (w/v) | ||
Yeast Extract | 0.5 % (w/v) | ||
NaCl | 10 mM | ||
KCl | 2,5 mM | ||
MgCl2 | 10 mM | ||
MgSO4 | 10 mM | ||
Glucose | 20 mM |
pH has to be regulated to pH=6.8-7.0
Full Medium (LB) for E.coli | |||
---|---|---|---|
Yeast Extract | 0.5 % (w/v) | ||
Trypton | 1 % (w/v) | ||
NaCl | 0.5 % (w/v) |
pH has to be regulated with NaOH to pH=7.5
Every cluture medium has to be autoclaved to be sterile.
Agar Plate
LBampicillin-Agar
Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C steril ampicillin is added. Plates were poured.
SCD-Agar
Add 2 % agar to SCD-medium. After autoclaving and cooling-down steril amino acid solution is added. Dependent on the respective selective medium Histidin (0.25 mM), Trypthophan (0.19 mM), Uracil (0.44 mM) or Leucin (0.35 mM) are added. Plates were poured.
YEPDG418-Agar
Add 2 % agar to YEPD-medium. After autoclaving and cooling-down sterile G418 (final concentration 2g/l) is added. Plates were poured.
Gel Electrophoresis
Agarose Gel (1x) | |||
---|---|---|---|
TAE puffer | 1x | ||
Agarose | 1 % (w/v) |
Solve agarose in TAE by boiling it. After cooling-down to 55-60 °C gel is poured.
TAE Puffer (50x) for Gel Electrophoresis | |||
---|---|---|---|
EDTA | 18,6 g | ||
Tris | 242g | ||
Glacial Acetic Acid | 57,2 ml | ||
Purified Water | 1000ml |
pH has to be regulated with glacial acetic acid to pH=8.
Kit
PCR Purification Kit from Qiagen
Gel Extraction Kit from Qiagen
Midi Plasmid Preparation Kit from Qiagen