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Team:Frankfurt/Notebook
From 2012.igem.org
(Difference between revisions)
| Line 54: | Line 54: | ||
* Formation of the mevalonate overexpression plasmid via gap repair | * 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 | ** 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 | + | ** 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 | ** inoculation of several clones of the third yeast transformation | ||
** plasmid preparation of the clones | ** plasmid preparation of the clones | ||
| Line 61: | Line 61: | ||
* Amplifying pSB1C3 for biobrick production | * Amplifying pSB1C3 for biobrick production | ||
** trials to amplify pSB1C3, whose blunt ends were ligated and transformed in ''E.coli'' | ** 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)<br> | + | ** pSB1C3 should be linearized by ''EcoRI'' and ''PstI'' : did not work (two fragments instead of one)<br> |
** preparative gel of the linear fragment: very low concentration of linear pSB1C3 (was not sufficient for ligation) | ** preparative gel of the linear fragment: very low concentration of linear pSB1C3 (was not sufficient for ligation) | ||
| Line 68: | Line 68: | ||
** GC analysis of the wild type CEN.PK2-1C (standard GGOH): as expected no GGOH could be observed | ** 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 | + | * 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) | ** 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<br> | + | ** Gibson assembly of the fragments of ''KO'' and ''KAH'': did not work<br> |
==September 2012== | ==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 | ||
==October 2012== | ==October 2012== | ||
Revision as of 12:11, 20 September 2012
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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
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 |
- 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
October 2012
Methods and Protocols
Plasmid Preparation
Plasmid Preparation of E.coli (Mini Preparation)
Plasmid Preparation of Saccharomyces cerevisia
Transformation
Yeast Transformation
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* | 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
Gel Extraction Kit
Midi Plasmid Preparation Kit
