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Team:Frankfurt/Notebook
From 2012.igem.org
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* Trials to get the genes, promoters and terminators via PCR | * Trials to get the genes, promoters and terminators via PCR | ||
==August 2012== | ==August 2012== | ||
| - | * PCR of the genes, promoters and terminators<br>all genes (without KO and KAH), promoters and terminators could be amplified<br> | + | * PCR of the genes, promoters and terminators<br>all genes (without ''KO'' and ''KAH''), promoters and terminators could be amplified<br> |
{| class="wikitable zebra" | {| class="wikitable zebra" | ||
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| Line 50: | Line 50: | ||
** plasmid isolation of ''E.coli'' clones<br> | ** plasmid isolation of ''E.coli'' clones<br> | ||
** control restriction of biobrick plasmids with ''EcoRI'' and ''SpeI''<br> | ** control restriction of biobrick plasmids with ''EcoRI'' and ''SpeI''<br> | ||
| + | * 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''<br> | ||
| + | * 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)<br> | ||
| + | ** preparative gel of the linear fragment: very low concentration of linear pSB1C3 (was not sufficient for ligation) | ||
| + | ** | ||
| + | |||
==September 2012== | ==September 2012== | ||
| + | |||
==October 2012== | ==October 2012== | ||
Revision as of 11:49, 20 September 2012
 | Home | Team | Project | Organisms | New Yeast RFC | Notebook | Registered Parts | Modeling | Safety | Attributions | Official Team Profile |
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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)
September 2012
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
