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Team:Groningen/Notebook/2 September 2009
From 2009.igem.org
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| + | '''New o.n. precultures''' | ||
| + | The colonies on the [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] plate were used to inocculate 5mL LB-kan<sub>50</sub>-IPTG medium to see if growth takes place. | ||
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| + | :→ The stock solution of IPTG was 0.5M (mol/L), and the concentration needed for maximum plasmid copy (~150) of [http://partsregistry.org/Part:pSB2K3 pSB2K3] is 100 μmol/L. This is 0.1 μmol/mL, which accounts for 0.2 μL/mL of medium used. | ||
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| + | '''New o.n. plates''' | ||
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| + | The plates of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3 were used to grow single colonies on new LB-amp<sub>100</sub> plates, to test this method of getting pure colonies. If it is not succesfull the old approach of first growing precultures, plating, growing single colonies, and growing new precultures has to be repeated. | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 07:56, 2 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → TODO Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3
- → TODO Choose colonies from plates for growth of E.coli TOP10 with [http://partsregistry.org/Part:pSB2K3 pSB2K3] plasmid from the registry (plate 1, 7C with RFP Coding Device [http://partsregistry.org/Part:BBa_J04450 BBa_J04450]
- → TODO Make glycerol stocks of pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid)
- → TODO Test control of bouyancy in Saline solution (grow plates with GVP constructs)
- → TODO Order synthetic DNA for GVP
- → TODO Order primer for PstI site removal
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Colonies on Plates
| Name | Plasmid Used | Antibiotics on Plasmid | No. of Colonies | Date |
| [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_J04450 BBa_J04450] (1,7C) | pSB2K3 | Kanamycin | 0 | 2/9 |
| [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_J04450 BBa_J04450] (1,7C)(conc.) | pSB2K3 | Kanamycin | 4 | 2/9 |
| [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] (1,7K) | pSB2K3 | Kanamycin | 0 | 2/9 |
| [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] (1,7K)(conc.) | pSB2K3 | Kanamycin | 0 | 2/9 |
- → The plates showed very low colony growth, and can be caused by the fact it is a low copy plasmid which can be induced by iptg to increase the copy number. Additional growth with IPTG should increase the amount of colonies and available plasmid.
- → From the plate with four colonies 5mL LB-amp100 medium was inoculated, and additional plates were swiped with the used colonies to see if single colony growth on plate can occur.
New o.n. precultures
The colonies on the [http://partsregistry.org/Part:pSB2K3 pSB2K3]-[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] plate were used to inocculate 5mL LB-kan50-IPTG medium to see if growth takes place.
- → The stock solution of IPTG was 0.5M (mol/L), and the concentration needed for maximum plasmid copy (~150) of [http://partsregistry.org/Part:pSB2K3 pSB2K3] is 100 μmol/L. This is 0.1 μmol/mL, which accounts for 0.2 μL/mL of medium used.
New o.n. plates
The plates of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3 were used to grow single colonies on new LB-amp100 plates, to test this method of getting pure colonies. If it is not succesfull the old approach of first growing precultures, plating, growing single colonies, and growing new precultures has to be repeated.
Transporters
Metal Accumulation
MBP-ArsR
- Check sequence
- Put promotors in front
- Put transporters in back
fMT
- Plasmid isolation & Restriction analysis
- Put promotors in front
SmtA
- Plasmid isolation & Restriction analysis
- Put promotors in front
Vectors
Dry
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