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Team:Groningen/Notebook/27 August 2009
From 2009.igem.org
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===GVP Cluster=== | ===GVP Cluster=== | ||
| - | :→ {{ | + | :→ {{done}} Check o.n. plates with transformed E.coli TOP10 cells |
| - | :→ {{ | + | :→ {{done}} Use colonies to grow o.n. precultures for plasmid isolation |
:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | :→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | ||
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|27/8 | |27/8 | ||
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| + | :→ The plates where old DNA fragments were used showed a lower amount of colonies, probably due to degradation of the DNA ends. In the case of the constitutive promoter BBa_J23109 no colonies at all were visible, and can be explained by both fragments being cut earlier. | ||
| + | |||
| + | :→ From the "new" plates four colonies of each construct will be used for preculture growth, and from the "old" fragments only two will be used. (12 in total) | ||
===Transporters=== | ===Transporters=== | ||
| + | |||
| + | ====HmtA==== | ||
| + | Colony pcr gave no positive response, samples 1.3.4.5.7.8.9.11 will be isolated for restriction check with NcoI that cuts in vector as wel as insert giving 2 fragments of 1814bp and 3261bp. An aditionnal control will be a pcr with VF2 and VR resulting in a fragment of 2222bp. | ||
| + | |||
| + | Below fisrt row 1-7, second row 8-12, F24,F2,F1 | ||
| + | |||
| + | [[Image:Clony-pcr-1-12%2Cphusion%2CpBAD24%2CF1%2CF2.jpg|200px]] | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | <center>[[Image:FMT_%26_MBP-ArsR_colony_PCR_to_check_presence_inducible_promotors_-_27august2009.JPG| 250px]]</center> | ||
| + | 1% Agarose (1xTBE) gel with Colony PCR reaction of fMT and MBP-ArsR with either low constitutive - or inducible Lac or Bad promotor. PCR was performed either using standard V<sub>R</sub>/V<sub>F</sub> primers to amplify entire region between prefix and suffix or the RBS oligo and fMT reverse primer. Expected size for fMT using V<sub>R</sub>/V<sub>F</sub> primers was ~500 bp and ~200 bp using fMT primers. MBP-ArsR should be ~1600 bp. | ||
===Vectors=== | ===Vectors=== | ||
Latest revision as of 07:23, 28 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → DONE Check o.n. plates with transformed E.coli TOP10 cells
- → DONE Use colonies to grow o.n. precultures for plasmid isolation
- → 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 |
| LAC-GVP (new) | pSB1AC3 | Ampicillin | ~80 | 27/8 |
| LAC-GVP (new, concentrated) | pSB1AC3 | Ampicillin | ~200 | 27/8 |
| LAC-GVP (old) | pSB1AC3 | Ampicillin | 3 | 27/8 |
| LAC-GVP (old, concentrated) | pSB1AC3 | Ampicillin | 11 | 27/8 |
| pBAD-GVP (new) | pSB1AC3 | Ampicillin | 18 | 27/8 |
| pBAD-GVP (new, concentrated) | pSB1AC3 | Ampicillin | ~80 | 27/8 |
| pBAD-GVP (old) | pSB1AC3 | Ampicillin | 4 | 27/8 |
| pBAD-GVP (old, concentrated) | pSB1AC3 | Ampicillin | 28 | 27/8 |
| pLow (constitutive)-GVP (old) | pSB3K3 | Kanamycin | 0 | 27/8 |
| pLow (constitutive)-GVP (old, concentrated) | pSB3K3 | Kanamycin | 0 | 27/8 |
| MQ (neg. control) | X | X | 0 | 27/8 |
| pHigh (constitutive)-RFP (pos. control) | J61002 | Ampicillin | ~2000 | 27/8 |
- → The plates where old DNA fragments were used showed a lower amount of colonies, probably due to degradation of the DNA ends. In the case of the constitutive promoter BBa_J23109 no colonies at all were visible, and can be explained by both fragments being cut earlier.
- → From the "new" plates four colonies of each construct will be used for preculture growth, and from the "old" fragments only two will be used. (12 in total)
Transporters
HmtA
Colony pcr gave no positive response, samples 1.3.4.5.7.8.9.11 will be isolated for restriction check with NcoI that cuts in vector as wel as insert giving 2 fragments of 1814bp and 3261bp. An aditionnal control will be a pcr with VF2 and VR resulting in a fragment of 2222bp.
Below fisrt row 1-7, second row 8-12, F24,F2,F1
Metal Accumulation
1% Agarose (1xTBE) gel with Colony PCR reaction of fMT and MBP-ArsR with either low constitutive - or inducible Lac or Bad promotor. PCR was performed either using standard VR/VF primers to amplify entire region between prefix and suffix or the RBS oligo and fMT reverse primer. Expected size for fMT using VR/VF primers was ~500 bp and ~200 bp using fMT primers. MBP-ArsR should be ~1600 bp.
Vectors
Dry
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