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Team:TU Delft/12 July 2010 content
From 2010.igem.org
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High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus we are planning to also measure the fluorescence assays with the BioBricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB with 100 μg/mL Ampicillin, awaiting plasmid isolation and plasmid swapping. | High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus we are planning to also measure the fluorescence assays with the BioBricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB with 100 μg/mL Ampicillin, awaiting plasmid isolation and plasmid swapping. | ||
| - | + | ==Emulsifier== | |
The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3. | The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3. | ||
Revision as of 19:25, 2 August 2010
Lab work
Characterization of Anderson RBS sequences
E.coli Top10 strains containing the following composite BioBricks in pSB1A2 had been obtained:
| BioBrick | Composed of: |
| [http://partsregistry.org/Part:BBa_K398500 K398500] | J23100 + J61100 + I13401 |
| [http://partsregistry.org/Part:BBa_K398501 K398501] | J23100 + J61101 + I13401 |
| [http://partsregistry.org/Part:BBa_K398502 K398502] | J23100 + J61107 + I13401 |
| [http://partsregistry.org/Part:BBa_K398503 K398503] | J23100 + J61117 + I13401 |
| [http://partsregistry.org/Part:BBa_K398504 K398504] | J23100 + J61127 + I13401 |
To prepare for the fluorescence assay measurements the strains were grown in 3 mL LB medium containing 100 μg/mL AMP as well as in M9 minimal medium containing 0.4% glucose and 100 μg/mL AMP.
High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus we are planning to also measure the fluorescence assays with the BioBricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB with 100 μg/mL Ampicillin, awaiting plasmid isolation and plasmid swapping.
Emulsifier
The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3.
Digested were performed according to the digestion protocol:
| # | Sample | Enzyme 1 | Enzyme 2 | Buffer | BSA | Needed fragment |
| 1 | 1.0 μg AlnA | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–AlnA–S’ |
| 2 | 1.0 μg OprG | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–OprG–S’ |
| 3 | 1.0 μg R0011 | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–R0011–S’ |
| 4 | 1.0 μg B0032 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–B0032–P’ |
| 5 | 1.0 μg B0015 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–B0015–P’ |
| 6 | 1.0 μg pSB1T3 | EcoRI | PstI | 2 (BioLabs) | ✓ | ‘E–linear pSB1T3–P’ |
The digestion products were ligated overnight:
| # | BioBrick | Fragment 1 | Fragment 2 | Recipient vector |
| 1 | K398202 | μL ‘E–R0011–S’ | μL ‘X–B0032–P’ | μL ‘E–linear pSB1T3–P’ |
| 2 | K398203 | μL ‘E–AlnA–S’ | μL ‘X–B0015–P’ | μL ‘E–linear pSB1T3–P’ |
| 3 | K398204 | μL ‘E–OprG–S’ | μL ‘X–B0015–P’ | μL ‘E–linear pSB1T3–P’ |
| 4 | negative control | - | - | μL ‘E–linear pSB1T3–P’ |
