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Team:TU Delft/12 July 2010 content
From 2010.igem.org
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Revision as of 14:11, 13 July 2010
Lab Work
Characterization of Anderson RBS sequences
E.coli TOP 10 strains containing the following composite biobricks in pSB1A2 had been obtained:
| BioBrick # | Composed of: |
| [http://partsregistry.org/Part:BBa_K398050 K398050] | J23100 + J61100 + I13401 |
| [http://partsregistry.org/Part:BBa_K398051 K398051] | J23100 + J61101 + I13401 |
| [http://partsregistry.org/Part:BBa_K398052 K398052] | J23100 + J61107 + I13401 |
| [http://partsregistry.org/Part:BBa_K398053 K398053] | J23100 + J61117 + I13401 |
| [http://partsregistry.org/Part:BBa_K398054 K398054] | J23100 + J61127 + I13401 |
To prepare for the fluorescence assay measurements the strains were grown in M9 minimal medium containing 0.4% glucose and ampicillin.
High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus the fluorescence assays will also be performed with the biobricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB-Amp, awaiting QIAGEN Midi kit 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.
Digestion substrates:
| Label | Content | Concentration (ng/ul) |
| D1 | Plasmid [http://partsregistry.org/Part:pSB1T3 pSB1T3] | 137.2 |
| D2 | AlnA | 357.4 |
| D3 | OprG | 197.3 |
| D4 | B0015 | 360.5 |
| D5 | R0011 | 42.0 |
| D6 | B0032 | 82.0 |
Digested according to the digestion protocol (Link needed!).
The digestion products were ligated over night to produce the following biobricks:
| Label | BioBrick # | Composed of: |
| L1 | Control | Plasmid |
| L2 | [http://partsregistry.org/Part:BBa_K398202 K398202] | R0011 + B0032 |
| L3 | [http://partsregistry.org/Part:BBa_K398203 K398203] | OprG + B0015 |
| L4 | [http://partsregistry.org/Part:BBa_K398204 K398204] | AlnA + B0015 |
