Team:TU Delft/12 July 2010 content


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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:
K398500 J23100 + J61100 + I13401
K398501 J23100 + J61101 + I13401
K398502 J23100 + J61107 + I13401
K398503 J23100 + J61117 + I13401
K398504 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 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 K398202 R0011 + B0032
L3 K398203 OprG + B0015
L4 K398204 AlnA + B0015