"

From 2010.igem.org

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 M9 minimal medium containing 0.4% glucose and 100 μg/mL Ampicillin.

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:

#	Digestion reaction	Needed fragment
1	1,0 μg AlnA + EcoRI + SpeI	‘E – AlnA – S’
2	1,0 μg OprG + EcoRI + SpeI	‘E – OprG – S’
3	1,0 μg R0011 + EcoRI + SpeI	‘E – R0011 – S’
4	1,0 μg B0032 + XbaI + PstI	‘X – B0032 – P’
5	1,0 μg B0015 + XbaI + PstI	‘X – B0015 – P’
6	1,0 μg pSB1T3 + EcoRI + PstI	‘E – ---- – P’

The digestion products were ligated overnight:

#	Ligation reaction
1	μL R0011 + μL B0032 + μL pSB1T3
2	μL AlnA + μL B0015 + μL pSB1T3
3	μL OprG + μL B0015 + μL pSB1T3
4	μL pSB3C5 (negative control)