"

From 2010.igem.org

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