"

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:
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 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’