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From 2012.igem.org

Tom

Incubating Bacillus in LB medium for overnight growth. Next day: growth curves in SMM medium.

Nisa

Possible promoter (inducible promoter)

1. Strong pBAD promoter (BBa_K206000)

2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)

Allows for strong promotion that can be:

a) repressed by lacI

b) induced by IPTG in E.coli DH5a over a >600 fold range

Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)

actvator: CII or luxI

... and CIII cd for promoter leakiness

For BBa_R0011 (pL hybrid -> inducible promoter)

Forward primer=prefix-Fprimer Reverse primer= Rprimer-Pactivator homology (pRE, pluxR)

For P actvator-Actvator-GFP

A= RBS+CII - Fwd primer homolgue to P A (pRE, PluxR), Rev primer homologue to RBS+GFP

= RBS+luxI - For RBS use BBA_537034 (exclude terminator)

B=RBS+GFP - Fwd primer homologue to (A), Rev primer+ suffix

Emeraldo

PCR AlsT (150F, 205F, 300F, 500F -R) and GFP Fwd and Rev using Phusion polymerase. Result=OK! Now we have fragments of 150bp, 250bp, 300bp ,500bp and 850bp.

Nanodrop concentrations (ng/ul):

150_alsT = 31,6

250_alsT = 20,5

300_alsT = 22

500_alsT = 27,6

GFP = 39