Team:Groningen/Notebook/Wetwork 18July2012

From 2012.igem.org

(Difference between revisions)
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Possible promoter (inducible promoter)
Possible promoter (inducible promoter)
 +
1. Strong pBAD promoter (BBa_K206000)
1. Strong pBAD promoter (BBa_K206000)
 +
 +
2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)
2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)
Allows for strong promotion that can be:
Allows for strong promotion that can be:
 +
a) repressed by lacI
a) repressed by lacI
 +
b) induced by IPTG in E.coli DH5a over a >600 fold range
b) induced by IPTG in E.coli DH5a over a >600 fold range
Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)
Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)
 +
actvator: CII or luxI
actvator: CII or luxI
 +
... and CIII cd for promoter leakiness
... 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

Revision as of 09:24, 24 July 2012




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