Team:Groningen/Notebook/Wetwork 18July2012

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Tom
 
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Incubating Bacillus in LB medium for overnight growth. Next day: growth curves in SMM medium.
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Nisa
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<p class="margin">
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Tom<br>
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Possible promoter (inducible promoter)
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<br>
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Incubating Bacillus in LB medium for overnight growth. Next day: growth curves in SMM medium.<br>
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1. Strong pBAD promoter (BBa_K206000)
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<br>
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<br>
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Nisa<br>
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2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)
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<br>
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Possible promoter (inducible promoter)<br>
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Allows for strong promotion that can be:
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1. Strong pBAD promoter (BBa_K206000)<br>
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2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)<br>
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a) repressed by lacI
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<br>
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Allows for strong promotion that can be:<br>
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b) induced by IPTG in E.coli DH5a over a >600 fold range
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a) repressed by lacI<br>
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b) induced by IPTG in E.coli DH5a over a >600 fold range<br>
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<br>
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Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)
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Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)<br>
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actvator: CII or luxI<br>
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actvator: CII or luxI
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... and CIII cd for promoter leakiness<br>
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<br>
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... and CIII cd for promoter leakiness
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For BBa_R0011 (pL hybrid -> inducible promoter)<br>
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<br>
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Forward primer=prefix-Fprimer<br>
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For BBa_R0011 (pL hybrid -> inducible promoter)
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Reverse primer= Rprimer-Pactivator homology (pRE, pluxR)<br>
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<br>
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Forward primer=prefix-Fprimer
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For P actvator-Actvator-GFP<br>
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Reverse primer= Rprimer-Pactivator homology (pRE, pluxR)
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<br>
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A= RBS+CII - Fwd primer homolgue to P A (pRE, PluxR), Rev primer homologue to RBS+GFP<br>
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or<br>
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For P actvator-Actvator-GFP
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A = RBS+luxI - For RBS use BBA_537034 (exclude terminator)<br>
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B=RBS+GFP - Fwd primer homologue to (A), Rev primer+ suffix<br>
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A= RBS+CII - Fwd primer homolgue to P A (pRE, PluxR), Rev primer homologue to RBS+GFP
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<br>
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<br>
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or
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Emeraldo<br>
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<br>
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A = RBS+luxI - For RBS use BBA_537034 (exclude terminator)
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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.<br>
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<br>
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B=RBS+GFP - Fwd primer homologue to (A), Rev primer+ suffix
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Nanodrop concentrations (ng/ul):<br>
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150_alsT = 31,6<br>
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250_alsT = 20,5<br>
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Emeraldo
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300_alsT = 22<br>
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500_alsT = 27,6<br>
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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.
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GFP = 39<br>
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<br>
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Nanodrop concentrations (ng/ul):
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<br>
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
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150_alsT = 31,6
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</p><br><br>
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</body>
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250_alsT = 20,5
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300_alsT = 22
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500_alsT = 27,6
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GFP = 39
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<html>
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
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</html>
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{{Template:SponsorsGroningen2012}}
{{Template:SponsorsGroningen2012}}

Latest revision as of 21:14, 25 September 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
or
A = 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


Back to notebook



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