Difference between revisions of "Part:BBa K091146:Experience"

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Further discussion of these results can be found [http://www.bio.davidson.edu/courses/genomics/2008/DeLoache/HonorsThesis.pdf here]. The raw data can be downloaded [http://gcat.davidson.edu/GcatWiki/images/c/cb/FluorescenceExperiment.xls here].
 
Further discussion of these results can be found [http://www.bio.davidson.edu/courses/genomics/2008/DeLoache/HonorsThesis.pdf here]. The raw data can be downloaded [http://gcat.davidson.edu/GcatWiki/images/c/cb/FluorescenceExperiment.xls here].
 
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Andrew Kirk, undergraduate, Penn State iGEM 2010
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Two Lux-inducible promoters that were already on the registry (K091107 and K091146) were characterized. Because these promoters require AHL and LuxR in order to activate, a construct was created that consisted of a Lux promoter followed by RFP, and a constitutive promoter followed by the LuxR gene. LuxR was expected to be present in excess so that the limiting factor would be AHL. The coding sequences for LuxR and RFP were preceded by the standard RBS B0034.  The LuxR part used was [https://parts.igem.org/wiki/index.php?title=Part:BBa_K376010 K376010], which was just added to the registry by Penn State 2010. Using the Voigt RBS calculator, it was predicted that the translation initiation rate for expression of this gene was 246720 au. 
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The AHL used was OC6-homoserine lactone. It was added to samples in a 96-well plate in concentrations of 0, .1, 1, 10, 100 and 1000nM.
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The results shown below and [http://2010.igem.org/Team:Penn_State/Project#Lux_Promoter_Characterization on our wiki] show no trend in protein expression based on concentration of OC6-homoserine lactone. More research is warranted to provide information about what are the best conditions for chemically inducing the Lux promoters.
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Latest revision as of 05:55, 4 November 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K091146

User Reviews

UNIQ160fd93177b17645-partinfo-00000000-QINU

No review score entered. Wideloache

This part was tested with the autoinducer molecules 3OC6 and 3OC12 in cells that constitutively expressed LuxR and LasR. It was expected that GFP would be expressed when 3OC12 was given to the cells but not when both 3OC12 and 3OC6 was given. This was because of the lux box that was put in the -35 to -10 region of the promoter. Binding of LuxR+3OC6 to this region was expected to result in repression of the promoter. I found that 3OC12 appeared to activate LuxR, which resulted in unexpected behavior. See the graphs below for my data.

This graph shows how the pLasLux promoter (K091146) is induced by 3OC12 and 3OC6. The pLas' promoter corresponds to BBa_K091117 and has no lux box in the -35 to -10 region of the promoter. The cell strain MC4100::K091206 expresses LuxR and LasR, while JM109 does not. I would have expected to see induction of pLasLux when only 3OC12 was added. We expected that the lack of induction is due to cross reactivity between 3OC12 and LuxR.
The Lux Receiver corresponds to K09100. The Las Receiver corresponds to K091134. This is evidence of cross reactivity between 3OC12 and LuxR, as 3OC12 is shown to induce the Lux Receiver.

Further discussion of these results can be found [http://www.bio.davidson.edu/courses/genomics/2008/DeLoache/HonorsThesis.pdf here]. The raw data can be downloaded [http://gcat.davidson.edu/GcatWiki/images/c/cb/FluorescenceExperiment.xls here].

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Andrew Kirk, undergraduate, Penn State iGEM 2010

Two Lux-inducible promoters that were already on the registry (K091107 and K091146) were characterized. Because these promoters require AHL and LuxR in order to activate, a construct was created that consisted of a Lux promoter followed by RFP, and a constitutive promoter followed by the LuxR gene. LuxR was expected to be present in excess so that the limiting factor would be AHL. The coding sequences for LuxR and RFP were preceded by the standard RBS B0034. The LuxR part used was K376010, which was just added to the registry by Penn State 2010. Using the Voigt RBS calculator, it was predicted that the translation initiation rate for expression of this gene was 246720 au.

The AHL used was OC6-homoserine lactone. It was added to samples in a 96-well plate in concentrations of 0, .1, 1, 10, 100 and 1000nM.

The results shown below and [http://2010.igem.org/Team:Penn_State/Project#Lux_Promoter_Characterization on our wiki] show no trend in protein expression based on concentration of OC6-homoserine lactone. More research is warranted to provide information about what are the best conditions for chemically inducing the Lux promoters.

1107 108.png

1107 116 tetR.png

146 108 tetR.png

146 116 tetR.png UNIQ160fd93177b17645-partinfo-00000002-QINU