Team:ETH Zurich/lab/biobrick/used1

From 2014.igem.org

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(Background information)
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== Background information ==
== Background information ==
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We used an ''E. coli'' TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first one contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter [http://parts.igem.org/Part:BBa_R0062 pLuxR (BBa_R0062)] followed by [http://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts [http://parts.igem.org/Part:BBa_B0040 BBa_B0040] and [http://parts.igem.org/Part:BBa_B0015 BBa_B0015] were used, respectively.
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We used an ''E. coli'' TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter [http://parts.igem.org/Part:BBa_R0062 pLuxR (BBa_R0062)] followed by [http://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts [http://parts.igem.org/Part:BBa_B0040 BBa_B0040] and [http://parts.igem.org/Part:BBa_B0015 BBa_B0015] were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, one of three promoters chosen from the [http://parts.igem.org/Promoters/Catalog/Anderson Anderson promoter collection] followed by [http://parts.igem.org/Part:BBa_C0062 luxR (BBa_C0062)].
=== Systems considered ===
=== Systems considered ===

Revision as of 15:26, 16 October 2014

<partinfo>BBa_R0062 AddReview 4</partinfo> ETH Zurich 2014

Contents

Characterization of the promoter's basal leakiness to LuxR in the absence of 3OC6-HSL

The amount of regulator LuxR (BBa_C0062) in the system was shown to influence the pLuxR promoter's basal expression or leakiness. By using the three different constitutive promoters BBa_J23100, BBa_J23109, and BBa_J23111 for the production of LuxR we have measured this effect in terms of fluorescence.

Background information

We used an E. coli TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter pLuxR (BBa_R0062) followed by RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts BBa_B0040 and BBa_B0015 were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, one of three promoters chosen from the Anderson promoter collection followed by luxR (BBa_C0062).

Systems considered

Modeling leakiness

Results

Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration

Background information

Systems considered

Modeling promoter's sensitivity

Results

Characterization of two-level crosstalk on the promoter

Background information

System considered

Modeling crosstalk

First-order crosstalk

First Level crosstalk: LuxR binds to different HSL and activates the promoter

ETH Zurich 1crosstalkPlux.png

Second Level crosstalk: other regulatory proteins, like LasR, bind to their natural HSL substrate and activates the promoter

ETH Zurich 2crosstalkPlux.png

Second order crosstalk: Combination of both cross-talk levels

Other regulatory proteins, like LasR, bind to different HSL and activates the promoter

ETH Zurich 3crosstalkPlux.png

Results

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