Difference between revisions of "Part:BBa K1893007"

Revision as of 16:09, 28 October 2016

Cin receiver with GFP (CinR+pCin+GFP)

The gene encoding the transcriptional activator CinR is downstream a constitutive Anderson promoter, j23101. CinR is activated by 3O-C14-HSL. Included in the construct is the CinR-activated promoter pCin upstream of a reporter sequence including GFP. This part was designed so that we could characterize the CinR quorum sensing system and determine activation ranges and crosstalk data.

Usage and Biology

Characterisation data

Figure 1. Characterisation of the Cin response device (BBa_K1893007). (A) Transfer function curve of normalised fluorescence against cognate inducer C14-AHL concentrations. (B) Heat map of normalised fluorescence of CinR-GFP system over a range of AHL concentrations: (i) Binding of CinR-GFP to its cognate AHL (3O-C14 AHL). (ii) Binding of RhlR-GFP to 3 non-cognate AHLs (C4 AHL, 3O-C6 AHL, 3O-C12 AHL). (C) Transfer function curves of normalised fluorescence against non-cognate inducer AHL (3O-C14 AHL) concentrations to investigate inducer AHL crosstalk: (i) C4-AHL (C4 AHL) of the Rhl system (ii) C6-AHL (3O-C6 AHL) of the Lux system (iii) C12-AHL (3O-C12 AHL) of the Las system. Experiments were performed in E. coli Top10 cell strain cultured at 37°C. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Fluorescence measurements were recorded at 180 minutes. Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation of these.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 611
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1698

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 ===Characterisation data===
 [[File:IC16_CinGraph.png|700px|center|]]
-Figure 1. Characterisation of the Rhl response device [https://parts.igem.org/Part:BBa_K1893003 (BBa_K1893003)]. (A) Transfer function curve of normalised fluorescence against cognate inducer C4-AHL concentrations. (B) Heat map of normalised fluorescence of RhlR-GFP system over a range of AHL concentrations: (i) Binding of RhlR-GFP to its cognate AHL (C4 AHL). (ii) Binding of RhlR-GFP to 3 non-cognate AHLs (3O-C6 AHL, 3O-C12 AHL, 3OH-C14 AHL). (C) Transfer function curves of normalised fluorescence against non-cognate inducer AHL (C4 AHL) concentrations to investigate inducer AHL crosstalk: (i) C6-AHL (3O-C6 AHL) of the Lux system (ii) C12-AHL (3O-C12 AHL) of the Las system (iii) C14-AHL (3O-C14 AHL) of the Cin system. Experiments were performed in E. coli Top10 cell strain cultured at 37°C. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Fluorescence measurements were recorded at 180 minutes. Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation of these.
+Figure 1. Characterisation of the Cin response device (BBa_K1893007). (A) Transfer function curve of normalised fluorescence against cognate inducer C14-AHL concentrations. (B) Heat map of normalised fluorescence of CinR-GFP system over a range of AHL concentrations: (i) Binding of CinR-GFP to its cognate AHL (3O-C14 AHL). (ii) Binding of RhlR-GFP to 3 non-cognate AHLs (C4 AHL, 3O-C6 AHL, 3O-C12 AHL). (C) Transfer function curves of normalised fluorescence against non-cognate inducer AHL (3O-C14 AHL) concentrations to investigate inducer AHL crosstalk: (i) C4-AHL (C4 AHL) of the Rhl system (ii) C6-AHL (3O-C6 AHL) of the Lux system (iii) C12-AHL (3O-C12 AHL) of the Las system. Experiments were performed in E. coli Top10 cell strain cultured at 37°C. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Fluorescence measurements were recorded at 180 minutes. Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation of these.
 <span class='h3bb'>Sequence and Features</span>