Difference between revisions of "Part:BBa K4054100"
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pLuxR is a common used induciable promoter. However, almost all of the teams characterized the pLuxR basing on AHL concentration. In our project this year, depending our <i>in vitro</i> system, we measure the promoter strenth changes at different concentration of LuxR.We had expressed LuxR by BL21(DE) and harvest it, which we used in the measurement by diluting LuxR to different concentrations and added into the cell free system. Through real-time detection by microplate reader, we achieved the result and confirmed the transition concerntration range. | pLuxR is a common used induciable promoter. However, almost all of the teams characterized the pLuxR basing on AHL concentration. In our project this year, depending our <i>in vitro</i> system, we measure the promoter strenth changes at different concentration of LuxR.We had expressed LuxR by BL21(DE) and harvest it, which we used in the measurement by diluting LuxR to different concentrations and added into the cell free system. Through real-time detection by microplate reader, we achieved the result and confirmed the transition concerntration range. | ||
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[[File:T--XJTLU-CHINA-RT.png|500px|thumb|alt=domestication.|Figrure 2.The result of real-time detection while the eGFP expression was reduced by different concentration of LuxR.]] | [[File:T--XJTLU-CHINA-RT.png|500px|thumb|alt=domestication.|Figrure 2.The result of real-time detection while the eGFP expression was reduced by different concentration of LuxR.]] | ||
[[File:T--XJTLU-CHINA-S Curve.png|500px|thumb|alt=domestication.|Figure 3. The trend of expression rate with the changes of LuxR concentration.]] | [[File:T--XJTLU-CHINA-S Curve.png|500px|thumb|alt=domestication.|Figure 3. The trend of expression rate with the changes of LuxR concentration.]] | ||
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<P>As the Figure 2 and 3 show, the eGFP expression perform a trend of approximate limear growrth, whrn its gexpression rate stabilized. Meanwhile, there is a rapid rate transition as the LuxR concentration between 1μM to 10μM in reaction system.</P> | <P>As the Figure 2 and 3 show, the eGFP expression perform a trend of approximate limear growrth, whrn its gexpression rate stabilized. Meanwhile, there is a rapid rate transition as the LuxR concentration between 1μM to 10μM in reaction system.</P> | ||
Revision as of 12:22, 20 October 2021
pLuxR_T7 RBS_GFP
This is a composite part used for eGFP expression. It consist of pLuxR, T7 RBS and ther coding sequence of reporter GFP As it include the component "pLuxR", the activator LuxR is required to start the transcription.
Introduction
In this year, XJTLU-CHINA characterized pLuxR promoter by this part. As the Figure 1 shows, the composite part consists of the promoter pLuxR (K4054017), the RBS (K4054017)，the coding sequence of eGFP (E0040) and the terminator K4054018.
The part was expressed in Cell Free systemm (in vitro transcription and translation system)， which allowed us to measure the GFP expression level under different LuxR concentration.
Experiment
pLuxR is a common used induciable promoter. However, almost all of the teams characterized the pLuxR basing on AHL concentration. In our project this year, depending our in vitro system, we measure the promoter strenth changes at different concentration of LuxR.We had expressed LuxR by BL21(DE) and harvest it, which we used in the measurement by diluting LuxR to different concentrations and added into the cell free system. Through real-time detection by microplate reader, we achieved the result and confirmed the transition concerntration range.
As the Figure 2 and 3 show, the eGFP expression perform a trend of approximate limear growrth, whrn its gexpression rate stabilized. Meanwhile, there is a rapid rate transition as the LuxR concentration between 1μM to 10μM in reaction system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 736