Difference between revisions of "Part:BBa K1980009"
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===Description=== | ===Description=== | ||
<p> This synthetic promoter system includes the response regulator CusR (part of the CusR/S copper sensitive two-component system found in <i>E. coli</i>) and the an RFP variant (mKate) expressed downstream of a copper sensitive promoter (pCusC). The fact that CusR is found downstream of the promoter it activates means that a positive feedback loop is created when sufficient copper is sensed in the environment</p> | <p> This synthetic promoter system includes the response regulator CusR (part of the CusR/S copper sensitive two-component system found in <i>E. coli</i>) and the an RFP variant (mKate) expressed downstream of a copper sensitive promoter (pCusC). The fact that CusR is found downstream of the promoter it activates means that a positive feedback loop is created when sufficient copper is sensed in the environment</p> | ||
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| + | <img src="https://static.igem.org/mediawiki/parts/b/bc/T--Oxford--pCusC_CusR_RFP.jpg"/> | ||
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===Experience=== | ===Experience=== | ||
| − | <p>We cloned pCusC CusR RFP from a gBlock into the shipping plasmid pSB1C3. <i>E. coli</i> strain MG1655 was transformed using the specific recombinant plasmid and a 5ml culture of a transformed colony was grown overnight. A plate with four repeats at 10 different copper concentrations (ranging from 0mM to 2mM) plus a negative control was made. The absorbance and fluorescence of each well was measured over time in order to assess the absolute fluorescence of the construct. </p> | + | <p>We cloned pCusC CusR RFP from a gBlock into the shipping plasmid pSB1C3. <i>E. coli</i> strain MG1655 was transformed using the specific recombinant plasmid and a 5ml culture of a transformed colony was grown overnight. A plate with four repeats at 10 different copper concentrations (ranging from 0mM to 2mM) plus a negative control was made. The absorbance and fluorescence of each well was measured over time in order to assess the absolute fluorescence of the construct.</p> |
| + | <p>This sort of positive feedback system was shown to be more responsive in a paper by Ravikumar S <i>et al.</i><sup>(2)</sup>. However after many attempts at cloning our only full length construct had a point mutation (Val to Ala) in the CusR gene. We tested this part to see if this mutation was tolerable, but found no evidence for a more sensitive system </p> | ||
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| + | <img src="https://static.igem.org/mediawiki/parts/3/36/T--Oxford--pCusC_CusR_RFP_data.jpg" width="50%"/> | ||
Revision as of 13:39, 20 October 2016
pCusC CusR RFP
pCusC CusR RFP
Description
This synthetic promoter system includes the response regulator CusR (part of the CusR/S copper sensitive two-component system found in E. coli) and the an RFP variant (mKate) expressed downstream of a copper sensitive promoter (pCusC). The fact that CusR is found downstream of the promoter it activates means that a positive feedback loop is created when sufficient copper is sensed in the environment
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Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 527
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]