Difference between revisions of "Part:BBa K1980009"

Revision as of 13:32, 20 October 2016

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 527
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 pCusC CusR RFP
-<!-- Add more about the biology of this part here
+===Description===
-===Usage and Biology===
+<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>
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1980009 SequenceAndFeatures</partinfo>
+<!-- Add more about the biology of this part here
+===Usage and Biology===
+<p> The second system E. coli uses to respond to copper is the CusS/CusR two-component system. This consists of the transmembrane histidine kinase enzyme: CusS in the bacterial cytoplasmic membrane and a cytoplasmic response regulator: CusR.
+When CusS binds periplasmic copper it transfers a phosphate group from ATP to CusR aspartate residue 51 via CusS histidine residue 271. Phosphorylated CusR can bind to DNA inverted repeat CusR boxes (AAAATGACAANNTTGTCATTTT) and activate gene expression.
+In <i>E. coli</i> this box is present between the promoters for CusCFBA operon which encodes a multi-protein pump that exports cytoplasmic and periplasmic copper from the cell and the CusRS operon which encodes the two component system (therefore acting as a positive feedback loop in vivo).<sup>(1)</sup></p>
+===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>
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