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 (mKate2) 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 (mKate2) 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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[[Image:T--Oxford--FCK_diagram_v2.jpg|400px|thumb|center|Schematic of the system]]
  
 
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<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>
 
<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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[[Image:T--Oxford--FCKDATA.jpeg|400px|thumb|center|Absolute fluorescence for the construct at four hours at various copper concentrations.]]
  
 
==References==
 
==References==

Revision as of 15:41, 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 (mKate2) 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.

Schematic of the system

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]

Usage and Biology

E. coli responds 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 E. coli 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).(1)

Experience

We cloned pCusC CusR RFP from a gBlock into the shipping plasmid pSB1C3. E. coli 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.

This sort of positive feedback system was shown to be more responsive in a paper by Ravikumar S et al.(2). 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

Absolute fluorescence for the construct at four hours at various copper concentrations.

References

(1) Yamamoto K, Ishihama A. (2005) “Transcriptional response of Escherichia coli to external copper.” Mol Microbiol. 2005 Apr;56(1):215-27.

(2) Sambandam Ravikumar, Van Dung Pham, Seung Hwan Lee, Ik-keun Yoo, Soon Ho Hong (2012) “Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop” Journal of Industrial Microbiology & Biotechnology June 2012, Volume 39, Issue 6, pp 861–868