Difference between revisions of "Part:BBa K2353001:Design"
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===Design Notes===
 
===Design Notes===
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The promoter and terminator are both part of different parts, which means tspurple is not expressed unless it is assembled with pLambdaR-LacI and r0011ClpXPCI.
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tsPurpleDAS is composed of a ribosomal binding site, tsPurple (a purple chromoprotein which functions as a reporter), and DAS (a moderately fast degradation tag). tsPurpleDAS is intended to be assembled with pLambdaR-LacI (BBa_K1911000),which is the promoter and regulatory system, and pLac-ClpXP-CI (BBa_K1911001),which is the non-lysosomal protease. The terminator is located at the beginning of pLac-ClpXP-CI. When fully assembled, p-lambda-r LacI transcribes tsPurple and LacI represses pLac, preventing transcription of ClpXP-CI. Upon induction with IPTG, LacI binds to IPTG which prevents repression of pLac. Therefore, ClpXP-CI is transcribed; ClpXP should recognize the DAS deg tag and degrade the tsPurple protein. The ensuing levels of expression could then be compared to that of BBa_K2353000 (tsPurple without a deg tag) to see the relative level of degradation experienced.
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IMPORTANT NOTE: The promoter and terminator are both part of different parts, which means tspurple is not expressed unless it is assembled with pLambdaR-LacI (BBa_K1911000). The protease complex of ClpX and ClpP is from part (BBa_K1911001)
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Revision as of 15:11, 1 November 2017


tsPurpleDAS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

tsPurpleDAS is composed of a ribosomal binding site, tsPurple (a purple chromoprotein which functions as a reporter), and DAS (a moderately fast degradation tag). tsPurpleDAS is intended to be assembled with pLambdaR-LacI (BBa_K1911000),which is the promoter and regulatory system, and pLac-ClpXP-CI (BBa_K1911001),which is the non-lysosomal protease. The terminator is located at the beginning of pLac-ClpXP-CI. When fully assembled, p-lambda-r LacI transcribes tsPurple and LacI represses pLac, preventing transcription of ClpXP-CI. Upon induction with IPTG, LacI binds to IPTG which prevents repression of pLac. Therefore, ClpXP-CI is transcribed; ClpXP should recognize the DAS deg tag and degrade the tsPurple protein. The ensuing levels of expression could then be compared to that of BBa_K2353000 (tsPurple without a deg tag) to see the relative level of degradation experienced. IMPORTANT NOTE: The promoter and terminator are both part of different parts, which means tspurple is not expressed unless it is assembled with pLambdaR-LacI (BBa_K1911000). The protease complex of ClpX and ClpP is from part (BBa_K1911001)


Source

The sequences for this part came from the registry.

References

Eurofins Genomics. (n.d.) Retrieved November 01, 2017, from https://www.eurofins.com
Chromogenic Protein Paintbox™ - E. coli – pMOTHER vectors. (n.d.) Retrieved November 01, 2017, from https://www.atum.bio/eCommerce/catalog/datasheet/526
Part:BBa_K1033906. (n.d.). Retrieved November 01, 2017, from https://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1033906