Difference between revisions of "Part:BBa M50006:Design"
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===Design Notes=== | ===Design Notes=== | ||
+ | Because we aimed to develop an Escherichia coli (E. coli) biosensor that glows in the presence of unhealthy lead concentration, we isolated the zraP promoter DNA sequence, which is activated by the endogenous E. coli ZraS/ZraR two-component system in the presence of extracellular lead(II) ions. | ||
+ | |||
+ | Other Note: | ||
No Bsa1 sites, less than 2,000 base pairs total | No Bsa1 sites, less than 2,000 base pairs total | ||
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− | |||
− | |||
− | |||
[[File:ZraS System NDM.png]] | [[File:ZraS System NDM.png]] | ||
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Our E. coli biosensor is built to function based on the ZraS/ZraR system, a two-component endogenous stimulus-sensing apparatus within E. coli. | Our E. coli biosensor is built to function based on the ZraS/ZraR system, a two-component endogenous stimulus-sensing apparatus within E. coli. | ||
− | We found the zraP promoter sequence from a 2014 online research paper from Maruthamuthu, Ganesh, Ravikumar, and Hong, and removed the forward and end primer sequences to isolate the promoter sequence. | + | We found the zraP promoter sequence from a 2014 online research paper from Maruthamuthu, Ganesh, Ravikumar, and Hong, and removed the forward and end primer sequences to solely isolate the promoter sequence. |
[[File:NanodropTheMicDeviceDesignNew.png]] | [[File:NanodropTheMicDeviceDesignNew.png]] | ||
− | We also replaced the ribosome binding site (RBS) provided in the sequence with DNA 2.0’s default high-strength RBS. Downstream of the zraP promoter | + | We also replaced the ribosome binding site (RBS) provided in the sequence with DNA 2.0’s default high-strength RBS. Downstream of the zraP promoter, we added Comet (GFP), which will be expressed when zraP is activated due to the extracellular concentration of lead(II) ions. A lead-binding peptide, OmpC, on the outer surface of E. coli cells is able to bind lead(II) ions, thus activating the cell response through the ZraS/ZraR two component system. |
===Source=== | ===Source=== |
Revision as of 07:20, 12 December 2016
zraP promoter for 2-component lead sensing system in E. coli
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Because we aimed to develop an Escherichia coli (E. coli) biosensor that glows in the presence of unhealthy lead concentration, we isolated the zraP promoter DNA sequence, which is activated by the endogenous E. coli ZraS/ZraR two-component system in the presence of extracellular lead(II) ions.
Other Note: No Bsa1 sites, less than 2,000 base pairs total
Our E. coli biosensor is built to function based on the ZraS/ZraR system, a two-component endogenous stimulus-sensing apparatus within E. coli.
We found the zraP promoter sequence from a 2014 online research paper from Maruthamuthu, Ganesh, Ravikumar, and Hong, and removed the forward and end primer sequences to solely isolate the promoter sequence.
We also replaced the ribosome binding site (RBS) provided in the sequence with DNA 2.0’s default high-strength RBS. Downstream of the zraP promoter, we added Comet (GFP), which will be expressed when zraP is activated due to the extracellular concentration of lead(II) ions. A lead-binding peptide, OmpC, on the outer surface of E. coli cells is able to bind lead(II) ions, thus activating the cell response through the ZraS/ZraR two component system.
Source
ZraP gene
References
ZraP:Gene. (2013, September 13). Retrieved October 25, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Gene
Maruthamuthu, M. K., et al. (2014, November 30). Evaluation of zraP gene expression characteristics and construction of a lead (Pb) sensing and removal system in a recombinant Escherichia coli. Biotechnol Lett, 37(2015), 659-664. doi:DOI 10.1007/s10529-014-1732-x
Escherichia coli K-12 substr. MG1655 Pathway: ZraSR Two-Component Signal Transduction System. (2016). Retrieved December 11, 2016, from https://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PWY0-1498
ZraP: Expression. (2015, July 21). Retrieved December 11, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Expression
ZraP DNA. (2011). Retrieved October 25, 2016, from http://ecogene.org/gene/EG11918/dnasequence?&type=coor