DNA
zraP

Part:BBa_M50006:Design

Designed by: Marilu Bravo, Janelle Kaneda, Katie Lee   Group: Stanford BIOE44 - S11   (2016-10-27)
Revision as of 06:58, 12 December 2016 by Katieblee (Talk | contribs) (→‎Design Notes)


zraP promoter for 2-component lead sensing system in E. coli


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

No Bsa1 sites, less than 2,000 base pairs total

We isolated the zraP promoter from the RBS and didn't use the RBS entirely and instead opted to use a DNA 2.0 RBS that was stronger and better for binding.

E. coli endogenous ZraS/ZraR two-component system

ZraS System NDM.png

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.

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 is 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