Difference between revisions of "Part:BBa M50006:Design"

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===Design Notes===
 
===Design Notes===
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No Bsa1 sites, less than 2,000 base pairs total
+
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 [1,2,3].
 +
 +
We also do not have any Bsa1 sites, and the sequence is less than 2,000 base pairs total.
  
−
We isolated the zraP promoter from the RBS and instead opted to use a DNA 2.0 RBS that was stronger and better for binding.
+
[[File:Figure 1 NDM.png]]
  
−
E. coli endogenous ZraS/ZraR two-component system
+
We found the zraP promoter sequence from a 2014 online research paper from Maruthamuthu, Ganesh, Ravikumar, and Hong [4] and removed the forward and end primer sequences to solely isolate the promoter sequence.
−
 
+
−
[[File: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.
+
  
 
[[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 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.
+
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 [2,3].
  
 
===Source===
 
===Source===
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===References===
 
===References===
  
−
ZraP:Gene. (2013, September 13). Retrieved October 25, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Gene
+
1. ZraP: Expression. (2015, July 21). Retrieved December 11, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Expression
 +
 
 +
2. Petit-Hartlein, I., et al. (2015, December 1). Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system. Biochemical Journal, 472(2), 205-216. doi:10.1042/bj20150827
 +
 
 +
3. 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
+
4. 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
+
5. 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
+
6. Lead and Copper Rule. (2016, October 13). Retrieved December 10, 2016, from https://www.epa.gov/dwreginfo/lead-and-copper-rule
  
−
ZraP DNA. (2011). Retrieved October 25, 2016, from http://ecogene.org/gene/EG11918/dnasequence?&type=coor
+
DNA Sequence Citation: ZraP DNA. (2011). Retrieved October 25, 2016, from http://ecogene.org/gene/EG11918/dnasequence?&type=coor

Latest revision as of 18:42, 12 December 2016


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    COMPATIBLE 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 [1,2,3].

We also do not have any Bsa1 sites, and the sequence is less than 2,000 base pairs total.

Figure 1 NDM.png

We found the zraP promoter sequence from a 2014 online research paper from Maruthamuthu, Ganesh, Ravikumar, and Hong [4] and removed the forward and end primer sequences to solely 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, 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 [2,3].

Source

ZraP gene

References

1. ZraP: Expression. (2015, July 21). Retrieved December 11, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Expression

2. Petit-Hartlein, I., et al. (2015, December 1). Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system. Biochemical Journal, 472(2), 205-216. doi:10.1042/bj20150827

3. ZraP:Gene. (2013, September 13). Retrieved October 25, 2016, from http://ecoliwiki.net/colipedia/index.php/zraP:Gene

4. 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

5. 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

6. Lead and Copper Rule. (2016, October 13). Retrieved December 10, 2016, from https://www.epa.gov/dwreginfo/lead-and-copper-rule

DNA Sequence Citation: ZraP DNA. (2011). Retrieved October 25, 2016, from http://ecogene.org/gene/EG11918/dnasequence?&type=coor