Difference between revisions of "Part:BBa K2086002"

(→‎Biology and Our Application)
(→‎PyeaR)
Line 14: Line 14:
 
===Biology and Our Application===
 
===Biology and Our Application===
 
===PyeaR===
 
===PyeaR===
 +
[[Image:T--UNebraska-Lincoln--Repression.png|400px|thumb|right|'''Figure 1:''' The biology of the yeaR promoter. In the presence of nitrate, nitrite, and nitrous oxide, the promoter is uninhibited and can code for 3-D-phosphogylcerate hydrogenase, an important enzyme in the serine biosynthesis pathway.]]
 
The yeaR-yoaG operon is found in <i>E. coli</i> and has been shown to be sensitive to nitrate concentrations as well as nitrite concentrations to a lesser degree [1]. The promoter of this operon is regulated by the Nar and NsrR repressors found. These repressors are deactivated in the presence of nitrate,nitrite, and nitric oxide to a lesser extent. This promoter is also unique in the fact that it is not repressed under aerobic conditions. The original BioBrick <partinfo>K216005</partinfo> was made by the 2009 Edinburgh iGEM team.
 
The yeaR-yoaG operon is found in <i>E. coli</i> and has been shown to be sensitive to nitrate concentrations as well as nitrite concentrations to a lesser degree [1]. The promoter of this operon is regulated by the Nar and NsrR repressors found. These repressors are deactivated in the presence of nitrate,nitrite, and nitric oxide to a lesser extent. This promoter is also unique in the fact that it is not repressed under aerobic conditions. The original BioBrick <partinfo>K216005</partinfo> was made by the 2009 Edinburgh iGEM team.
  

Revision as of 19:50, 17 October 2016


PyeaR - SerA This part is a composite part of the promoter BBa_K216005 with the serine coding sequence BBa_K2086002 as well as a standard RBS BBa_B0030 and terminator BBa_B0015. It is designed to act as a nitrate sensitive kill-switch by utilizing a nitrate sensitive promoter coordinated with an essential gene to complement an auxotrophic strain of E. coli only under high nitrate conditions.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 422
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Biology and Our Application

PyeaR

Figure 1: The biology of the yeaR promoter. In the presence of nitrate, nitrite, and nitrous oxide, the promoter is uninhibited and can code for 3-D-phosphogylcerate hydrogenase, an important enzyme in the serine biosynthesis pathway.

The yeaR-yoaG operon is found in E. coli and has been shown to be sensitive to nitrate concentrations as well as nitrite concentrations to a lesser degree [1]. The promoter of this operon is regulated by the Nar and NsrR repressors found. These repressors are deactivated in the presence of nitrate,nitrite, and nitric oxide to a lesser extent. This promoter is also unique in the fact that it is not repressed under aerobic conditions. The original BioBrick BBa_K216005 was made by the 2009 Edinburgh iGEM team.

[1] HSIA-YIN LIN, PEGGY J. BLEDSOE, AND VALLEY STEWART: Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168752/

Serine

Figure 1: The serine biosynthesis pathway. Provided by: EcoCyc, a member of the BioCyc database collection http://ecocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=SERSYN-PWY&show-citations=NIL

Serine is an amino acid produced in E. coli K12 through the metabolic pathway shown in Figure 1. The SerA gene codes for D-3-Phosphoglycerate Dehydrogenase, the enzyme responsible for catalyzing the committed step of serine biosynthesis. Without SerA, E. coli are unable to grow without sufficient supplementation of other amino acids [2].

Taking advantage of the bacteria's dependence on serine, we planned to create a safety kill switch by controlling the production of the amino acid. By obtaining an auxotrophic strain missing the SerA strain (JW2880 [3]) we were able to create a complement SerA plasmid to rescue the strain when grown in media without supplementary amino acids. Our kill-switch (BBa_K2086002) was incorporated into our final project design.

[2] PAULA D. RAVNIKAR AND RONALD L. SOMERVILLE: Genetic Characterization of a Highly Efficient Alternate Pathway of Serine Biosynthesis in Escherichia coli. http://jb.asm.org/content/169/6/2611.full.pdf

[3] Coli Generic Stock Center: JW2880. http://cgsc.biology.yale.edu/Strain.php?ID=108515

SerA Supplementation

Figure 2: The growth curve of E. coli JW2880 complemented with a plasmid (SerA + native promoter). OD600 measured every 12.5 minutes to indicate cell growth grown in our M9 minimal medium.
Figure 3: Qualitative results from testing our composite BioBrick BBa_K2086002. Both tubes contain JW2880, complemented with our PyeaR-SerA kill switch, grown anaerobically in our M9 minimal medium. The left bottle contains no potassium nitrate, while the right contains 4 mM potassium nitrate.

Before interpreting the results from our composite part including SerA, it's important to first consider whether our selected auxotroph (JW2880) was able to be rescued with a supplemental SerA gene. By complementing our auxotroph with a plasmid containing SerA and its native promoter, the growth curve in Figure 2 was obtained. The ability of our isolated SerA gene to rescue our auxotroph was confirmed by testing our composite BioBrick kill switch (BBa_K2086002), which expresses the SerA gene when nitrate concentrations reach a certain threshold. The qualitative results from one of these experiments are pictured in Figure 3.