Difference between revisions of "Part:BBa K2086001"

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(→‎Plasmid Synthesis)
 
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[[Image:T--UNebraska-Lincoln--Biosynthesis.gif|450px|thumb|right|'''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]]
 
[[Image:T--UNebraska-Lincoln--Biosynthesis.gif|450px|thumb|right|'''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 <i>E. coli</i> K12 through the metabolic pathway shown in <b>Figure 1</b>. The SerA gene codes for D-3-Phosphoglycerate Dehydrogenase, the enzyme responsible for catalyzing the committed step of serine biosynthesis. Without SerA, <i>E. coli</i> are unable to grow without sufficient supplementation of other amino acids [1].  
 
Serine is an amino acid produced in <i>E. coli</i> K12 through the metabolic pathway shown in <b>Figure 1</b>. The SerA gene codes for D-3-Phosphoglycerate Dehydrogenase, the enzyme responsible for catalyzing the committed step of serine biosynthesis. Without SerA, <i>E. coli</i> are unable to grow without sufficient supplementation of other amino acids [1].  
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<p>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 [2]) we were able to create a complement SerA plasmid to rescue the strain when grown in media without supplementary amino acids. </p>
+
<p>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 [2]) we were able to create a complement SerA plasmid to rescue the strain when grown in media without supplementary amino acids. Our kill-switch (<partinfo>K2086002</partinfo>) was incorporated into our final project design.</p>
  
 
[1]. PAULA D. RAVNIKAR AND RONALD L. SOMERVILLE: Genetic Characterization of a Highly Efficient Alternate Pathway of
 
[1]. PAULA D. RAVNIKAR AND RONALD L. SOMERVILLE: Genetic Characterization of a Highly Efficient Alternate Pathway of
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[2] Coli Generic Stock Center: JW2880. http://cgsc.biology.yale.edu/Strain.php?ID=108515
 
[2] Coli Generic Stock Center: JW2880. http://cgsc.biology.yale.edu/Strain.php?ID=108515
  
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===Isolation from Genomic DNA===
+
===Synthesis of SerA Gene===
 +
We identified the SerA gene from the serine biosynthesis sequence present in <i>E. coli</i> MG1655, a laboratory bacteria strain with minimal genetic manipulation [1]. We made a single mutation in the DNA sequence, while conserving the amino acid sequence, in order to eliminate an illegal BioBrick restriction enzyme site. We then added the standard BioBrick prefix and suffix to our DNA sequence and ordered our gene from Integrated DNA Technologies [2]. We used this DNA sequence in the preparation of our submitted plasmid, as well as in a composite BioBrick used in our project (<partinfo>K2086002</partinfo>).
  
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===SerA Supplementation===
+
[1] Kyoto Encyclopedia of Genes and Genomes: MG1655. http://www.genome.jp/kegg-bin/show_organism?org=eco
 +
<p>[2] Integrated DNA Technologies: http://www.idtdna.com/site
  
 +
===SerA Supplementation===
 +
[[Image:SerASupplementationGrowthCurve.jpg|450px|thumb|left|'''Figure 2:''' The growth curve of <i>E. coli</i> JW2880 complemented with a plasmid (SerA + native promoter). OD600 measured every 12.5 minutes to indicate cell growth grown in our M9 minimal medium.]]
 +
[[Image:QualitativeSerASupplemenation.jpg|450px|thumb|Right|'''Figure 3:''' Qualitative results from testing our composite BioBrick <partinfo>K2086002</partinfo>. Both tubes contain <i>JW2880</i>, 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 <b>Figure 2</b> was obtained.
 +
The ability of our isolated SerA gene to rescue our auxotroph was confirmed by testing our composite BioBrick kill switch (<partinfo>K2086002</partinfo>), which expresses the SerA gene when nitrate concentrations reach a certain threshold. The qualitative results from one of these experiments are pictured in <b>Figure 3</b>.
  
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===Plasmid Creation===
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===Plasmid Synthesis===
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[[Image:UNebraska_Lincoln_SerAPlasmidPrep.jpg|450px|thumb|left|'''Figure 3:''' The gel electrophoresis results of the two components of our SerA plasmid]]
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[[Image:UNebraska_Lincoln_SerAPlasmidPrep.jpg|450px|thumb|left|'''Figure 4:''' The gel electrophoresis results of the two components of our SerA plasmid]]
−
After testing, we were able to synthesize a basic SerA ORF plasmid, biobrick compatible with a wide variety of plasmids, promoters, and ribosome binding sites. In <b>Figure 3</b>, you can see the results of our gel electrophoresis of the two components of our plasmid:
+
After testing, we were able to synthesize a basic SerA ORF plasmid, biobrick compatible with a wide variety of plasmids, promoters, and ribosome binding sites. In <b>Figure 4</b>, you can see the results of our gel electrophoresis, used to ligate the two components of our plasmid:
 
*pSB1C3 : 2070 bp
 
*pSB1C3 : 2070 bp
 
*SerA : 1233 bp  
 
*SerA : 1233 bp  

Latest revision as of 00:55, 17 October 2016


Serine Repeat Antigen (SerA)

The E. coli serA gene encodes the D-3-phosphoglycerate dehydrogenase, which catalyzes the first committed step in the biosynthesis of serine. Serine is an essential amino acid for E. coli growth in minimal medium. Deletion of the serA gene leads to a serine auxotroph, which can be rescued either by the expression of a protein with the same catalytic activity as SerA or by the addition of serine in the growth media.


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 293
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Biology and Our Application

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 [1].

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 [2]) 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.

[1]. 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

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

Synthesis of SerA Gene

We identified the SerA gene from the serine biosynthesis sequence present in E. coli MG1655, a laboratory bacteria strain with minimal genetic manipulation [1]. We made a single mutation in the DNA sequence, while conserving the amino acid sequence, in order to eliminate an illegal BioBrick restriction enzyme site. We then added the standard BioBrick prefix and suffix to our DNA sequence and ordered our gene from Integrated DNA Technologies [2]. We used this DNA sequence in the preparation of our submitted plasmid, as well as in a composite BioBrick used in our project (BBa_K2086002).

[1] Kyoto Encyclopedia of Genes and Genomes: MG1655. http://www.genome.jp/kegg-bin/show_organism?org=eco

[2] Integrated DNA Technologies: http://www.idtdna.com/site

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.

Plasmid Synthesis

Figure 4: The gel electrophoresis results of the two components of our SerA plasmid

After testing, we were able to synthesize a basic SerA ORF plasmid, biobrick compatible with a wide variety of plasmids, promoters, and ribosome binding sites. In Figure 4, you can see the results of our gel electrophoresis, used to ligate the two components of our plasmid:

  • pSB1C3 : 2070 bp
  • SerA : 1233 bp