Difference between revisions of "Part:BBa K2573000"

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===Characterization of MetE Coding Device===
 
===Characterization of MetE Coding Device===
  
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We transformed the BioBrick into the JT2-E. coli strain and grew it in methionine deficient media. Since methionine is essential for E.coli growth and JT2 does not contain MetE in its genome, seeing growth indicates that the part does indeed work as intended as the JT2 cells, lacking the proper methionine synthesis pathway, would not have grown otherwise.
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We transformed this BioBrick into E. coli strain JT2. JT2 has MetE in its genome, under the CcaS/R promoter. However, this strain does not contain genes encoding CcaS/R, meaning it is unable to express MetE.
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The BioBrick in JT2 was grown in M9 medium with and without methionine. Since methionine is essential for bacterial growth, and JT2 is a methionine knockout, growth of JT2 in a methionine deficient medium indicates that the MetE BioBrick functions as intended.
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Empty JT2 cells were also grown in M9 with and without methionine, to confirm that the MetE gene in the JT2 genome is non-functional. E. coli strain DH5a cells, which contain MetE in the genome, were also grown in the same media as a control to show that bacteria are able to grow in the M9 prepared.  
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[[File:UWaterloo BioBrick grown in methionine deficient media.jpeg.png|500px|BioBrick grown in methionine deficient media]]
 
[[File:UWaterloo BioBrick grown in methionine deficient media.jpeg.png|500px|BioBrick grown in methionine deficient media]]
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Figure 1: BioBrick in JT2, empty JT2, and DH5a cells were grown in LB. Overnight cultures of BioBrick in JT2, empty JT2, and DH5a were rinsed 3x with M9 to remove residual LB.
  
  
 
[[File:UWaterloo_BioBrick_characterization.png|500px]]
 
[[File:UWaterloo_BioBrick_characterization.png|500px]]
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Figure 2: Each strain (BioBrick in JT2, empty JT2, DH5a) was inoculated in M9 with and without methionine.

Revision as of 04:34, 17 October 2018


MetE Coding Device

Usage and Biology

Methionine is an essential amino acid for E.coli growth, and the MetE gene is essential for methionine synthesis. It encodes for an enzyme that catalyzes the final step of de novo methionine biosynthesis without using an intermediate methyl carrier. For optimal MetE function, vitamin B12 should not be present, as it functions as a MetE repressor.

Biosynthesis of Methionine Mechanism


This part is the MetE gene cassette under the inducible LacI promoter, designed to be a MetE coding device.

A 2.4kb fragment containing the MetE gene from plasmid pSKA397 was cloned into Bba_J04450 via PCR and NEBuilder HiFi DNA Assembly.


REFERENCES: Pejchal, Robert, and Martha L Ludwig. “Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel That Evolved by Gene Duplication.” PLOS ONE, Public Library of Science, journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.0030031.

“Escherichia Coli K-12 Substr. MG1655 MetE.” MetaCyc Parathion Hydrolase, biocyc.org/gene?orgid=ECOLI&id=EG10584.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1864
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization of MetE Coding Device

We transformed this BioBrick into E. coli strain JT2. JT2 has MetE in its genome, under the CcaS/R promoter. However, this strain does not contain genes encoding CcaS/R, meaning it is unable to express MetE.

The BioBrick in JT2 was grown in M9 medium with and without methionine. Since methionine is essential for bacterial growth, and JT2 is a methionine knockout, growth of JT2 in a methionine deficient medium indicates that the MetE BioBrick functions as intended. Empty JT2 cells were also grown in M9 with and without methionine, to confirm that the MetE gene in the JT2 genome is non-functional. E. coli strain DH5a cells, which contain MetE in the genome, were also grown in the same media as a control to show that bacteria are able to grow in the M9 prepared.


BioBrick grown in methionine deficient media Figure 1: BioBrick in JT2, empty JT2, and DH5a cells were grown in LB. Overnight cultures of BioBrick in JT2, empty JT2, and DH5a were rinsed 3x with M9 to remove residual LB.


UWaterloo BioBrick characterization.png Figure 2: Each strain (BioBrick in JT2, empty JT2, DH5a) was inoculated in M9 with and without methionine.