Part:BBa_K1218007

HisC E. coli (Ancestral)

HisC is a gene that makes a phosphate aminotransferase essential to the production of histidine. Histidine does not grow abiotically, so this part was characterized in a pUC19 expression vector by rescuing HisC knockout strains grown on M9 minimal plates using IPTG as an inducer.

We gathered data on extant HisC genes using the seed sequences from the PFAM data base to create a phylogenetic tree using PhyML. We then used ProtTest to determine that the best ancestral reconstruction model was the WAG model, which we performed using PAML with Lazarus. See the Stanford-Brown 2013 iGEM wiki for process details and authenticity. The resulting phosphate aminotransferase is 39.1% similar to the extant protein in E. coli.

Usage and Biology

These graphs represent the growth curves in LB of our rescued HisC positive control cells (top) and rescued HisC ancestral gene (bottom). Six separate growth curves were taken with each type of rescued cell. As you would expect from selective pressures, the extant genes used as controls promoted growth sooner.

Stanford-Brown iGEM 2013 tested and confirmed the functionality of this part. We used the pUC19 expression vector to express the amino acid production genes we had created/isolated in HisC knockout cells. We grew these cells on plates of M9 minimal media, so that no amino acids could be picked up from the media. This means that the cells would only survive if they picked up the gene to produce the essential amino acid (HisC in this case) they could no longer make themselves. We BioBricked the extant HisC gene to use as a positive control for our ancestral HisC tests. We used an empty pUC19 plasmid as the negative control for both. Below you can see a zoomed in image of these plates. The colonies on these plates were sequenced to confirm that our ancestral genes successfully rescued their respective cells.

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  Negative HisC control plated on M9 minimal media (Exhibits few misshaped colonies)

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  Positive Control HisC plated on M9 minimal media (Exhibits many regularly spaced, healthy colonies)

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  Ancestral HisC plated on M9 minimal media

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 433
  
• 1000
  COMPATIBLE WITH RFC[1000]