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Part:BBa_K1218005

Designed by: Trevor Kalkus, Gordon Wade, Alissa Greenberg   Group: iGEM13_Stanford-Brown   (2013-08-28)

CysE (Ancestral)

CysE is a gene that makes a serine acetyltransferase essential to the production of cysteine. The serine acetyltransferase is responsible for the acetylation of serine to give O-acetylserine, a precursor for the production of cysteine. The ancestral version of the N terminal of the protein has been reconstructed 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.

Due to limited data, we did not have enough information to create an ancestral version of the complete CysE gene. As a result, only the N terminus of the protein was reconstructed and is 53.3% similar to the modern one. We fused this to the extant E. coli C terminus. The C terminus is responsible for binding to other CysE generated proteins to make a heximer, whereas the N terminus is actually responsible for the reaction that creates cysteine.

Usage and Biology

These graphs represent the growth curves in LB of our rescued CysE positive control cells with the Trento2012 gene (top) and rescued CysE 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 CysE 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 (CysE 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.

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 478
    Illegal AgeI site found at 684
  • 1000
    COMPATIBLE WITH RFC[1000]


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