Coding
ButCoAT

Part:BBa_K2052018

Designed by: Burak Kızıl, Seniz Yuksel   Group: iGEM16_METU_HS_Ankara   (2016-09-24)
Revision as of 15:48, 16 October 2016 by Ipekkargin01 (Talk | contribs) (→‎Characterization)


Butyryl-CoA:acetate CoA-transferase

ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate.) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) (KEGG Reaction No. : R01179) is given down below :

Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA

ButCoaTStructure.gif

Figure 1: Crystal Structures of Acetobacter aceti Succinyl(Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)

Butyrate

Butyrate is a short chain fatty acid which is formed by bacterial fermentation of carbohydrates (Sengupta et al., 2005).

Modeling

caption

The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

caption

The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.

Design Notes

E.coli is synthesized suitable for Codon Bias.

In the expression of pet28a (expression vector) , stop codon is removed for the addition of HisTag.

DNA Gel Analysis

Characterization

PCR Conformation of ButCoAT + pSB1C3

ButCoaTGelImage.jpeg

We have loaded the uncut version of K2052015 (RBS + ButCoAT) next to EcoRI single digested one. In the third lane, RBS ligated ButCoat (K2052018) was loaded with its single digested version. Digested ones gave sharp lane at 3600 bp as we expected.

Source

Roseburia intestinalis L1-82


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


References

Weinstock G., Sodergren E., Clifton S., Fulton L., Fulton B., Courtney L., Fronick C., Harrison M., Strong C., Farmer C., Delahaunty K., Markovic C., Hall O., Minx P., Tomlinson C., Mitreva M., Nelson J., Hou S. Wilson R.K. (2009 , August ). UniProtKB - C7GB37 (C7GB37_9FIRM). Retrieved from http://www.uniprot.org/uniprot/C7GB37?sort=score

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