Part:BBa_K1211001

Clostridum propionicum propionate CoA transferase

This is the DNA coding for the enzyme propionate CoA transferase from Clostridum propionicum. The enzyme normally catalyzes the reaction: acetyl-CoA + propanoate <--> acetate + propanoyl-CoA. This mutated sequence has four point mutations and one amino acid substitution (compared to the wild type enzyme) in order to produce (D)-Lactyl-CoA.
We used this enzyme as a the first enzyme in the pathway to create PLA or Polylactic acid. However, this biobrick it produces (D)-Lactyl-CoA which is the precursor to all PHA, this biobrick can be used in a wide range of projects.

Data

Here is a gel showing our assembly of this gene. In order project we attached a second terminator to the gene as well.

• Our project used this gene along with a Pseudomonas resinovorans PHA synthase gene to produce PLA. We tested the ability of our E. coli to produce PLA by using Nile red
  • Nile red is an intercellular lipid strain
  • Nile red does not affect the growth of bacteria, and its fluorescence is quenched in water

• We then proceeded to test our plasmid in the plate reader.
  • Cells were grown for 24 hours with both enzymes induced and in the presence of Nile red. The cells were washed and re-suspended in PBS. The readings were normalized for optical density.

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To read more about our project [http://2013.igem.org/Team:Yale/Project_Overview click here]

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]