![](https://parts.igem.org/images/partbypart/icon_coding.png)
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.
![PCTgel.png](/wiki/images/6/6b/PCTgel.png)
![PCT.png](/wiki/images/9/9f/PCT.png)
- 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.
![EcNR2 LB x20 wiki.jpg](/wiki/images/2/2f/EcNR2_LB_x20_wiki.jpg)
This is a sample with sample is the all oligs. This is a combination of both the RBS oligos and the KO oligos. This sample has 98 cells within the gate, which is a six and a half fold increase. | ![]() |
Due to the fact the last sample with all the oligos had so many cells within the gate we decided to make a stringent gate. This gate was moved to only select the top 0.1% of the cells. This corresponded to 7/10,000. These cells were collected as well as the cells from the three previous graphs | 400px |
To read more about our project [http://2013.igem.org/Team:Yale/Project_Overview click here]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Unknown
- 1000COMPATIBLE WITH RFC[1000]
None |