Difference between revisions of "Part:BBa K1211001"
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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. <br> | 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. <br> | ||
− | We used this enzyme as a the first enzyme in the pathway to create PLA or Polylactic acid. However, this biobrick | + | <br> |
− | + | '''We used this enzyme as a the first enzyme in the pathway to create PLA or Polylactic acid. However, this biobrick produces (D)-Lactyl-CoA, which is the precursor to all PHAs, and thus this biobrick can be used in a wide range of projects in the future.''' | |
Revision as of 22:53, 5 October 2013
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 produces (D)-Lactyl-CoA, which is the precursor to all PHAs, and thus this biobrick can be used in a wide range of projects in the future.
Data
Here is a gel showing our assembly of this gene. In order project we attached a promoter and 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. However, other teams can add other heterologous enzymes and produce different PHA. 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 strain with out 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. Clearly, the one with our plasmid is more fluorescent indicating PLA production.
![EcNR2 LB x20 wiki.jpg](/wiki/images/2/2f/EcNR2_LB_x20_wiki.jpg)
- After creating diversity using MAGE or Multiplex automated genome engineering, we needed a way to sort out those with the highest levels of fluorescence indicating greater PLA production. We decided to use FACS or FLuorescence activated cell sorting, to select those cells with the highest levels of fluorescence.
To read more about our project [http://2013.igem.org/Team:Yale/Project_Overview click here]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]