Difference between revisions of "Part:BBa K1211001"
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− | *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 | + | *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 is an intercellular lipid strain | ||
**Nile red does not affect the growth of bacteria, and its fluorescence is quenched in water | **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. | + | *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. | + | **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. |
<center>[[File:EcNR2 LB x20 wiki.jpg|400px]]</center><br><br> | <center>[[File:EcNR2 LB x20 wiki.jpg|400px]]</center><br><br> | ||
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− | |The first sample is the wild type cells. These do not have any heterologous enzymes and thus should not show any significant Nile red fluorescence, since they are not producing PLA | + | |The first sample is the wild type cells. These do not have any heterologous enzymes and thus should not show any significant Nile red fluorescence, since they are not producing PLA. To keep all the control in place, this strain was grown overnight with the inducers and in the presence of Nile red to strain the PLA (this is the same procedure for all later strains as well). |
|style="padding-left: 20px; padding-right: 20px;"|[[File:wildtypea.jpg|400px]] | |style="padding-left: 20px; padding-right: 20px;"|[[File:wildtypea.jpg|400px]] | ||
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− | |This is EcNR2 with our plasmid containing both the PCT and PHA gene | + | |This is EcNR2 with our plasmid containing both the PCT and PHA gene. The gate (labeled P2) was chosen to select those with the highest levels of fluorescence. There is clearly a bimodal distribution that appeared when our construct was added. We predict that this smaller peak represents those cells who were able to produce enough PLA to be detected by FACS. |
|style="padding-left: 20px; padding-right: 20px;"|[[File:baselinereal.jpg|400px]] | |style="padding-left: 20px; padding-right: 20px;"|[[File:baselinereal.jpg|400px]] | ||
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Revision as of 22:46, 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 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 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)
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]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]