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. | + | 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. |
− | To read about our project [http://2013.igem.org/Team:Yale/Project_Overview click here] <br> | + | |
+ | |||
+ | |||
+ | |||
+ | |||
+ | == Data == | ||
+ | Here is a gel showing our assembly of this gene. In order project we attached a second terminator to the gene as well.<br> | ||
+ | <br> <center>[[File:PCTgel.png|150px]] [[File:PCT.png|200px]]</center><br> | ||
+ | |||
+ | |||
+ | *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. | ||
+ | <center>[[File:EcNR2 LB x20 wiki.jpg|400px]]</center><br><br> | ||
+ | |||
+ | |||
+ | {| | ||
+ | |- | ||
+ | |The first sample is the baseline. This is EcNR2 with our plasmid containing both the PCT and PHA gene. This strain was grown overnight with the cells induced and in the presence of Nile red to strain the PLA (this is the same procedure for all later strains as well). The gate (labeled P2) was chosen to select those with the highest levels of fluorescence. Due to the abnormally high levels of fluorescence in some cells in the baseline, the gate was chosen to include some baseline cells, but only those with very high levels of fluorescence. | ||
+ | |style="padding-left: 20px; padding-right: 20px;"|[[File:Baseline both.jpg|400px]] | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | |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. | ||
+ | |style="padding-left: 20px; padding-right: 20px;"|[[File:66-All both.jpg|400px]] | ||
+ | |} | ||
+ | |||
+ | ---- | ||
+ | |||
+ | {| | ||
+ | |- | ||
+ | |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 | ||
+ | |style="padding-left: 20px; padding-right: 20px;"|[[File:66-All high both.jpg|400px]] | ||
+ | |} | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | To read more about our project [http://2013.igem.org/Team:Yale/Project_Overview click here] <br> | ||
[[File:PLA pathway2.jpg|600px]] | [[File:PLA pathway2.jpg|600px]] | ||
Revision as of 22:20, 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 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]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]