![](https://parts.igem.org/images/partbypart/icon_coding.png)
Coding
Part:BBa_K1211002
Designed by: Joel Sher Group: iGEM13_Yale (2013-08-16)
Pseudomonas resinovorans Polyhydroxyalkanoate synthase
This is the coding sequence for a Polyhydroxyalkanoate synthase from Pseudomonas resinovorans. The enzyme takes the monomer (D)-lactyl-CoA and creates the PLA homopolymer. This sequence includes four mutations which were found to increase yields of PLA (Yang et al.)
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.
![PCTgel2.png](/wiki/images/a/a6/PCTgel2.png)
![PCT3.png](/wiki/images/2/25/PCT3.png)
- Our project used this gene along with a propionate CoA transferase from Clostridum propionicum 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 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
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1411
Illegal PstI site found at 25
Illegal PstI site found at 118
Illegal PstI site found at 208
Illegal PstI site found at 280
Illegal PstI site found at 742 - 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1411
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1411
Illegal PstI site found at 25
Illegal PstI site found at 118
Illegal PstI site found at 208
Illegal PstI site found at 280
Illegal PstI site found at 742 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1411
Illegal PstI site found at 25
Illegal PstI site found at 118
Illegal PstI site found at 208
Illegal PstI site found at 280
Illegal PstI site found at 742
Illegal NgoMIV site found at 1167
Illegal NgoMIV site found at 1212
Illegal AgeI site found at 448
Illegal AgeI site found at 853
Illegal AgeI site found at 1486 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 251
Contribution
- Group: iGEM Evry 2016
- Author: Toky Ratovomanana
- Summary: The part contained an error that has affected its functionality, one of the four amino acid mutation should be on Serine 477 instead of Serine 475. Moreover, the new part BBa_K1211001, is condon optimized for pseudomonas putida which is a safe organism reported to be efficient for polymerization.
- the documentation of your improvement (text, graphs, pictures, tables, etc.)
[edit]
Categories
Parameters
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