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 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



  • 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.
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). Wildtypea.jpg


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. Baselinereal.jpg


This is a sample with sample is the all oligos. 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. This means that there was diversity in our population and thus MAGE worked to increase the amount of PLA that our E. coli could produce. 66-All both.jpg




To read more about our project click here

PLA pathway3.jpg

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE WITH RFC[12]
    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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1411
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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_K2042001, is codon optimized for pseudomonas putida which is a safe organism reported to be efficient for polymerization.
  • Protein alignment of BBa K1212002 translation and BBa_K2042001 translation.
Alignement-BBa K1212002-BBa K2042001.png
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