Team:Yale/Project Validate

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Revision as of 02:11, 28 August 2013

Project Overview	Validate PLA synthesis	Develop bioassay	Apply MAGE	Introduce export system	Make a bioplastic

Aims for the Project

Engineer strains of E. coli to validate PLA synthesis
Develop bioassay to screen PLA production
Apply MAGE to optimize PLA production, guided by FBA
Introduce type 1 secretion system to export and extract PLA
Make a bioplastic

Engineer strains of E. coli to validate PLA synthesis

Synthesizing and Assembling the Heterologous Enzymes

In order to reproduce the results of the Lee group we needed to insert the two heterologous genes.

Clostridum propionicum propionate CoA transferase (denoted PCT)
Pseudomonas resinovorans polyhydroxyalkanoate synthase (denoted PHA)

This was our plan in order to validate PLA synthesis. In order to save money we ordered each of the two heterologous genes in four pieces from IDT giving us 8 GBlocks. Each gene had an inducible promoter in front so we could tightly regulate the expression of the enzyme. The terminators were amplified from other sources and given homology to the appropriate Gblocks.

Here is a schematic of what the entire construct looks like with both promoter, genes and terminators

Each fragment was amplified using PCR (according to the protocol here!!!!!!!!!!), to give homology to adjacent fragments. Using gibson assembly our plan was to assemble all 10 fragments into one construct.

In order to facilitate the process, we gibson assembled each gene separately. Here is a gel of the first 4 Gblocks (labeled PLA1-4), along with the first terminator (labeled T1), and then the 5 pieces assembled together.

Here is a gel of the assembly of the last 4 Gblocks, along with the second terminator.

Here is a gel of the assembly of the entire construct.

Sequencing the construct

Keck Biotechnology Resource Laboratory kindly sequenced our construct for us
- Using our Geneious license we aligned the sequencing results with the desired sequence of our construct
- Here is a zoomed in pictures of the results

This is the results of an allignment all 8 fragments with the desired sequence. Ignoring the mismatches at the beginning and end of the sequencing fragments, along with those mismatches covered by the complementary fragment, there appears to only be one legitimate error in our construct. It is denoted below with the red arrow. Luckily this happens to fall in a noncoding region of the construct (after the first terminator before the second promoter.

Inserting via Plasmid

In order to insert the two heterologous gene into E. Coli we used Gibson Assembly to add our construct onto a plasmid with KanR as a selectable marker

Inserting into the Genome

TolC Negative Selection

We used a strain with TolC located at 21B, a highly recombinogenic site in the genome (Isaacs et al.2011). Our plan was to replace TolC with our construct using double stranded DNA recombination. Then use Colicin E1 negative selection to pick desired cells (DeVito 2007).

Positive Selection with KanR

Screen Shot 2013-08-05 at 10.43.48 AM.png

@@ Line 73: / Line 73: @@
 <center>[[File:PZE plasmid.JPG|400px]]</center><br>
 <center>[[File:EcNR2transformPLA.png|600px]]</center><br>
+=== Inserting into the Genome ===
+==== TolC Negative Selection ====
+*We used a strain with TolC located at 21B, a highly recombinogenic site in the genome (Isaacs et al.2011).  Our plan was to replace TolC with our construct using double stranded DNA recombination.  Then use Colicin E1 negative selection to pick desired cells (DeVito 2007).
+<center>[[File:TolCnegative.JPG|400px]]</center><br>
+====Positive Selection with KanR====
+<center>[[File:KanR positive select.JPG|400px]]</center><br>
+<center>[[File:Screen Shot 2013-08-05 at 10.43.48 AM.png|400px]]</center><br>