"

From 2013.igem.org

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

Aims for the Project

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

Engineer strains of E. coli to validate PLA synthesis

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

1. Clostridum propionicum propionate CoA transferase (denoted PCT)
2. 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.

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.