Introduction

The first mechanism that we have decided to investigate is the interdependency between two cell types. In order to have a stable relationship in which the host-endosymbiont relationship is maintained through generations, the host and endosymbionts must depend on each other for their continued survival.

Within the endosymbiotic relationship, we envision a resource based cross-network between host and symbiont. The exchange of vital metabolites, necessary for proliferation, would ensure the coexistence of the symbiotic pair, whilst also ensuring mutual demise in case of the host’s or the symbiont’s perishment. Our goal would be that the pair would survive only if the condition of endosymbiosis is met.

This is important, as our project is about laying the foundation for stable modular endosymbiosis. We are working to establish a dependency between free living cells, as a groundwork for a later endosymbiotic relationship. Natural endosymbiotic relationships have been found between fungi and bacteria in mycorrhiza strains (Bianciotti 2000), and we will be working with two cells from these two phylogenetic kingdoms in our project; a tryptophan auxotrophic yeast AM94 and E. coli.

Experiments

Overview

• Synthetically produce a codon-optimised version of yddG
• Amplify trpE and aroG with point mutations
• Multi-gene constructs inserted in vector

Serial growth of E. coli and yeast

• Identify a minimal media supporting both E. coli and yeast growth
• Analyse auxotrophic yeast growth in the minimal media both with and without tryptophan added
• Grow auxotrophic yeast in media only containing tryptophan produced by E.coli

Gene expression and tryptophan production

• Western blotting to check expression of his-tagged proteins
• HPLC-MS measurements of tryptophan production

Verifications

We have used gel electrophoresis in the majority of our experiments and sub-projects to continually monitor the progress.

All transformations in interdependency and protein import were done with USER cloningand the cells were grown ON on LB agar with the appropriate selection (Ampicillin or Chloramphenicol depending on the resistance marker). Colony PCRs were performed to pick colonies with the correct inserts for inoculation in liquid culture.

Subsequently, plasmids were cured from the cultures and PCR amplification and/or restriction digestion were used to check for correct insertion. Cells with appropriately sized inserts were sent to sequencing for sequence validation.

Biobrick creation

We amplified our biobricks with primers carrying the biobrick prefixes and suffixes as overhangs, and purified them with gel extraction. Subsequently, we used the standard iGEM protocol for its insertion into the pSB1C3 submission vector.

All biobricks were sequenced in the expression vector, and aroG, trpE, CPP and CPP-YFP were also sequenced following their insertion into the submission vector.

Vector design

A modified version of the pET102 vector was used for expression of all genes in this sub-project. See protein import for details on design and creation of the vector.

Codon optimization and synthetic yddG

Following problems with amplifying yddG from the genomic DNA, we decided to order a synthetically made version, and codon optimized it in the process.

Point mutations for feedback resistance in trpE and aroG

We amplified the aroG and trpE genes using genomic DNA from E.coli strain MG1655. The amplification was done using two sets of primers per gene, with overhangs allowing for insertion of a point mutation in each gene and for cloning with USER assembly.

The mutations were designed in accordance with previous work by Gu et al 2012. In TrpE, position 293 is changed from methionine to threonine, and for AroG position 150 is changed from proline to leucine.