Team:ETH Zurich/Wetlab/Overview

In order to approach our goal of creating an E. coli carrying a minimal genome, there are three main tracks we are currently working on:

o genome reduction: show that in vivo restriction and religation is possible

o chemostat selection: introduce a limitation that confers a growth advantage to organisms with smaller genomes

o pulse generator: produce a biobrick that provides for short-term synthesis of the desired gene products

To prove that in vivo restriction and religation is possible is fundamental to our project which relies on the short-term expression of a restriction enzyme and a ligase. While the restriction enzyme will randomly cut DNA, the simultaneous or shortly delayed expression of the ligase should religate the DNA. If the DNA is cut at several sites, relegation will lead to exclusion of chromosomal fragments in a random manner.

In the continuous culture of a chemostat, those organisms with the highest rate of proliferation will overgrow those with a smaller growth rate. In order to bypass the need of manually selecting for those E. coli which have successfully reduced their genomes, we therefore need to introduce a constraint that confers a growth advantage to organisms with smaller genomes. We have chosen to introduce mutations in the nucleotide synthesis pathway to achieve this goal. This will render DNA replication the rate-limiting step of proliferation and therefore be advantageous to organisms with small genomes.

Finally, it is important to restrict the expression of the restriction enzyme to a short period of time. Otherwise, the DNA damage induced would almost certainly kill the organism. Therefore, we want to pulse the expression of the restriction enzyme, with each pulse resulting in one round of random deletion of chromosomal fragments.

On the following pages, we will show a detailed description of how we are trying to achieve these three goals.