Team:Amsterdam/Project/Stability

The starting point

There is an elephant in the room

Background

A big challenge in biotechnology and synthetic biology today is genetic instability. It has been called the elephant in the room which nobody could (or wanted) to see (Jones, 2014). A lot of the applications in synthetic biology work because an organism contains a certain gene construct which contains a set of genes which, when expressed, produces enzymes responsible for the synthesis of certain products.
These products are then excreted from the cell to be used by other organisms (us humans for example). A lot of parts in the part registry of iGEM also work based on this 'plug-and-play' rationale. After we, iGEM teams, find such an interesting set of genes, we register it, we characterize it and we envision big scale applications. But in reality a lot of these applications wouldn't work. This has everything to do with genetic instability.
In most circumstances in the lab where organisms are cultivated, or in bioreactors of any form, species are under a constant selection pressure for growth rate. This means that mutants with a slightly higher growth rate will take over an entire population within a few generations. An organism which produces a compound and transports it out of the cell, always has to divide the resources it has between growth and the production of the compound. The producing cells continuously diverge a part of the mass out of the cell. This means it can no longer use this mass for growth. So a hypothetical mutant which loses this producing activity will have a (slightly) higher growth rate, and thus soon take over the culture, making it loose its production. So if an organism can easily accumulate mutations such that it will loose its production, it will be genetically very unstable. And sometimes all it takes for a producing strain to lose a gene activity is a single mutation in the promoter.
Certainly in large scale applications the costs and effort to re-inoculate a culture, because the production of the old one has stopped can add up to significant amounts. This makes these applications not only economically less feasible, it is also less sustainable, since a lot of recourses are needed to re-inoculate.
We see it as a major challenge in synthetic biology to improve this stability. So this was also the first question we had in the wet lab. How do we make a Synechocystis strain which stably produces a carbon compound?

Our first stumbling block

Our initial plan was to develop Synechocystis strains that produce glucose, lactate, and glycerol, and to compare their performances in a consortium with E. coli. After after the initial results of lactose production came in, however, we decided to make genetic stability the central focus of our carbon fixation efforts: engineering a Synechocystis that produces a carbon compound - acetate, based on our modelling results - in the most genetically stable way possible such that it can be implemented in industrial settings for the constant conversion of CO2 to carbon product.

A new strategy

Explain here the growth coupled thingy

A stable producer

Here talk and link about the algorithm and the acs knockout.