There is a need in the field of synthetic biology of a standardized, stable and quickly expandable multi-plasmid system (A system in which we have different, unique plasmid groups in cell culture, and no plasmid group is lost during cell replication). Due to plasmid incompatibility*, current commercially available systems offer up to 5 different plasmids in cell using different origin of replication (ori) sequences that do not interfere with each other. Yet these systems are often unstable and unpredictable, also limited due to the fact that ori sequences are taken from plasmids that originate from different organisms. It is hard to expand the compatible ORI sequence list because, at the moment, we would need to discover the new sequences in nature, which would require a considerable amount of effort and time. Also, mentioned systems do not offer any way to control plasmid copy number of each plasmid group separately.

With that in mind we have set ourselves several goals we want to reach in our project:

Development of synthetic plasmid origin of replication (ori) site, based on ColE1 replicon, which would tackle the problem of incompatibility by using uniquely barcoded RNA I/RNA II gene pairs for each group of plasmid and show that we are able to stably co-maintain at least six of our newly made plasmids in E. coli. Using this strategy we also gain an opportunity to control the copy number of each plasmid group separately by adjusting RNA I gene expression of each group separately.

Construction of a discrete, recombinase based multi-level synthetic switch system that would allow scientists to precisely control the copy number and ratio between different groups of plasmids, also eliminating the need of periodical addition of inducers to the growth medium.

Because our system will contain different plasmids, we are doing research to develop alternatives to antibiotics (for purposes of biosafety) in order to apply external selection pressure,to ensure that any cell that loses a single group of plasmid would not survive.

In parallel with wet-lab experiments we plan to and currently began writing a mathematical model to describe various aspects of our system. Our dynamical plasmid replication models will allow us to predict theoretical outcomes of the system with various different parameters. We also aim to predict the probabilities of plasmid loss during the cell division and parameters that may influence that loss in order to minimize the depletion.

Our system would open the door to various new applications, such as larger synthetic metabolic pathways, drug synthesis, biological computing and much more!

*Plasmid incompatibility is defined as the failure of two different co-resident plasmids to be stably inherited together. It mainly arises because of fact that some plasmids share and compete for replication factors. For example, plasmids that replicate faster due to their small size would outgrow other plasmids really fast in a culture of cells, and the plasmid group with lesser copy number would have a much higher chance to be lost during cell division compared to the dominating group of plasmids.

Team:Vilnius-Lithuania

Vilnius-Lithuania

Controllable and flexible multi-plasmid system

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