Key Plasmid Design

Plasmid design

Our plasmid design aims to facilitate simultaneous expression of various proteins at different levels. In our project as a proof of concept we used three fluorescent proteins: Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), and Cyan Fluorescent Protein (CFP). However, in future other proteins could be used, not only fluorescent one, but also any other type of proteins which expression can be detected. We decided to use two plasmid system to obtain different phenotypes. First plasmid would contain fluorescent protein/proteins and dCas9 protein, which would repress a given promoter. This is achieved by the expression of a short guide RNA (gRNA), corresponding to this promoter, which are present on the second plasmid. dCas9 uses the gRNA to repress levels of the reporter by steric hindrance to RNAP between the -10 and -35 region. pReporter plasmid is low copy number to minimise toxic effects of dCas9 and to maximise the difference in gRNA inhibition and pgRNA plasmid is high copy number to have an excess of gRNAs to ensure full targeting of promoters on the other plasmid. We aimed to construct a promoter-gRNA library, then 3 of these promoters to a reporter and test the effect of the corresponding gRNA on repression levels. In further work, we have designed an assembly method where a pool of promoters could be used so that the promoter used would be random.

The idea behind the design of Key. coli is to create random combinations of products at different levels. Initially, we systematically construct bricks containing a set promoter with a fluorescent protein and terminator but later we can use random ligation reactions to produce random combinations due to the modularity of the design. We can place any promoter with any fluorescent protein, and place it anywhere in the plasmid to maximise combinations. Once randomly assorted, one plasmid is transformed into E. coli, which results in random combination of expression. Each colony will have a randomly selected expression level, similar to a random number generator. This process ensures that bacterial colony used as a key have unique characteristics increasing safety of Key. coli security system.

Random Brick Formation (components of plasmid)

Each individual Promoter-Reporter-Terminator brick contains interchangeable parts. The three parts are linked together with Bsa1 sites so that there is no preference for any part when ligating together. This allows randomness to be added later. This method is used also for the construction of Promoter-gRNA-Terminator bricks so that this could be randomised in the future. The bricks are then flanked by a prefix and suffix, and these are flanked by restriction sites ABCD on either end. Digestion of bricks with A+B, B+C, and C+D allows any brick to be placed in any position within the plasmid but it would be pre-determined. This means that the no one promoter-reporter-terminator brick would be limited to one specific place in the plasmid, which allows another level of randomness in assembly as we would not know which reporter was being placed where, which could also affect expression levels.

The arrangement of the DCBA restriction system means that any brick can be placed in any position in the plasmid which allows expansion of possibilities whilst maintaining randomness of insertion later. Bricks can be joined together via amplifying each randomly assembled brick through common amplification sites and then cutting them using a set of restriction enzymes which give each plasmid a specific order of bricks, depending on which are cut and then ligated together.