Design Process

Although our project is applied in the idea of Key. coli, we are actually looking at creating a general system for creating combinations of metabolites and reporters. We knew that we wanted to design a method where an expandable range of products can be expressed at various levels in order to create a large amount of combinations with randomness coming from the assortment of these promoters to different reporters.

Initially, we thought of the idea of using transposons to shuffle different strength promoters to assort them randomly to various reporters to give various levels of products. EXPLAIN IDEA HERE. Although we thought this would be really interesting to test, we came accross difficulties with sourcing the TsnD subunit of the Tn7 transposon. As expression and purification of this subunit was not realistic within the timeframe we had so we decided a new method would be needed.

Next we looked at using RNAi or CRISPRi. After a literature search, CRISPRi seemed to be more reliable and predictable than RNAi so we decided to use it. We came up with an idea that a variety of reporters within a plasmid could be under the control of promoters which can be targeted by guide RNAs. CRISPR interference (CRISPRi) uses dCas9 which is a nuclease-deficient enzyme that uses the RNA-guided DNA binding of Cas9 but represses expression by interfering with RNA polymerase binding instead of cutting the DNA. By linking gRNA targeted promoters up to the genes for various reporters we can control the level of expression of these reporters by providing a targeting or non-targeting gRNA to give an ON/OFF switch.

Proof of concept

Initially we will 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.

Each individual Promoter-Reporter-Terminator brick contains interchangable 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-sgRNA-Terminator bricks so that this could be randomised later on. 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.

Future work

We can think of a few ways by which we could expand the possible combinations for our system:

Introducing more possible reporters/products into the bricks

Characterising more and more promoter-gRNA combinations

Using gRNAs with single point mutations in the seed region which could give different levels of repression