The idea how to make bacteria which could be used as a security system evolved over several weeks. One of the first ideas how it could be achieved was the use of transposons. Transposable elements are DNA fragments that can change its position within a genome. This creates mutations resulting in different levels/suppression of expression of certain genes. The use of transposons would yield bacteria with various phenotypes, which could be used in Key. coli security system. A target site-specific Tn7 transposon could be used for this purpose. This bacterial mobile DNA segment inserts at high-frequency into a single specific site, called attTn7 in E. coli. However, further research revealed several disadvantages of this idea. The mechanisms of Tn7 recombination are complicated are requiring large number of proteins to work. Although we thought this would be interesting to test, we came across 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. The next idea to obtain different phenotypes of bacteria was the use of RNA interference. In this process RNA molecules inhibit gene expression. It is possible thanks to siRNA molecules which target specific mRNA strands. After that protein complex is formed and it breaks down mRNA preventing its translation into protein. An extensive literature search revealed that the CRISPR interference system seems to be more reliable and predictable than RNAi so we decided to use it.

The main difference between CRISPRi and RNAi is the level on which they control protein expression. RNAi regulates this on the translational level by interfering with mRNA and CRISPRi influences gene expression primarily at the transcriptional level.

CRISPRi allows sequence-specific control of gene expression. This method utilizes CRISPR pathway and catalytically inactive Cas9 (dCas9) protein, as well as single guide RNA (sgRNA), which are specific to chosen DNA regions. 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. dCas9 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. The complex consisting of dCas9 and sgRNA binds to complementary DNA and represses the expression of target genes by blocking the elongation by RNA polymerase. 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.