Preliminary approaches

Riboregulator

In bacteria, several factors affect translation initiation, including ribosomal recognition of the mRNA’s RBS and the start codon (AUG). Recognizing the importance of RNA interactions between the ribosome and RBS, and based on work on endogenous riboregulators, Isaacs et al. [8] sought to regulate bacterial gene expression by interfering with ribosomal docking at the RBS. From the outset, their objective was to create a modular post transcriptional regulation system that could be integrated into biological networks and implemented with any promoter or gene.

Figure 10. taRNA/RBS (riboregulator) regulated expression of protein 3. Genes 1 and 2 are on the conjugative plasmid present in all cells. S1: signal molecule. Gene 3 is present on the SDP. RBS: ribosome binding site. Cr: piece of RNA strand in front of RBS that is complementary to RBS. taRNA: trans-activating RNA, could also be small interference RNA, as long as it is complementary to cr. Product 3: Could be either GFP/luciferase (for testing) or endonuclease.

Either gene 1 or gene 2 is constitutively expressed (never both!) where the other is inducible. In the picture, gene 2 is constitutive and gene 1 is inducible by signal molecule S1. Gene 2 expresses constitutively the crRNA, on which the cr part of the RNA binds the RBS directly after transcription, causing RBS to be blocked for the ribosome and therefore blocks the RNA from translation. When the promoter of gene 1 is induced by S1, it produces the taRNA molecule which is complementary to the cr part of the crRNA. When the taRNA binds the crRNA it opens the RBS, which makes the crRNA ready for translation. The resulting product (the green folded protein 2) can then induce gene 3 to produce end-product 3.

Small interfering RNA

This approach is based on the gene silencing by small interfering RNA [9] and is given in the following scheme.

Figure 11. Genes 1 and 3 are present in the self destructive plasmid. Gene 2 is present in the cell and is always produced.

Protein 1 is always produced in the cell and is controlled by the constitutive promoter on gene 2. This protein must be selected carefully since the gene 1 producing the siRNA must be decided based on this protein and the siRNA must be specific for silencing the mRNA of protein 1 without disturbing the other mRNAs of the host organism. The protein 1 will be in excess concentration and hence will inhibit the production of protein 2 (restriction enzyme or fluorescence protein) by repressing the promoter of gene 3. When the production of siRNA is induced by activating the promoter of gene 1, on reaching a threshold it will interfere with the translation of protein 1 and hence the repression of gene 3 is released to produce the protein 2.

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