The Problem

The most common way to regulate gene expression in use today is by regulatable promoters. These are placed in front of a gene of interest and lets you express the gene when a specific condition is met in the media, eg. a sugar is present.

This method, though usefull, has a number of limitations. There are a limited number of "good" regulatable promoters, they are often "leaky" that is transcribed in the off-state, and most importantly extensive chromosomal engineering is needed to bring genes that are naturally unregulated under regulation.

The idea

We believe it is possible to overcome these limitations using a system found in E. coli, namely the chitobiose system, as an engineering framework for trans-acting RNA regulation. Since previous work on small RNAs in bacteria show that it is possible control the target of a small RNA by changing its sequence ???CITE???, this system could act as a universal tool for easy and specific gene silencing, both outside and within the Biobrick standard.

The chitobiose system in E. coli is regulated in a highly interesting way: A small RNA regulates the expression in a manner that is conceptually similar to the highly versatile miRNAs of Eukaryotes. The RNA selectively targets and facilitates the degradation of the mRNA of its target gene. The region used for targeting is easily identified upon inspection of the small RNA sequence, as it is highly similar to the targeted Shine-Dalgarno.

As an added bonus this system would work in addition to any previously employed promoter based regulation, and due to the existence of a second small RNA to regulate the first, highly advanced schemes of regulation such as pulses can be achieved.

In short we believe this to be a "Swiss army knife" of gene silencing.

The advantages

There are many reasons for using our system instead of, or in addition to, the more commonly used solutions. Here are a few:

The biggest reason is ease of use: Design the system in silico, order it and transform it. Done... You have now knocked down a gene.

This can ease engineering of other systems. Picked a promoter that is too strong? Use our system, and only induce the small RNA a little, thus lowering the amount of mRNA without knocking it completely out. Thus you can fine-tune a system that has already been designed, or you can just design your system with the strongest promoter there is, and use our system to tune it.

Since the small RNA (as indicated by the name) is small, this is not a big expense.

The second big reason is area of applicability. Any organism that is:

• Sequenced
• Has known stable plasmids
• Has hfq
• Is transformable

Is a potential target for our system. This includes many organisms that are difficult or impossible to chromosomally engineer.

Within the controlled environment of Biobricks the possibilities become even greater. Design your target gene with a unique Shine-Dalgarno and you can target just that gene, or use the same Shine-Dalgarno for all you genes and turn off your entire construct at once.

For the scientists there are a few more advantages: You can easily knock down genes in the middle of a gene, you can temporarily knock down essential genes and transcription factors. And all of this without changing the chromosome, your strain remains wild-type up until the moment you knock down the targeted gene.

The trap-RNA system can target any gene

The trap-RNA system provides unique flexibility for gene silencing in prokaryotes enabling control and tuning of gene expression. The specificity of the system depends on base pair complementarity. Therefore it can be designed to target any gene of interest by simply altering the sequences to match the target gene. Furthermore multiple trap-RNA systems can applied to the same biological circuit without interfering. Implementing sRNA and the trap-RNA into biological constructs they can be introduced by constitutive promoters or inducible promoters.