Team:DTU-Denmark/Vision
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'''Advantage 4''': The two small RNA components of the system potentially provides the ability to fine-tune gene expression levels. Because the natural system indicates a large dynamic range. Thus the proposed system could provide explicit '''control''' of gene expression over a large dynamic range. | '''Advantage 4''': The two small RNA components of the system potentially provides the ability to fine-tune gene expression levels. Because the natural system indicates a large dynamic range. Thus the proposed system could provide explicit '''control''' of gene expression over a large dynamic range. | ||
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== Examples of application == | == Examples of application == |
Revision as of 11:44, 21 September 2011
Vision
Contents |
Problems of modern biology
Problem 1: Gene silencing in bacteria is fundamental to basic and applied research. Gene knockouts are widely used to investigate gene function, but have severe limitations. Gene knockout requires technology which is only developed for a handful of organisms and can only be used to study non-essential genes. Following the explosion of known bacterial species new paradigms for research on genes are needed.
Problem 2: In bioengineering gene expression is often regulated by inducible promoters, but there exist only a few good inducible promoters. Most of these promoters are leaky, they express genes in the off state, and laborious chromosomal engineering is needed to place a promoter in front of natural genes.
The idea
Gene silencing and induction could be improved by developing a system inspired by chitobiose regulation in E. coli. The system provides an engineering framework for gene silencing by utilizing trans-acting RNA regulation.
Chitobiose regulation contains unique and very interesting features. A small RNA regulates gene expression analogously to the highly versatile miRNAs of eukaryotes. The RNA selectively aims and facilitates the degradation of the mRNA of a target gene. A short sequence confers specificity enabling to genetically engineer the system to target any gene.
In addition the chitobiose regulations system contains a second small RNA that regulates the first, enabling highly advanced schemes of regulation such as pulses. Having two small RNA components significantly increases the options to fine-tune an control gene expression. In conclusion the system has the potential to be a universal tool for easy and specific gene silencing or in other words a "Swiss army knife" of gene silencing.
Advantages of proposed system
Advantage 1: The proposed system could be superior to commonly used solutions for gene silencing by being easy to use. The system can simply be designed in silico, synthesized and transformed into cells to silence any gene of interest.
Advantage 2: Another advantage is increased area of applicability. Any organism that is sequenced, contains Hfq and have known stable plasmids can be subject gene silencing. A criteria which is satisfied for most bacteria.
Advantage 3: The proposed system is complementary to promoter based regulation and multiple systems can coexists without interfering. Thus the system provides a high degree of flexibility in designing experiments as well as biological circuits.
Advantage 4: The two small RNA components of the system potentially provides the ability to fine-tune gene expression levels. Because the natural system indicates a large dynamic range. Thus the proposed system could provide explicit control of gene expression over a large dynamic range.
Examples of application
Although the number of possible applications is huge we have chosen two, to show how this can be used in two very different settings: a basic research project and a huge industrial protein production.
Knocking down essential genes and transcription factors
Knocking out essential genes is difficult since they are needed by the cells and thus cannot simply be removed from the chromosome.
Knocking down transcription factors (TF's) is also problematic, although not necessarily lethal. It usually changes the phenotype of the cells. Chromosomally engineering a TF promoter will only show you the steady state achieved under these new conditions, and not the process of getting to the steady state.
One way to overcome this problem could be to place an repressible promoter in front of the gene, and only knock it out when needed. This has a number of issues. It involves chromosomally engineering the bacteria in question and thus changing the wild-type. Secondly, it is disruptive to remove the repressor once it has been added to the medium.
We propose a simple solution using our system:
Place the sRNA under control of the tetracycline promoter, and the trap-RNA under the control of the arabinose promoter. Target the sRNA to the TF or essential gene. When you want to knock down (or down-regulate) the gene, add anhydro-tetracycline. But when you want to restore function add arabinose. Should you want to disrupt once more, add glucose.
With one single transformation you get complete control over the genes. You can knock them down down-regulate them and restore them in whatever temporal manner you desire.
Production of a (toxic) protein
When using bacteria for production of proteins on an industrial scale, a tradeoff is often required. One can use a strong promoter to express a protein, which leads to the increase of the production per cell, but may lead to a lower cell density as well. Lowering the strength of the promoter has the opposite effect: productivity drops, but cell density rises. This is especially true for proteins that are toxic to the cells.
Another problem is that cells using the majority of their energy on protein production is under a heavy metabolic load, and tend to grow slowly.
To overcome this some experiments have been done using inducible promoters, which were only induced once the cell density is high. The downside is that these inducers are often expensive making the process unfeasible.
We propose another solution: place the gene of interest under the control of a strong constitutive promoter, constitutively express the small RNA, so the mRNA of the protein is degraded, and place the trap-RNA under the control of a stationary phase promoter. While growing, no energy will be wasted on producing protein, and upon reaching stationary phase the production of protein will be fully induced. The stationary phase promoter need not to be strong since even small amounts of the trap-RNA can degrade the sRNA.
In this way the production of protein will only be active when it is most effective, without using expensive inducers.