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BACKGROUND
Advances in our understanding of genetics and ability to manipulate gene expression have expanded the field of synthetic biology quite dramatically over the years. As a result, an increasing amount of people are looking at synthetic biology as a means of achieving a variety of objectives. For example, the field provides a basis for research on gene expression, an efficient way to biosynthesize materials of desirable qualities, potential solutions to environmental issues, and a multitude of other applications. Essentially, a world of possibilities can be found right at our micropipette tips in synthetic biology.
INTRODUCTION
As the field of synthetic biology expands, there exists considerable public concerns over the possibility of genetically modified organisms (GMOs) escaping from the laboratory setting and into the outside environment. If the GMO has a selective advantage over the species naturally present in the environment, it can potentially wreak havoc upon the ecosystem it escapes into. In addition to this, many people are afraid of the possibility that a GMO in the environment may have negative consequences for public health. In order to counter these fears and concerns and increase public support for synthetic biology, our project’s goal is to develop a “kill switch” device that activates cell apoptosis when a cell escapes the laboratory setting.
The switch will be activated through exposure to UV radiation and the cause of death will be the protein expression of ccdB - which is a naturally occurring toxin that interferes with vital processes such as DNA replication and RNA transcription. In other words, the device will utilize inducible promoters that respond to exposure to UV radiation as well as the ccdB coding gene. To control the expression of ccdB, a riboregulators that consists of a cis-repressive sequence and a trans-activating sequence will be utilize.
RIBOREGULATORS
Riboregulators are system composed of RNA that regulates expression of itself or another nucleic acid in response to a signal. The cis-repressive and trans-activating system is a system engineered by Farren J Isaacs and co, described in “Engineered riboregulators enable post-transcriptional control of gene expression”, that allows for post-transcriptional regulation in Escherichia coli by silencing or activating gene expression.
Under normal prokaryotic gene expression, a promoter (P) drives the expression of a gene and generates a messenger RNA (mRNA) with a ribosome binding site (RBS). Subsequently, a ribosome docks onto the RBS of the mRNA and initiates translation of a functional protein.
Taken from “Engineered riboregulators enable post-transcriptional control of gene expression” by Farren J. Isaacs, Daniel J. Dwyer, Chunming Ding, Dmitri D Pervouchine, Charles R. Cantor, & James J. Collins in Nature Biotechnology.
Under the cis and trans riboregulators system, however, a short cis repression (cr) sequence that is complementary to the RBS is inserted between the promoter and RBS coding region. The promoter and cis repression sequence is referred to as Pcr. With the Pcr, transcription of the gene generates an mRNA with a hairpin formation at the 5’ end. This hairpin prevents ribosomes from binding onto the RBS, thereby blocking protein expression. This cis-repressed RNA (crRNA) can be activated with a trans-activating RNA (taRNA) sequence. The taRNA is short noncoding RNA that targets crRNA with high specificity and whose transcription from DNA is facilitated by a promoter that is referred to as Pta.
When both crRNA and taRNA are present, a linear loop interaction occurs that exposes the RBS. As a result, ribosomes can now dock on it and allow for the expression of a functional protein. The figure shown reveals the basic steps of normal prokaryotic gene expression in the dotted box and the cis-repression and trans-activation riboregulators at work with green fluorescent protein (GFP) as the gene of interest.
