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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.
THE KILL GENE: ccdB
The gene that will be cis-repressed in our kill switch is the ccdB gene. The ccdB gene is part of a naturally occurring toxin-antitoxin system in which the ccdB gene produces a toxin and a ccdA gene produces an antitoxin. ccdB acts as a toxin by interfering with processes that involves DNA helicase, such as DNA replication and RNA transcription. During these processes, DNA helicase unwinds DNA and creates positive supercoils on DNA. When left alone, the supercoils slow down helicase activity or destroy the DNA itself. Another enzyme called DNA gyrase helps to relieve the tension caused by the positive supercoiling by breaking apart the double strand, adding a negative supercoiling, and rejoining the strands of DNA. When gyrase and helicase are together, gyrase acts ahead of helicase so that the positive supercoils can be neutralized before problems arise.
ccdB is able to disrupt helicase activity by binding onto the GyrA subunit of gyrase and messing up its protein structure. This binding causes the gyrase to stay clamped onto the DNA after it cleaves it. As a result, it can no longer add negative supercoils onto the positively supercoiled DNA. Thus, the cell can no longer perform the vital processes of DNA replication and RNA transcription. ccdA acts as an antitoxin by binding onto ccbD, allowing GyrA to be freed up and restoring the gyrase’s protein structure. However, if the ccdA gene is not present, then the ccdB will act on GyrA unhindered, ultimately causing cell death. [3] As a result, our choice of poison is none other than the ccdB gene.
THE Pta PROMOTERS: UV & recA
In order to control the expression of taRNA, the gene coding for it will be placed downstream of an inducible promoter. Since the condition for cell apoptosis in the death switch is exposure to UV radiation, the inducible promoter would have to be one that is related to this condition. As a result, the promoters of choice are the UV and recA promoter. The UV promoter, as the name implies, is a promoter that is induced by the presence of UV radiation, making it a very straight forward choice for the kill switch.
The recA promoter, on the other hand, does not respond directly to UV radiation. Instead, it responses to the presence of single stranded DNA (ssDNA). ssDNA is in abundance when DNA gets damaged. Since UV radiation, especially short UV radiation, causes damage to DNA, the recA promoter is also a suitable promoter for the expression of taRNA.
THE CIS-REPRESSED CONSTRUCT
The ccdB gene will be placed downstream of the crRNA sequence. This will subsequently be placed under the control of a constitutive promoter – which is constantly turned on. As a result, an inactive form of the ccdB mRNA will be produced constantly. This will allow for rapid cell death once the mRNA is activated by the taRNA and is expressed as a functional ccdB protein. One of the constitutive promoters of choice is the T7 promoter, which can produce high levels of transcription in E. coli. The other constitutive promoter is the pTet promoter, which is repressed by the presence of tetracycline repressor protein (TetR) (TetR is not present within our system so pTet is effectively always on). The two constructs will look as follows:
The two cis-repressed constructs with ccdB as the gene of interest and the respective constitutive promoter. It is to be noted that the construct containing pTet contains a double terminator because it was synthesized that way to ensure that transcription stops right after RNA polymerase passes the gene coding for ccdB.
Additionally, we will be placing a GFP translational unit downstream the T7 and pTet promoter and crRNA sequence. The reasoning for this is that this construct would allow us to characterize the efficiency of the cis-repressed and trans-activating constructs. The expression of the ccdB protein will end up killing the cell once the crRNA is activated by the taRNA; however, GFP will not have this effect and has the added benefit of an observable effect, making it ideal for characterization purposes. The construct will be similar to the one above and is as follows:
