Team:StuyGem NYC/Project

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        <h3> INTRODUCTION </h3>

            <p> 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. </p> 

      <h3> WHY A KILL SWITCH? </h3>
       <p> 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 death when a cell escapes the laboratory setting. </p>
 
        <h3>RIBOREGULATORS</h3>
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            <p>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.<a href = "#one"><sup>[1]</sup></a></p>
            <p>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. </p>
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            <img src="https://static.igem.org/mediawiki/2014hs/6/6d/RIBOREGULATORS.jpg" />
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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. 
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        <p>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.  </p>
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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.
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        <h3>THE KILL GENE: ccdB</h3>
 
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        <p>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. <a href = "#two"><sup>[2]</sup></a></p>
 
        <p>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 <a href = "#three"><sup>[3]</sup></a>. As a result, our choice of poison is none other than the ccdB gene. </p>
 
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        <h3>THE Pta PROMOTERS: UV & recA</h3>
 
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        <p>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.  </p>
 
        <p> 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.</p>
 
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        <h3>THE CIS-REPRESSED CONSTRUCT</h3>
 
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        <p>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:</p>
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            <img src="https://static.igem.org/mediawiki/2014hs/a/ac/T7_crRNA_ccdB.PNG" />
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<img src="https://static.igem.org/mediawiki/2014hs/8/8b/Ptet_crRNA_ccdB_dterm.PNG" />
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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.
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        <p> 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:</p>
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<img src="https://static.igem.org/mediawiki/2014hs/b/bd/T7_crRNA_GFP_Translational_Unit.PNG" />
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<img src="https://static.igem.org/mediawiki/2014hs/5/57/PTet_crRNA_GPF_Translational_Unit.PNG" />
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The two cis-repressed constructs with the GFP translational unit as the gene of interest and the respective constitutive promoters. 
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        <h3>THE TRANS-ACTIVATING CONSTRUCT</h3>
 
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        <p>The taRNA coding sequence will be placed under the control of the UV and recA promoter as stated above. It is to be noted that the taRNA sequence available to us had two double terminators attached to the end of it already, allowing for more accurate transcription of taRNA. The constructs will look as follows: </p>
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<img src="https://static.igem.org/mediawiki/2014hs/3/32/UV_taRNA_dterm.PNG" />
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<img src="https://static.igem.org/mediawiki/2014hs/6/6e/RecA_trRNA_dterm.PNG" />
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The trans-activating construct under two different promoters. There is a double terminator at the end of the taRNA sequence because the part was only available in that form.
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        <h3>THE “SIMPLE DEVICE”</h3>
 
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        <p>Now it may be asked why we did not simply place the inducible promoter right in front of the ccdB gene, thereby avoiding the entire crRNA and taRNA scenario and giving us a “simple” device. Our hypothesis is that the cis-repressive and trans-activating system will be better since riboregulators allow for more control of expression. In order to test this hypothesis, we will be comparing the efficiency of the two system using ccdB and the GFP translational unit as the genes of interest. The “simple devices” we will be examining will be as follows:</p>
<img src="https://static.igem.org/mediawiki/2014hs/c/cd/UV_ccdB.PNG" /><img src="https://static.igem.org/mediawiki/2014hs/4/47/RecA_ccdB.PNG" />
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<img src="https://static.igem.org/mediawiki/2014hs/3/39/UV_GFP_Translational_Unit.PNG" /><img src="https://static.igem.org/mediawiki/2014hs/b/bf/RecA_GFP_Translational_Unit.PNG" />
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The constructs above that will be compared with those in the cis-repressive and trans-activating system. The four constructs are each composed of the target gene (either ccdB or GFP translational unit) downstream of an inducible promoter. Effectively, these constructs are “simpler” versions of their riboregulator counterparts. 
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        <h3>ALTERNATIVES: THE ARABINOSE DEATH SWITCH</h3>
 
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        <p>The beauty about death switches is that there is more than one way to design one. As a result, we have decided to design another death switch still using ccdB as the kill gene. However, the condition for cell apoptosis will be the lack of arabinose in the surrounding. The death switch we came up with is a bit more complicated than the cis-repressive and trans-activating system as it involves two repressor for a less leaky system (meaning ccdB is not expressed when it is not supposed to) that is based off the naturally occurring arabinose operon. To fully understand this device, let us examine the way the device is designed.  </p>
<img src="https://static.igem.org/mediawiki/2014hs/7/72/AraC_complex.PNG" />
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<img src="https://static.igem.org/mediawiki/2014hs/c/c3/PCI_complex.PNG" />
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A kill switch based off of the naturally occurring arabinose operon. The parts used are all available as BioBricks in the iGEM registry. It is to be noted that the GFP portion of the device serve no use in this design. It was originally intended to show a successful ligation of a promoter upstream of it, but the pAraC promoter initiates the expression of araC in the reverse direction so GFP is not expressed.  
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<p>In the device, a promoter for araC (pAraC) initiates the expression of the protein araC. On its own, araC is a repressor for pBAD promoter, preventing the expression of Cubitus interruptus (CI). CI is a transcription factor that would repress the pCI promoter. However, without CI, pCI functions uninhibited and codes for ccdB, thereby killing off the cell. At least, this will be what occurs in a non-laboratory setting. In the laboratory setting, scientists can add L(+) arabinose to the media used to grow the bacteria that contains the kill switch described. L(+) arabinose is a commonly occurring form of the monosaccharide arabinose that is harmless and quite easy to obtain. With the presence of L(+) arabinose (a concentration of 0.001% to 0.02% is more than sufficient), L(+) arabinose will bind onto araC and change its conformation <a href = "#four"><sup>[4]</sup></a>. As a result, the pBAD promoter is no longer being repressed, so CI is now expressed. With the expression of CI, the pCI promoter is subsequently repressed so that ccdB is no longer expressed. Thus, the arabinose death switch will be extremely effective as long as scientists add L(+) arabinose to media in the laboratory setting.  </p>
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REFERENCES
<a name = "one">[1] Isaacs, Farren J., Daniel J. Dwyer, Chunming Ding, Dmitri D. Pervouchine, Charles R. Cantor, and James J. Collins. "Engineered Riboregulators Enable Post-transcriptional Control of Gene Expression." Nature Biotechnology22.7 (2004): 841-47. Web.</a><br>
<a name = "two">[2] "Action of DNA Gyrase." Molecular Biology . McGraw HIll, n.d. Web. 15 June 2014.</a><br>
<a name = "three">[3] Wikipedia contributors. "CcdA/CcdB Type II Toxin-antitoxin system." Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia, 14 May. 2014. Web. 15 Jun. 2014.</a><br>
<a name = "four">[4] Herbring, Valentina, Sebastian Palluk, and Andreas Schmidt. "Part:BBa_K808000 - AraC-Pbad - Arabinose Inducible Regulatory Promoter/repressor Unit." Part:BBa K808000. IGEM, 11 Aug. 2012. Web. 19 June 2014.</a>
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