Team:Toronto/Project

From 2014.igem.org

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<h1><span>Design</span></h1>
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<h4>Design criteria for plasmid loss genetic safeguard systems</h4>
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<p>
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An effective plasmid loss genetic safeguard system will need to meet the following three design criteria:
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<ol>
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<li>
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Ability to inactivate/destroy plasmid, or increase rate of plasmid loss
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<ul>
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<li>If the efficiency of plasmid loss is low, the system will not be effective at biocontainment </li>
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</ul>
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</li>
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<li>
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Does not affect cell viability.
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<ul>
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<li>If the mechanism causes death or impairs the cell's ability to grow and reproduce, then it is no different from a kill switch.</li>
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</ul>
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</li>
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<li>
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Inducible/repressible expression
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<ul>
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<li>The synthetic organism needs its plasmid so that it can do its intended job. If plasmid loss occurs too early, the synthetic organism would be unusable for its original purpose.</li>
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</ul>
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</li>
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</ol>
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</p>
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<h3>What is CRISPR?</h3>
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<p>
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CRISPR � Clustered Regularly Interspaced Short Palindromic Repeats � is a long name for a characteristic DNA locus that is responsible for the adaptive immunity in most bacteria and in many species of archaea. CRISPRs have been divided into three types (I-III), of which type II is the best understood. A CRISPR type II locus consists of three components: tracrRNA, the Cas genes, and the �array�.
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</p>
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<p>
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<i>Cas gene(s):</i> The �signature gene� of a type II CRISPR system is Cas9, an endonuclease. Its original purpose in bacteria is to degrade foreign DNA.
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</p>
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<p>
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<i>Array:</i> By incorporating bits of foreign DNA into this array, the bacteria gains immunity to it in the future. Those bits, which are called spacers, are the instructions that tell Cas9 where to cut. They are complementary to part of the DNA of a previous invader; the exact sequence it matches is called the protospacer. So, the array follows a pattern of �spacer � repeat � spacer � repeat�, where the repeats are palindromic sequences that aid in identification of the individual spacers. When foreign DNA enters the bacterium, if the bacterium possesses a complementary spacer to it, that spacer is transcribed separately from its surrounding parts as what is called a crRNA.
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</p>
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<p>
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<i>TracrRNA:</i> Binds to repeats to help with transcription of the array to the currently needed crRNA. crRNA + tracrRNA together is called guideRNA (gRNA) and it is this that directs Cas9 to where it needs to cut.
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</p>
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<h3>The viability of CRISPR tech in biosafety systems</h3>
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<p>
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There are have been dozens of solutions proposed to solve the biocontainment problem. The 2012 Paris-Bettencourt iGEM team created <a href="http://parts.igem.org/Biosafety?title=Biosafety">a very useful page</a> that lists, amongst other things, all parts that have been created so far tagged in the �biosafety� category and divides them into 3 types: <b>Semantic Containment</b>, <b>Kill Switch</b>, and <b>XNase</b>.
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</p>
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<h4>Semantic Containment</h4>
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<p>
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The genetic construct can only be �understood� in the synthetic cell and can�t confer its advantages to wild-type population partners (if the construct were to be shared by horizontal gene transfer) because the cell expresses a �nonsense suppressor� i.e. a mutated form of tRNA. Paris-Bettencourt 2012 is the only team to have a created a part of this type.
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</p>
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<p>
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<b>Disadvantages:</b> Prevents the horizontal transfer of synthetic genes but doesn't do anything about the escaped synthetic organisms themselves. Provides an additional line of defence, but may not be enough on its own.
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</p>
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<h4>Kill Switch</h4>
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<p>
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This system boils down to a change in protein expression induced by a specific signal, perhaps a compound like arabinose. That change in protein expression (ie production of a toxic product) kills the organism.
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</p>
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<p>
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Disadvantage: Imposes selective pressure on the population to evolve mutations that disable the kill switch mechanism.
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</p>
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<h4>XNase</h4>
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-->
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<h3>CRISPR as a plasmid loss genetic safeguard</h3>
 +
<p>
 +
The CRISPR system is a suitable mechanism for implementing a plasmid loss genetic safeguard because it can cause plasmid loss within the bacteria cell. In particular, the precise targeting of Cas9 to a specific protospacer makes it possible to target the synthetic plasmid only, without causing damage to the cell's own genomic DNA. This is important because if we harmed the host cell in the process of removing the synthetic plasmid, it would be no different from the kill-switch scenario, where there is a fitness cost on cells to have an intact genetic safeguard.
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</p>
 +
<p>
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Another incidental benefit of using CRISPR results from the ease with which it can retargetted. By changing the spacer, the inducible CRISPR system we are making for our project can be repurposed by future iGEM teams for applications other than biocontainment.
 +
</p>
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 +
<h3>Existing CRISPR BioBrick</h3>
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<p>
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The CRISPR system already exists in the registry as <a href="http://parts.igem.org/Part:BBa_K1218011">BBa_K1218011</a>, a biobricked version of the pCas9 plasmid described in Jiang et al, 2013. pCas9 was designed to allow expression in E. coli of the type II CRISPR-Cas system originally from Streptococcus pyogenes. It contains in one convenient package the minimum essential components to allow the CRISPR/Cas9 system to work, including the tracrRNA, Cas9 gene, and crRNA array. A useful feature of pCas9 is that the spacer in the CRISPR array has been replaced with a placeholder sequence containing BsaI sites, allowing it to be easily swapped out using the <a href="http://www.addgene.org/static/cms/files/Marraffini_pCas9_protocol.pdf">Marrafini lab pCas9 protocol</a> Golden Gate assembly for any desired spacer sequence.
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</p>
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<p>
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However, pCas9 did not fully meet our requirements because its components are constitutively expressed (<a href="http://parts.igem.org/Template:2014_Toronto_pCas9_sources">see emails from Jiang and Marrafini</a>). In addition, the location of the Cas9 gene in the middle of the sequence makes it difficult to swap out the constitutive promoter for an inducible one.
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</p>
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<h3>Re-engineering pCas9</h3>
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<p>
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In order to facilitate the inducible expression of Cas9 while retaining benefits of having the essential components of the CRISPR/Cas9 system in one biobrick, we decided to re-engineer pCas9 by rearranging the order of its components. Our new design (<a href="http://parts.igem.org/Part:BBa_K1559002">BBa_K1559002</a>) puts the Cas9 right at the beginning (upstream end) of the biobrick, so that it is removed from the control of the existing constitutive promoters originally from <i>Streptococcus pyogenes</i>, and is instead exposed to transcriptional control from promoters added upstream of the biobrick.
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</p>
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<p>
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<img src="https://static.igem.org/mediawiki/2014/e/ee/CRISPR_rearrangement.png" width="600"><br>
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<b>Figure 2:</b> A schematic of the rearrangement of pCas9 to expose Cas9 directly to the control of an upstream promoter.
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</p>
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<p>
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Conceptually, this new design could be constructed by dividing the existing pCas9 part <a href="http://parts.igem.org/Part:BBa_K1218011">BBa_K1218011</a> in two sections, one containing the tracrRNA and bidirectional constitutive promoter region (<a href="http://parts.igem.org/Part:BBa_K1559000">BBa_K1559000</a>) and another containing the Cas9 coding sequence and CRISPR array (<a href="http://parts.igem.org/Part:BBa_K1559001">BBa_K1559001</a>). By joining the Cas9-array section to the upstream end of the tracrRNA section, we would have effectively moved Cas9 to the front of the part (see <a href="http://parts.igem.org/Part:BBa_K1559002">BBa_K1559002</a>). The expression of the tracrRNA and crRNA array are unaffected because they have not been moved relative to their original promoters.
 +
</p>
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 +
<h3>Testing Cas9 expression with various promoters</h3>
 +
<p>
 +
In order to test the effect of various levels of Cas9 expression, we planned on putting our reengineered CRISPR/Cas9 system under the control of Anderson constitutive promoters of various strengths
 +
(<a href="http://parts.igem.org/Part:BBa_K1559003">high</a>,
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<a href="http://parts.igem.org/Part:BBa_K1559004">medium</a>,
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<a href="http://parts.igem.org/Part:BBa_K1559005">low</a>).
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</p>
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<p>
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We also planned on testing various inducible promoters
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(<a href="http://parts.igem.org/Part:BBa_K1559006">pBad</a>,
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<a href="http://parts.igem.org/Part:BBa_K1559007">pLac</a>,
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<a href="http://parts.igem.org/Part:BBa_K1559008">pRha</a>)
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with our reengineered CRISPR/Cas9 system.
 +
</p>
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<p>
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One problem encountered by previous iGEM teams (eg: <a href="https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme">Paris Bettencourt 2012</a>) was leaky expression. We will be expressed our CRISPR/Cas9 system in a low-copy plasmid (<a href="BBa_K1559011">modified pTRC99A</a>) to reduce the effect of leaky expression. As an extension to our project, we also planned on testing tet-off (<a href="http://parts.igem.org/Part:BBa_K1061012">BBa_K1061012</a>, <a href="http://parts.igem.org/Part:BBa_K1061013">BBa_K1061013</a>) as a low leaky expression alternative for the inducible control of the CRISPR/Cas9 system.
 +
</p>
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<h3>Assembly Strategy</h3>
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We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:
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<ol type="A">
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<li>biobrick prefix, promoter, and rbs (synthesized as gblock, overlaps with C and B)</li>
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<li>Cas9 and crRNA array (PCR from <a href="http://parts.igem.org/Part:BBa_K1218011">BBa_K1218011</a>)</li>
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<li>tracrRNA and biobrick suffix (synthesized as gblock, overlaps with B and D)</li>
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<li>the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from <a href="http://www.addgene.org/vector-database/4402/">pTRC99A</a>)</li>
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</ol>
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 +
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<h3>CRISPR activity assay</h3>
 +
<p>
 +
We designed a CRISPR activity assay order to measure the activity of our CRISPR/Cas9 system under the control of various constitutive and inducible promoters.
 +
</p>
 +
<p>
 +
This assay involves two plasmids, a �target� plasmid, containing a protospacer and PAM, as well as the CRISPR plasmid itself (promoter, cas9, crRNA array, tracrRNA), with the corresponding spacer sequence inserted into the spacer insertion site of the crRNA array. The two plasmids must have different antibiotic selectable markers.
 +
</p>
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<p>
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In our case, we chose to use <a href="http://parts.igem.org/Part:BBa_J04450">RFP</a> in pSB1C3 as our target plasmid because it has an easily recognizable phenotype (red colonies), as well as a different antibiotic resistance marker (Chloramphenicol resistance) than our CRISPR plasmids (which are Ampicillin resistant). To make our CRISPR plasmids target the RFP plasmid, we found a <a href="http://parts.igem.org/Part:BBa_K1559010">suitable protospacer</a> within the sequence of RFP to insert into the spacer insertion site of our CRISPR plasmids.
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</p>
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<p>
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Summary of the two plasmids:
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<ul>
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<li><i>Target plasmid:</i> constitutively active RFP (red colonies), Chloramphenicol resistance</li>
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<li><i>CRISPR plasmid:</i> Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.</li>
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</ul>
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<p>
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Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid.
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</p>
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<p style="float: right; width: 200px; padding: 10px">
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For target plasmids that do not exhibit a visible phenotype like red fluorescence, the CFU of cells that retained the target plasmid even after successful transformation of the CRISPR plasmid can be determined by plating on both antibiotics (eg Choramphenicol and Ampicillin). This value can then be divided by the CFU of a plate with only the CRISPR plasmid antibioc resistance (eg Ampicillin only) and subtracted from one to determine the rate of CRISPR activity.
 +
</p>
 +
<p>
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In our assay, these target-carrying cells are transformed with our CRISPR plasmid and plated on agar plates containing Ampicillin (the antibiotic marker for the CRISPR plasmid). Colonies growing on the Ampicillin plates result from cells that were successfully transformed with our CRISPR plasmid. Since all of these cells were known to carry the target RFP plasmid at the start of the assay, we can reasonably conclude that any colonies that are no longer red after being successfully transformed with the CRISPR plasmid had lost the target RFP plasmid due to cleavage by the CRISPR/Cas9 system. The percentage efficiency of the CRISPR/Cas9 system can be determined by dividing the CFU of non-red colonies by the total CFU on the Ampicillin plate.
 +
</p>
 +
<p>
 +
In the case of inducible promoters, the assay can be repeated with and without the inducer present in the medium, to determine the �on� and �off� rates of CRISPR activity.
 +
</p>
 +
<p>
 +
<b>Note:</b> Our CRISPR activity assay requires the CRISPR/Cas9 system and the protospacer it targets to be on two different plasmids. However this is not intended to be the actual configuration of our plasmid-loss system. We envision our final product to be a plasmid on which the CRISPR/Cas9 system, controlled by an inducible promoter, and the protospacer targeted by the CRISPR are both present on the same plasmid.
 +
</p>
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<h3>References</h3>
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<ol>
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<li>
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Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 � 239. doi:10.1038/nbt.2508
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</li>
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</ol>
</div>
</div>
</div>
</div>

Revision as of 03:56, 18 October 2014


Background

The need for biocontainment

Synthetic biology has the potential for a variety of applications outside the lab. For example, many biosensor and bioremediation systems developed using synthetic biology entail the release of genetically modified organisms into an outdoor environment.

The potential impact of synthetic organisms on the natural environment is difficult to predict. However, as demonstrated by the devastating impact of invasive species on ecosystems around the world today, the introduction of new organisms into the environment poses significant risks. Thus, the development of reliable means of biocontainment will be essential for synthetic biology to be considered safe for routine use.

Conventional genetic safeguards for biocontainment

Many of the biocontainment systems being developed today are genetic safeguards that work by conditionally expressing a gene that leads to the host cell's death (Moe-Behrens et al. 2013). Such systems are called "kill switch" systems.

The problem with kill switches

At first glance, the use of kill switches seems to be a simple and effective solution to the problem of biocontainment. However, when considered from an evolutionary perspective, kill switches may not be as effective in practice as they are conceptually. There are two related issues with kill switches: mutation and selection

Mutation

Unfortunately organisms aren't as well behaved as machines. You may have a kill switch set up to contain your synthetic organisms, but chances are, there are going to be some cells that have mutations that break your kill switch. There has been evidence that in many implementations of kill switch systems, a small (but not insignificant) proportion of individuals in a population are able to survive the activation of the kill switch due to spontaneous mutations that render the kill switch ineffective (Knudsen and Karlstrom, 1991). These cells are not going to get killed when you turn on the toxic gene, so they could escape and have negative consequences.

Selection

The other problem with kill-switches is that they actually encourage mutations that inactivate kill switches. Since kill-switches kill off cells that have working kill switches, they impose selective pressure on population to evolve resistance to the kill switch. You're essentially saying to the cells: "mutate or die", and the cells that end up mutating are going to be the ones that eventually dominate the population.

A new approach: Plasmid loss genetic safeguard system

The way we've approached this issue is to make synthetic organisms lose their synthetic plasmids, instead of killing them outright. When we turn on a plasmid-loss system, most individuals in a population of synthetic organisms would lose the genes responsible for the engineered trait and revert to a wild-type genotype and phenotype. As in the kill-switch systems, a small proportion of individuals could still retain the engineered trait due to mutation inactivation of the genetic safeguard system. However, while we may not be able to get rid of mutation, we can remove the selective pressure on the non-mutants so that the mutants can't dominate the population.



Figure 1: The effect of (Top) kill-switch and (Bottom) plasmid loss genetic safeguard systems on a hypothetical population of synthetic organisms, containing a small proportion of mutant individuals resistant to the effect of the genetic safeguard. Not shown: the effect of genetic drift on the mutant sub-population over time.

Although resistant mutants are present in both the kill switch and plasmid loss systems, their evolutionary outcomes differ:

  • Kill-switch system: Once the system is turned on, all individuals with an intact genetic safeguard die, leaving a new population consisting entirely of mutant cells resistant to the kill switch. In evolutionary terms, the resistance allele has gone from a very low allele frequency to fixation (100% allele frequency). Since the non-mutants in the population are all dead, the mutant survivors have unimpeded access to all the resources available in the environment and are able to quickly grow and replicate.
  • Plasmid loss system: The allele frequency of the inactivated genetic safeguard does not change. This is because the individuals with an intact genetic safeguard do not die, they simply revert back to the wild-type genotype and phenotype. In the new population, the proportion of resistant individuals remains small due to competition from the majority of wild-type individuals.

The effect of genetic drift

Genetic drift is the effect of random chance on proportions of alleles in a population. In a population containing both mutant and wild type cells, the proportions of the two will fluctuate slightly every generation simply due to random variability. In most cases the effect of genetic drift is small. However, in a situation where there are very few mutants and many wild-type cells, the "luck" of these few mutant cells will have a disproportional effect on the overall proportion of mutants in the population.

In the kill switch scenario, the population of mutant cells increases quickly, so they are no longer susceptible to extinction by genetic drift. On the other hand, in the plasmid loss scenario, the number of mutants is kept low, so there is a greater chance for genetic drift to lead to extinction of the mutant allele.

The big-picture view of biocontainment

Ultimately, as noted by Paris Bettencourt's recommendations on biosafety, no containment system can be perfect.

In the case of our plasmid loss system, It is still possible that the mutant cells, carrying the engineered plasmid, could have enough of a fitness advantage to out-compete the wild-type individuals, who have lost the engineered plasmid. However, this outcome is no worse than the worst-case scenario of kill-switch systems. We expect that this worst-case scenario will be unlikely to occur, since plasmids impose a metabolic burden on their host cells. In addition. Even if the engineered trait do confer a slight fitness advantage to the resistant individuals, it may not be enough of a selective advantage to overcome the effect of genetic drift.

From the perspective of the fault-tree analysis proposed by Paris Bettencourt, our plasmid-loss system is more robust than the previous kill-switch approaches. While kill switches are only one mutation away from failure, our plasmid loss system tolerates mutations and reduces the chance of failure. Because of this, even if our plasmid loss system does not work 100% of the time, it is still an improvement to the overall safety of synthetic biology.

References

  1. Moe-Behrens, G. H., Davis, R., & Haynes, K. A. (2013, 12). Preparing synthetic biology for the world. Frontiers in Microbiology, 4. doi: 10.3389/fmicb.2013.00005
  2. Knudsen, S. M., and Karlstrom, O. H. (1991). Development of efficient suicide mechanisms for biological containment of bacteria. Appl. Environ. Microbiol. 57, 85:92.
    1. Design

      What is CRISPR?

      CRISPR � Clustered Regularly Interspaced Short Palindromic Repeats � is a long name for a characteristic DNA locus that is responsible for the adaptive immunity in most bacteria and in many species of archaea. CRISPRs have been divided into three types (I-III), of which type II is the best understood. A CRISPR type II locus consists of three components: tracrRNA, the Cas genes, and the �array�.

      Cas gene(s): The �signature gene� of a type II CRISPR system is Cas9, an endonuclease. Its original purpose in bacteria is to degrade foreign DNA.

      Array: By incorporating bits of foreign DNA into this array, the bacteria gains immunity to it in the future. Those bits, which are called spacers, are the instructions that tell Cas9 where to cut. They are complementary to part of the DNA of a previous invader; the exact sequence it matches is called the protospacer. So, the array follows a pattern of �spacer � repeat � spacer � repeat�, where the repeats are palindromic sequences that aid in identification of the individual spacers. When foreign DNA enters the bacterium, if the bacterium possesses a complementary spacer to it, that spacer is transcribed separately from its surrounding parts as what is called a crRNA.

      TracrRNA: Binds to repeats to help with transcription of the array to the currently needed crRNA. crRNA + tracrRNA together is called guideRNA (gRNA) and it is this that directs Cas9 to where it needs to cut.

      CRISPR as a plasmid loss genetic safeguard

      The CRISPR system is a suitable mechanism for implementing a plasmid loss genetic safeguard because it can cause plasmid loss within the bacteria cell. In particular, the precise targeting of Cas9 to a specific protospacer makes it possible to target the synthetic plasmid only, without causing damage to the cell's own genomic DNA. This is important because if we harmed the host cell in the process of removing the synthetic plasmid, it would be no different from the kill-switch scenario, where there is a fitness cost on cells to have an intact genetic safeguard.

      Another incidental benefit of using CRISPR results from the ease with which it can retargetted. By changing the spacer, the inducible CRISPR system we are making for our project can be repurposed by future iGEM teams for applications other than biocontainment.

      Existing CRISPR BioBrick

      The CRISPR system already exists in the registry as BBa_K1218011, a biobricked version of the pCas9 plasmid described in Jiang et al, 2013. pCas9 was designed to allow expression in E. coli of the type II CRISPR-Cas system originally from Streptococcus pyogenes. It contains in one convenient package the minimum essential components to allow the CRISPR/Cas9 system to work, including the tracrRNA, Cas9 gene, and crRNA array. A useful feature of pCas9 is that the spacer in the CRISPR array has been replaced with a placeholder sequence containing BsaI sites, allowing it to be easily swapped out using the Marrafini lab pCas9 protocol Golden Gate assembly for any desired spacer sequence.

      However, pCas9 did not fully meet our requirements because its components are constitutively expressed (see emails from Jiang and Marrafini). In addition, the location of the Cas9 gene in the middle of the sequence makes it difficult to swap out the constitutive promoter for an inducible one.

      Re-engineering pCas9

      In order to facilitate the inducible expression of Cas9 while retaining benefits of having the essential components of the CRISPR/Cas9 system in one biobrick, we decided to re-engineer pCas9 by rearranging the order of its components. Our new design (BBa_K1559002) puts the Cas9 right at the beginning (upstream end) of the biobrick, so that it is removed from the control of the existing constitutive promoters originally from Streptococcus pyogenes, and is instead exposed to transcriptional control from promoters added upstream of the biobrick.


      Figure 2: A schematic of the rearrangement of pCas9 to expose Cas9 directly to the control of an upstream promoter.

      Conceptually, this new design could be constructed by dividing the existing pCas9 part BBa_K1218011 in two sections, one containing the tracrRNA and bidirectional constitutive promoter region (BBa_K1559000) and another containing the Cas9 coding sequence and CRISPR array (BBa_K1559001). By joining the Cas9-array section to the upstream end of the tracrRNA section, we would have effectively moved Cas9 to the front of the part (see BBa_K1559002). The expression of the tracrRNA and crRNA array are unaffected because they have not been moved relative to their original promoters.

      Testing Cas9 expression with various promoters

      In order to test the effect of various levels of Cas9 expression, we planned on putting our reengineered CRISPR/Cas9 system under the control of Anderson constitutive promoters of various strengths (high, medium, low).

      We also planned on testing various inducible promoters (pBad, pLac, pRha) with our reengineered CRISPR/Cas9 system.

      One problem encountered by previous iGEM teams (eg: Paris Bettencourt 2012) was leaky expression. We will be expressed our CRISPR/Cas9 system in a low-copy plasmid (modified pTRC99A) to reduce the effect of leaky expression. As an extension to our project, we also planned on testing tet-off (BBa_K1061012, BBa_K1061013) as a low leaky expression alternative for the inducible control of the CRISPR/Cas9 system.

      Assembly Strategy

      We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:
      1. biobrick prefix, promoter, and rbs (synthesized as gblock, overlaps with C and B)
      2. Cas9 and crRNA array (PCR from BBa_K1218011)
      3. tracrRNA and biobrick suffix (synthesized as gblock, overlaps with B and D)
      4. the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from pTRC99A)

      CRISPR activity assay

      We designed a CRISPR activity assay order to measure the activity of our CRISPR/Cas9 system under the control of various constitutive and inducible promoters.

      This assay involves two plasmids, a �target� plasmid, containing a protospacer and PAM, as well as the CRISPR plasmid itself (promoter, cas9, crRNA array, tracrRNA), with the corresponding spacer sequence inserted into the spacer insertion site of the crRNA array. The two plasmids must have different antibiotic selectable markers.

      In our case, we chose to use RFP in pSB1C3 as our target plasmid because it has an easily recognizable phenotype (red colonies), as well as a different antibiotic resistance marker (Chloramphenicol resistance) than our CRISPR plasmids (which are Ampicillin resistant). To make our CRISPR plasmids target the RFP plasmid, we found a suitable protospacer within the sequence of RFP to insert into the spacer insertion site of our CRISPR plasmids.

      Summary of the two plasmids:

      • Target plasmid: constitutively active RFP (red colonies), Chloramphenicol resistance
      • CRISPR plasmid: Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.

      Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid.

      For target plasmids that do not exhibit a visible phenotype like red fluorescence, the CFU of cells that retained the target plasmid even after successful transformation of the CRISPR plasmid can be determined by plating on both antibiotics (eg Choramphenicol and Ampicillin). This value can then be divided by the CFU of a plate with only the CRISPR plasmid antibioc resistance (eg Ampicillin only) and subtracted from one to determine the rate of CRISPR activity.

      In our assay, these target-carrying cells are transformed with our CRISPR plasmid and plated on agar plates containing Ampicillin (the antibiotic marker for the CRISPR plasmid). Colonies growing on the Ampicillin plates result from cells that were successfully transformed with our CRISPR plasmid. Since all of these cells were known to carry the target RFP plasmid at the start of the assay, we can reasonably conclude that any colonies that are no longer red after being successfully transformed with the CRISPR plasmid had lost the target RFP plasmid due to cleavage by the CRISPR/Cas9 system. The percentage efficiency of the CRISPR/Cas9 system can be determined by dividing the CFU of non-red colonies by the total CFU on the Ampicillin plate.

      In the case of inducible promoters, the assay can be repeated with and without the inducer present in the medium, to determine the �on� and �off� rates of CRISPR activity.

      Note: Our CRISPR activity assay requires the CRISPR/Cas9 system and the protospacer it targets to be on two different plasmids. However this is not intended to be the actual configuration of our plasmid-loss system. We envision our final product to be a plasmid on which the CRISPR/Cas9 system, controlled by an inducible promoter, and the protospacer targeted by the CRISPR are both present on the same plasmid.

      References

      1. Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 � 239. doi:10.1038/nbt.2508