Team:Imperial College London/test7

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<h1>Testing</h1>
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<h1>Main Results</h1>
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<h2>1. Expression of indole 3-acetic acid (IAA) by our modified bacteria</h2>
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<p>Horizontal gene transfer is currently an increasingly pressing topic in the field of Synthetic Biology. It is the main limiting factor which is halting the release of GMO's to the environment as while we know what our genetic construct will do in our own chassis we are unsure what it will do in another organism. In order to prevent this issue of genetic containment we have designed a novel toxin/anti-toxin system, the Gene Guard.</p>
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<h3>Salkowski assay</h3>
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<p>In this module we wanted to first test the necessity of this system by performing experiments to see how long E. coli would retain the plasmid in soil as well as under optimal conditions. Then we tested whether the Gene Guard was assembled correctly at each stage. Finally, we wanted to perform an experiment where we transform control cells and our Gene Guard cells with the holin-endolysin plasmid. Is Gene Guard necessary? Moreover, does it work? Stay tuned to find out.</p>
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<p>We grew up cultures of our IAA-producing <i>E. coli</i> as well as a control culture overnight and performed a colourimetric assay on the cell filtrate in order to determine if there is a difference in the level of IAA. Our results showed that our Auxin Xpress cells produced more IAA than the control (see Figure 1 and 2).</p>
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<div class="technology">1. <i>E.coli</i> survivability in soil and plasmid retainment</div>
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<h3>1.1 Survivability in soil<a href="a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Switch"><img src="https://static.igem.org/mediawiki/2011/5/58/ICL_ProtocolIconDark.png" width="140px" align="right"/></a></h3>
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<img class="border" style="border-color:#B2B2B2;" src="https://static.igem.org/mediawiki/2011/f/f8/ICL_salkowskiAuxinproduction.png" width=500px/>
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<p><i>Figure 1: Results from the Salkowski assay with cell filtrate of IAA-producing </i>E. coli<i> DH5α.(Data by Imperial College London iGEM team 2011).</i></p>
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<p><i>Figure 1: Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil  (Data by Imperial College London iGEM team 2011).</i></p>
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<p><i>Figure 2: Visual results correlating with OD measurements. The eppendorf on the right contains IAA producing </i>E. coli<i> DH5α and the eppendorf on the left contains control </i>E. coli<i> DH5α. (Data by Imperial College London iGEM team 2011). </i></p>
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<h3>Plant response</h3>
 
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<p>We exposed <i>Arabidopsis</i> seedlings to our IAA-producing bacteria to observe their effect on the roots. For this, we used a reporter line that expresses YFP in response to indole 3-acetic acid. As controls, we used a culture of seedlings that was not incubated with any bacteria and a control that was indicated with <i>E. coli</i> not expressing IAA. Fluorescence was brightest in the roots incubated with IAA-producing bacteria (Video 1).</p>
 
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We set up a soil experiment to test how long our <i>E. coli</i> chassis can retain its plasmid in soil. We initially transformed chemically competent <i>E. coli</i>DH5α cells with superfolder GFP. These cells were inoculated on small filter discs (about 0.5 cm diameter), which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of seven weeks.</p>
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<p>After seven weeks, we were able to recover fluorescent bacteria from sterilised and non-autoclaved soil (Figure 1).
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<img class="border" src="http://partsregistry.org/wiki/images/9/9c/Soil_digest_gel.png" width="250" />
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<p><i>Figure 2: Gel digests of bacteria displaying colony morphology typical of <i>E. coli</i> recovered from non-sterilised and sterilised soil. These bacteria exhibited colony morphologies typical of </i>E. coli.<i> (Data by Imperial College iGEM team 2011).</i></p>
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<p>As is visible from these plates, fluorescence was present in bacteria recovered from both sterile and non-sterile soil. A control plate grown from a filter disc inoculated in non-sterilised soil without fluorescent bacteria showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the <i>E. coli</i>-like colonies from the non-sterile sample had retained a plasmid, we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Figure 2).</p>
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<p>The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that no mutations had taken place in the superfolder GFP gene contained in the bacteria inoculated in non-sterile and sterile soil.This result was obtained in three separate replicates.<p>In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.
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<p><i>Video 1. Stack images taken by confocal microscopy converted into three-dimensional videos of </i>Arabidopsis thaliana DR5:3VENUS<i>roots (data and imaging by Imperial College London iGEM team 2011).</i></p>
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<p>All three samples, the sample from the sterile plate, and the two from the non-sterile plate showing <i>E. coli</i>-like and non-<i>E. coli</i>-like morphologies will be used for 16S ribosomal RNA sequencing (using commonly used primers <sup>[1]</sup>) to determine the bacterial species.
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<p>This result is extremely important as it shows that plasmids can be retained in <i>E. coli</i> for a very long period of time even in the presence of competition when inoculated in soil. This gives us an indication of the life-span our chassis would have in the soil in its implementation stage. In addition, long retainment of the plasmid means that the chance for horizontal gene transfer increases, rendering this result very important for the Gene Guard module.
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<h3>1.2 Plasmid retainment<a href="a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Switch"><img src="https://static.igem.org/mediawiki/2011/5/58/ICL_ProtocolIconDark.png" width="140px" align="right"/></a></h3>
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<p>After observing survaviblity and growth of <i>E. coli</i> in soil, we assumed that the plasmid with sfGFP (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515105">BBa_K515105</a>) has been retained in the cells for 7 weeks either due to static state or very slow growth of cells. Therefore we wanted to test the retainment of plasmid within our chassis in optimal growth conditions without the presence of antibiotics. For this observation we required cells, which would be continuously dividing. As experimental set up we have grown cells in LB for a number of days with supplying fresh LB each day to a number of bacteria, which would then each day go through the growth phases of bacterial maturation. Also each day a small proportion of the culture was plated on a petri dish containing antibiotic and grown overnight to observe the extent of plasmid loss. </p>
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<p><i>Figure 2: </i>E. coli<i> cells grown in LB without antibiotic and subsequently plated on the selective plate have been imaged using Fujifilm LAS 3000 Imager. The samples have been plated after a) 1 day, b) 14 days. In sample after 1 day growth single colonies can not be observed due to high cell density which was plated. In sample after 14 days of growth, decrease in the bacterial density and decrease in fluorescence can be seen. The bacteria were imaged using GFP excitation and emission wavelengths and the picture was kept in the original greyscale format.(Data by Imperial College iGEM team 2011).</i></p>
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<p>We could observe that even after fourteen days of continuous growth in optimal conditions without the presence of antibiotics, our chassis still managed to express sfGFP and therefore contained the plasmid. However considerable loss of fluorescence can be seen due to progressive loss of plasmid. Also decrease in number of colonies can be observed. The lower number of colonies is due to the bacteria losing the plasmid. The loss of plasmid can be attributed to the replication rate of the bacteria being faster than the replication rate of the plasmid.</p>
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<p>In order to quantify the expression of fluorescence in the plants exposed to different treatments we processed the images in ImageJ and analysed fluorescence intensity in individual particles. The relative fluorescence intensity of roots exposed to our IAA-secreting bacteria is 1.93x that of roots exposed to no bacteria and 2.9x that of roots exposed to non-IAA-producing bacteria. It therefore appears that bacteria normally suppress production of IAA in root tips. However, the bacteria we have engineered to express IAA produce enough of the compound to not only overcome this limitation but to also increase expression of the reporter gene. </p>
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<h2>2. Uptake of bacteria into plant roots</h2>
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<p>We observed uptake of GFP-expressing bacteria into plant roots (Figure 3). The fact that (at least some) plants can actively break up bacteria and use them for nutrients was only reported last year<sup>[1]</sup>. This finding is of extreme importance to our project as the exposure of roots to indole 3-acetic acid will vary significantly if the uptake of the compound takes place inside the roots themselves rather than from the outside. This also presents a novel platform of indirectly engineering plants as delivery of a whole range of compounds will be possible using this method.</p>
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<div class="technology">2. Anti-holin expression</div>
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<p>We have completed stage 1 of the assembly of the Gene Guard. A protein gel showed a clear band of the appropriate size right at the bottom of the gel when compared to a control cell. Therefore, we have sequence verified and shown that the <b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515104">BBa_K515104</a></b> expresses a protein of the appropriate size (Figure 3).</p>
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<p><i>Figure 3: </i>Escherichia coli<i> cells expressing superfolder GFP (sfGFP) can be seen inside an Arabidopsis thaliana root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root. (Data and imaging by Imperial College London iGEM team 2011).</i></p>
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<p><i>Figure 3: Protein gel showing an over-expression band (higher in intensity) of a small protein. Lane 1 and 2 are control and lane 3 and 4 are the anti-holin expressing cells.(Data by Imperial College London iGEM team 2011).</i></p>
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<p>To ensure that the bacteria we imaged were inside the roots, we took stack images that were later converted to three-dimensional views of the roots containing bacteria (Video 2).</p>
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<div class="technology">3. Gene integration of the anti-holin</div>
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<h4 style="text-align:center;background-color:#FF944D;color:#000000;">New since regional jamboree</h4>
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<p>Once we knew that the anti-holin construct was alright and seemed to be producing a small protein we had to excise the insert and ligate it into the CRIM plasmid. This was complicated as the high copy number plasmid was placing a heavy metabolic burden on the cells making them grow incredibly slowly (something that is not desirable for when you have to be working for a deadline). Not only that, the cells seemed to be losing the plasmid after four days on a plate in a cold room and some of the colonies managed to alter the plasmid in a way that made it lose the sfGFP and allowed the cells to grow quickly and out-compete any cells with the correct plasmid. However, after a lot of trial and error we managed to isolate eight plasmids that were possible candidates for the plasmid we wanted to integrate.</p>
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<img class="border" src="https://static.igem.org/mediawiki/2011/4/41/ICL_Anti-holin_genome_integration_PCR.PNG" width="280" />
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<p><i>Figure 4: Gel of colonies suspected of containing anti-holin in the genome. The amplified bands of all the colonies correspond to around 300 bp which is approximately the size of the anti-holin gene. The lane furthest to the right is a control in which the anti-holin gene is contained in a pSB1C3 plasmid.(Data by Imperial College London iGEM team 2011).</i></p>
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<p>In order to test these eight colonies we first did an EcoRI and PstI digest and then looked at the size of the insert compared to a control which only contained sfGFP in the CRIM. According to our results, three of the colonies contained an insert that was larger than that of the control. However, the gel red we used to stain the gel seems to do something funny with the buffer we were using to digest making all the bands run higher than they should be running. Therefore, to conclude that the plasmid contained the inserts we wanted we looked for a restriction site that is unique on the anti-holin and the CRIM. We found that the anti-holin construct contains a ClaI site that is also present on the CRIM vector. We performed this digest in another buffer and obtained the expected bands for the three colonies confirming that they contain the anti-holin on the CRIM.</p>
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<p>Then we performed the genome integration step which involves the transformation of a cell line containing the helper plasmid<sup>[2]</sup>. In order to test whether the colonies had integrated the CRIM in the correct location we ordered the primers that were used in the original CRIM paper and performed a colony PCR on all the colonies. A few of the colonies had integrated the CRIM twice which could be clearly seen under a blue box. However, the rest of the colonies had a single integration event which could also be seen by their phenotype under blue light.</p>
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<p>Finally, we had to prove that the anti-holin gene was now present within the colonies. In order to achieve this we performed numerous PCR's with all kinds of buffers and polymerases until we finally got one to work. Using Taq polymerase and Barnes' buffer we managed to amplify a band of the appropriate size using the same primers that we used to initially amplify the anti-holin from its origin.</p>
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<p><i>Video 2. Confocal stack imaging showing sfGFP-expressing bacteria inside plant roots showing blue autofluorescence (data and imaging by Imperial College London iGEM team 2011).</i></p>
 
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<h2>3. Chemotaxis of our chassis expressing PA2652 construct towards malate</h2>
 
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<p><i>Figure 4: Dependence of bacterial chemotaxis to varied malate concetrations. Cells containing PA2652 (<a href="http://partsregistry.org/Part:BBa_K515102">BBa_K515102</a>) have shown inreased number of cells in capillaries with inreasing malate concentration, with a peak at 1 mM. The number of cells drops sharply after 1 mM due to saturation. Negative control were cells without contruct. The cell count for negative control in each of the capillaries with increasing attractant concentration has not increased. Data collected by Imperial iGEM 2011.</i></p>
 
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<p>In the Phyto-Route module of our system we have expressed the malate-reponsive chemoreceptor PA2652 in our chassis. The result of quantitative assay demonstrates chemotaxis of <i>E. coli</i> expressing PA2652 towards L (-) malic acid. The response towards different concentrations of malate has been quantified and the strongest response can be seen at 1 mM. These results show functionality of the Phyto-Route construct that enables movement of our bacteria towards plant root exudate malate. Once the bacteria are attracted towards roots, then they can be naturally taken up into the plant's roots.</p>
 
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<h2>4. <i>E. coli</i> survivability in soil</h2>
 
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<p>To assess how well our chassis <i>Escherichia coli</i> is able to survive in soil and for how long a plasmid can be retained, we set up an experiment that consisted of inoculating <i>E. coli</i> into sterile and non-sterile soil. After six weeks of incubation, samples grown up from sterile cultures still expressed fluorescence. Cultures exhibiting <i>E. coli</i>-like morphologies that were grown from non-sterile plates had lost fluorescence (Figure 5).
 
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<p><i>Figure 5: Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil  (Data by Imperial College London iGEM team 2011).</i></p>
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<p>A restriction digest revealed that cultures from sterile and non-sterile plates that display <i>E. coli</i>-like morphologies have both retained the plasmid. Sequencing of the insert revealed a frameshift mutation in the coding region of the sfGFP gene in the colonies from non-sterile soil. Both samples will be sent off for 16S rRNA sequencing to identify the bacterial species. However, it seems very likely that our GM <i>E. coli</i> have been able to survive in soil and retain their plasmid for six weeks despite competition and selective pressure against the plasmid.<p>
 
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<div class="technology">4. Cell survival of holin-endolysin construct</div>
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<h2>5. Tracking of bacterial metabolic activity using Dendra2</h2>
 
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<p>Dendra2 is a photoconvertable protein<sup>[2]</sup> that normally exhibits green fluorescence but can be switched, using single photon activation, to fluoresce red. This conversion cannot be reversed. Dendra2 is therefore very useful for observing the metabolism of cells: if all protein in a cell is converted to red fluorescence and it subsequently expresses green fluorescence, the cell has to be metabolically active. As we wanted to know if our cells are metabolically active once they have been taken up into root cells, we performed another plant uptake experiment.</p>
 
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<p>We have made a novel fluorescent protein, Dendra2 available as a BioBrick. Dendra2 normally fluoresces green but can be irreversibly converted to red fluorescence. This is very useful for tracking the viability of cells in different circumstances (for instance, after they have been taken up into root cells). We did a plant uptake experiment with Dendra2-expressing <i>E. coli</i> and converted the protein using a 405 nm laser on a confocal microscope (Figure 6 and Video 3).</p>
 
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<p style="padding-left:0px"><i>Figure 6: Dendra2 photoconversion in bacteria taken up inside plant roots. The graph on the top displays averaged fluorescence over the entire photoconverted area and the amount of brightfield light (the background light used to see the outline of the roots) recorded is therefore very high. The graph on the bottom displays emission at green and red fluorescence over the same time span. (Data by Imperial College iGEM team 2011).</i></p>
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<p><i>Video 3. This video shows the photoconversion of Dendra2 within </i>E. coli<i> cells that have been taken up into the plant roots as a time-lapse of pictures taken after each round of bleaching at 405 nm. The targeted area of cells being photoconverted corresponds to the top graph in Figure 5. There is a single bacterium visible on the right that was not targeted for photoconversion and serves as a control (data and imaging by Imperial College iGEM team 2011).</i></p>
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<h2>References:</h2>
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<div class="technology">5. References</div>
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<p>[1] Paungfoo-Lonhienne, C. et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One, 5(7), e11915.
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<p>[2] Gurskaya N et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology 24: 461-465.
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<p>[1] Tanner M et al. (2000) Molecular phylogenetic evidence for noninvasive zoonotic transmission of <i>Staphylococcus intermedius</i> from a canine pet to a human. <i>Journal of Clinical Microbiology</i> <b>38(4):</b> 1628-1631.</p>
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<p>[2] Haldimann and Wanner, 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology 183(21) p. 6384-6393.</p>
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M3: Assembly
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M3: Future Work
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Latest revision as of 17:40, 28 October 2011




Module 3: Gene Guard

Containment is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent horizontal gene transfer of the genes we are expressing in our chassis, we have developed a system based on the genes encoding holin, anti-holin and endolysin. We are engineering anti-holin into the genome of our chassis, where it acts as an anti-toxin, and holin and endolysin on plasmid DNA. In the event of horizontal gene transfer with a soil bacterium, holin and endolysin will be transferred without anti-holin, rendering the recipient cell non-viable and effectively containing the Auxin Xpress and Phyto-Route genes in our chassis.




Testing

Horizontal gene transfer is currently an increasingly pressing topic in the field of Synthetic Biology. It is the main limiting factor which is halting the release of GMO's to the environment as while we know what our genetic construct will do in our own chassis we are unsure what it will do in another organism. In order to prevent this issue of genetic containment we have designed a novel toxin/anti-toxin system, the Gene Guard.

In this module we wanted to first test the necessity of this system by performing experiments to see how long E. coli would retain the plasmid in soil as well as under optimal conditions. Then we tested whether the Gene Guard was assembled correctly at each stage. Finally, we wanted to perform an experiment where we transform control cells and our Gene Guard cells with the holin-endolysin plasmid. Is Gene Guard necessary? Moreover, does it work? Stay tuned to find out.

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1. E.coli survivability in soil and plasmid retainment

1.1 Survivability in soil


Figure 1: Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil (Data by Imperial College London iGEM team 2011).


We set up a soil experiment to test how long our E. coli chassis can retain its plasmid in soil. We initially transformed chemically competent E. coliDH5α cells with superfolder GFP. These cells were inoculated on small filter discs (about 0.5 cm diameter), which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of seven weeks.

After seven weeks, we were able to recover fluorescent bacteria from sterilised and non-autoclaved soil (Figure 1).

Figure 2: Gel digests of bacteria displaying colony morphology typical of E. coli recovered from non-sterilised and sterilised soil. These bacteria exhibited colony morphologies typical of E. coli. (Data by Imperial College iGEM team 2011).










As is visible from these plates, fluorescence was present in bacteria recovered from both sterile and non-sterile soil. A control plate grown from a filter disc inoculated in non-sterilised soil without fluorescent bacteria showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the E. coli-like colonies from the non-sterile sample had retained a plasmid, we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Figure 2).

The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that no mutations had taken place in the superfolder GFP gene contained in the bacteria inoculated in non-sterile and sterile soil.This result was obtained in three separate replicates.

In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.

All three samples, the sample from the sterile plate, and the two from the non-sterile plate showing E. coli-like and non-E. coli-like morphologies will be used for 16S ribosomal RNA sequencing (using commonly used primers [1]) to determine the bacterial species.

This result is extremely important as it shows that plasmids can be retained in E. coli for a very long period of time even in the presence of competition when inoculated in soil. This gives us an indication of the life-span our chassis would have in the soil in its implementation stage. In addition, long retainment of the plasmid means that the chance for horizontal gene transfer increases, rendering this result very important for the Gene Guard module.

1.2 Plasmid retainment


After observing survaviblity and growth of E. coli in soil, we assumed that the plasmid with sfGFP (BBa_K515105) has been retained in the cells for 7 weeks either due to static state or very slow growth of cells. Therefore we wanted to test the retainment of plasmid within our chassis in optimal growth conditions without the presence of antibiotics. For this observation we required cells, which would be continuously dividing. As experimental set up we have grown cells in LB for a number of days with supplying fresh LB each day to a number of bacteria, which would then each day go through the growth phases of bacterial maturation. Also each day a small proportion of the culture was plated on a petri dish containing antibiotic and grown overnight to observe the extent of plasmid loss.


Figure 2: E. coli cells grown in LB without antibiotic and subsequently plated on the selective plate have been imaged using Fujifilm LAS 3000 Imager. The samples have been plated after a) 1 day, b) 14 days. In sample after 1 day growth single colonies can not be observed due to high cell density which was plated. In sample after 14 days of growth, decrease in the bacterial density and decrease in fluorescence can be seen. The bacteria were imaged using GFP excitation and emission wavelengths and the picture was kept in the original greyscale format.(Data by Imperial College iGEM team 2011).


We could observe that even after fourteen days of continuous growth in optimal conditions without the presence of antibiotics, our chassis still managed to express sfGFP and therefore contained the plasmid. However considerable loss of fluorescence can be seen due to progressive loss of plasmid. Also decrease in number of colonies can be observed. The lower number of colonies is due to the bacteria losing the plasmid. The loss of plasmid can be attributed to the replication rate of the bacteria being faster than the replication rate of the plasmid.

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2. Anti-holin expression

We have completed stage 1 of the assembly of the Gene Guard. A protein gel showed a clear band of the appropriate size right at the bottom of the gel when compared to a control cell. Therefore, we have sequence verified and shown that the BBa_K515104 expresses a protein of the appropriate size (Figure 3).


Figure 3: Protein gel showing an over-expression band (higher in intensity) of a small protein. Lane 1 and 2 are control and lane 3 and 4 are the anti-holin expressing cells.(Data by Imperial College London iGEM team 2011).

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3. Gene integration of the anti-holin

New since regional jamboree

Once we knew that the anti-holin construct was alright and seemed to be producing a small protein we had to excise the insert and ligate it into the CRIM plasmid. This was complicated as the high copy number plasmid was placing a heavy metabolic burden on the cells making them grow incredibly slowly (something that is not desirable for when you have to be working for a deadline). Not only that, the cells seemed to be losing the plasmid after four days on a plate in a cold room and some of the colonies managed to alter the plasmid in a way that made it lose the sfGFP and allowed the cells to grow quickly and out-compete any cells with the correct plasmid. However, after a lot of trial and error we managed to isolate eight plasmids that were possible candidates for the plasmid we wanted to integrate.

Figure 4: Gel of colonies suspected of containing anti-holin in the genome. The amplified bands of all the colonies correspond to around 300 bp which is approximately the size of the anti-holin gene. The lane furthest to the right is a control in which the anti-holin gene is contained in a pSB1C3 plasmid.(Data by Imperial College London iGEM team 2011).

In order to test these eight colonies we first did an EcoRI and PstI digest and then looked at the size of the insert compared to a control which only contained sfGFP in the CRIM. According to our results, three of the colonies contained an insert that was larger than that of the control. However, the gel red we used to stain the gel seems to do something funny with the buffer we were using to digest making all the bands run higher than they should be running. Therefore, to conclude that the plasmid contained the inserts we wanted we looked for a restriction site that is unique on the anti-holin and the CRIM. We found that the anti-holin construct contains a ClaI site that is also present on the CRIM vector. We performed this digest in another buffer and obtained the expected bands for the three colonies confirming that they contain the anti-holin on the CRIM.

Then we performed the genome integration step which involves the transformation of a cell line containing the helper plasmid[2]. In order to test whether the colonies had integrated the CRIM in the correct location we ordered the primers that were used in the original CRIM paper and performed a colony PCR on all the colonies. A few of the colonies had integrated the CRIM twice which could be clearly seen under a blue box. However, the rest of the colonies had a single integration event which could also be seen by their phenotype under blue light.

Finally, we had to prove that the anti-holin gene was now present within the colonies. In order to achieve this we performed numerous PCR's with all kinds of buffers and polymerases until we finally got one to work. Using Taq polymerase and Barnes' buffer we managed to amplify a band of the appropriate size using the same primers that we used to initially amplify the anti-holin from its origin.

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4. Cell survival of holin-endolysin construct
5. References

[1] Tanner M et al. (2000) Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. Journal of Clinical Microbiology 38(4): 1628-1631.

[2] Haldimann and Wanner, 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology 183(21) p. 6384-6393.

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M3: Assembly M3: Future Work