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$("#Content1").html("<h2>Designing a Switch</h2>We designed multiple RNA structures by changing the variable regions of the synthetic guide RNA. Using computational modelling from NUPACK, we designed constructs that would be unfolded by complementary binding of a target sense RNA (sRNA) (Fig. 1). <img src='https://static.igem.org/mediawiki/2018/4/48/T--Warwick--riboresults1AA.png' style='display: inline; width: 50%;'><img src='https://static.igem.org/mediawiki/~~parts~~/e/e7/T--Warwick--~~2018-riboswitch-alpha-maxx~~.png' style='display: inline; width: 50%;'> Figure 1: Computational modelling of the secondary RNA structure of the Alpha construct (BBa_K2841503). The dCas9 targeting region is circled red. (A) Alpha construct secondary RNA structure in its normal, closed state. The targeting region is occluded by its complementary “clamp” region. (B) The full RNA of legiolysin bound to the Alpha construct. Here, the dCas9 targeting region is exposed such that dCas9 is able to bind its target.");

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$("#Content1").html("<h2>Designing a Switch</h2>We designed multiple RNA structures by changing the variable regions of the synthetic guide RNA. Using computational modelling from NUPACK, we designed constructs that would be unfolded by complementary binding of a target sense RNA (sRNA) (Fig. 1). <img src='https://static.igem.org/mediawiki/2018/4/48/T--Warwick--riboresults1AA.png' style='display: inline; width: 50%;'><img src='https://static.igem.org/mediawiki/2018/8/8b/T--Warwick--riboresults2.png' style='display: inline; width: 50%;'> Figure 1: Computational modelling of the secondary RNA structure of the Alpha construct (BBa_K2841503). The dCas9 targeting region is circled red. (A) Alpha construct secondary RNA structure in its normal, closed state. The targeting region is occluded by its complementary “clamp” region. (B) The full RNA of legiolysin bound to the Alpha construct. Here, the dCas9 targeting region is exposed such that dCas9 is able to bind its target.");

$("#Content2").html("<h2>Primary or Secondary Structure Design?</h2>Construct Alpha had a variable loop region that was built by selecting regions of the primary sequence of the target RNA and complementing these regions with our variable regions (in text format). We then tested these sequences for hairpin loop-like secondary structure using NUPACK. Construct Gamma had a variable loop region that was built by selecting regions of the secondary structure of the target RNA and complementing these regions with our variable regions (from NUPACK-generated structures). We then tested these sequences for hairpin loop-like secondary structure using NUPACK. We measured the fluorescence/OD600 of the two constructs, Alpha (BBa_K2841503) and Gamma (BBa_K2841501), in a 96-well plate reader (TECAN F500) to observe which method gives the best output (Fig. 2). Results were blanked against M9 minimal media and M9 minimal media with ATC (to be subtracted from data for results where we induced with ATC only). Results of fluorescence/OD600 at a time point of 18 hours (maximum OD600) shows: • Construct Alpha shows 2.1-2.5-fold upregulation of GFP when induced • Construct Gamma shows 1.8-fold upregulation of GFP when induced • The Gamma construct cells did not achieve an OD600 as high as that of other cells upon induction with ATC. <img src='https://static.igem.org/mediawiki/2018/8/8e/T--Warwick--riboresults3.png'> <img src='https://static.igem.org/mediawiki/2018/d/dd/T--Warwick--riboresults4.png'> <img src='https://static.igem.org/mediawiki/2018/8/8f/T--Warwick--riboresults5.png'> Figure 2: Induction by ATC shows differences in fluorescence for constructs Alpha (AI + AN) and Gamma (LI + LN). Error bars show standard deviation. Number of replicas = 3. The positive control were cells constitutively expressing GFP (due to a deletion of lacI in the chromosome), the negative control were cells containing pSB1AK8 with appropriate antibiotics. (A) Fluorescence/OD600 over time. (B) OD600 over time. (C) Fluorescence over time. A Shapiro-Wilk test for normality showed the data were not normally distributed (p = 0.000) (Fig. 3A). A Mann-Whitney U test was conducted to test for statistical significance. The data was statistically significant for both constructs, Alpha and Gamma (p = 0.000) (Fig. 3B). <img src='https://static.igem.org/mediawiki/2018/4/46/T--Warwick--riboresults6.png'><img src='https://static.igem.org/mediawiki/2018/7/71/T--Warwick--riboresults7.png'> <img src='https://static.igem.org/mediawiki/2018/4/4e/T--Warwick--riboresults8.png'> <img src='https://static.igem.org/mediawiki/2018/0/08/T--Warwick--riboresults9.png'> Figure 3: Statistical analysis of the raw fluorometry data for constructs Alpha and Gamma. (A) Shapiro-Wilk test for normality showed data were not normally distributed. Therefore, we needed to run a non-parametric test. (B) A Mann-Whitney U test was conducted to test for the statistical significance of our data. The data shows both constructs expressed statistically significant data (p = 0.000). (C) A Mann-Whitney U test for categories across constructs shows statistical significance, indicating that the exact sequence of our variable sRNA-binding regions gives significantly different data based on the primary sequence of these regions. Taken together these data show that the synthetic guide RNA constructs, Alpha and Gamma, are capable of significant induction of gene regulation via their ability to guide dCas9 to its target DNA to alter gene expression. Our data also show statistically significant differences in fluorescence/OD600 based on the construct that is used; even if targeting different regions of the same RNA (in our case, legiolysin) (Fig. 3C) (p = 0.000). This indicates that there is a significant difference in fluorescence highly dependent on the primary sequence of the variable regions of the guide RNA. It is also worth noting that the data across constructs for non-induction are also statistically significant. Thus, there is significant differences in background fluorescence and in induced fluorescence based on the primary sequence of the guide RNA.");

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$("#inorgIMG").css("background-color", "grey");

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$("#Content1").html("<h2>Did we get our plasmids in?</h2>GELS: To assess whether the transformations we carried were correct, we amplified the identified and ran gels to compare the lengths of the DNA present in the plasmids present in the cells. Though we did not do this for every transformation, some of the most exciting one which we ran gels of were: * The first of our transformations was inserting JDE131 with GvpA into DH5A (E. coli). As can be seen from the gel, the bands amplified were at just under 500bp which is close to the expected length of the amplicon which would be 468bp. <img src='https://l.messenger.com/l.php?u=http%3A%2F%2F2018.igem.org%2FFile%3AT--Warwick--2018-FMX1.png&h=AT0L2PEQieLC2g1emnsiShdv2dP-E5lpl4A-F36SnIuGP72NTj-JyXzhR69Du3VpycZ06AVjP3nE7444rrahtLczJdDyFTRgw0hEX3FOCzUoMRibXiLC2ZZekQm0lFVr52UCKg'> * The next of our transformations was inserting ECE174 with GvpC into DH5A (E. coli). As can be seen from the gel, the bands amplified were at around 500bp which is close to the expected length of the amplicon, which only amplified the GvpC gene, which is 500bp. <img src=''> Another one our transformations was inserting JDE131 with CFP into DH5A (E. coli). As can be seen from the gel, the bands amplified were at … which is close to the expected length of the amplicon which would be 961bp. <img src='https://static.igem.org/mediawiki/2018/9/9d/T--Warwick--2018-FMX3.png'>");

+

$("#Content2").html("<h2>Plate growth shows that the plasmids did get in as they survived in antiobiotics</h2> * Our plasmids have antibiotic resistant genes to ensure that the cell retains the associated genes, and thus can be grown on antibiotic plates. * We observed LBA Ampicillin plates with GvpA E. coli and CFP E. coli grown on them. * We also observed LBA Spectinomycin with GvpA B. subtilis grown on them and LBA Chloramphenicol plates with GvpC B. subtilis grown on them. <h2>Sequencing the data of plasmids</h2>We were also able to miniprep or PCR purify the following inserts to sequence verify them: GvpA E. coli, GvpC E. coli, GvpA B. sub, CFP E. coli. The sequencing results can be seen below, and show that they were correctly inserted without significant mutations. GvpA E. coli <img src=''> GvpC E. coli <img src=''> CFP E. coli <img src=''> GvpA B. subtilis <img src=''>");

+

$("#Content3").html("<h2>Did we get our plasmids to work?</h2>In order to characterise our promoters, we inserted a yellow fluorescent protein after both of the bacillus promoters (Hyperspank and Popp). We collected results that suggested that these promoters were successful in transcribing the fluorescent proteins and were thus functional in Bacillus subtilis. The data showed that the higher level of IPTG present, the higher level of fluorescence in the inducible promoter (Hyperspank). The data also compares the difference between the inducible (Hyperspank) and the constitutive (Popp) promoters fluorescence, showing that Popp has almost three-fold expression. The full data for our characterisation is available on our registry, as well as extensively detailed on our models page. We also got addgene mega cluster into E. coli and conducted several floating experiments to see whether or not the cells floated. We saw qualitative preliminary data, that suggested gas vesicles may have been formed. This is shown in example, by the image below which shows the formation of bubbles at the top of the culture based on the level of IPTG concentration: at 0ul IPTG and 200ul IPTG there were no bubble present; this is likely because the promoters were not induced and over-worked respectively. 2ul shows the highest amount of bubbles, this is expected number at which the T7 promoter would be most efficient and, 20ul has a few bubbles, as, again, it is likely overworked. As mentioned, this is extremely preliminary data, but was replicated and showed similar qualitative results.<img src=''>");

}

Difference between revisions of "Team:Warwick/Results"

Revision as of 03:48, 18 October 2018

Results

Contact Us

igem@warwick.ac.uk

Sponsors

@@ Line 132: / Line 132: @@
          $("#inorgIMG").css("background-color", "white");
-         $("#Content1").html("<h2>Designing a Switch</h2><p>We designed multiple RNA structures by changing the variable regions of the synthetic guide RNA. Using computational modelling from NUPACK, we designed constructs that would be unfolded by complementary binding of a target sense RNA (sRNA) (Fig. 1).</p><br><br><img src='https://static.igem.org/mediawiki/2018/4/48/T--Warwick--riboresults1AA.png' style='display: inline; width: 50%;'><img src='https://static.igem.org/mediawiki/parts/e/e7/T--Warwick--2018-riboswitch-alpha-maxx.png' style='display: inline; width: 50%;'><br><br><p>Figure 1: Computational modelling of the secondary RNA structure of the Alpha construct (BBa_K2841503). The dCas9 targeting region is circled red. (A) Alpha construct secondary RNA structure in its normal, closed state. The targeting region is occluded by its complementary “clamp” region. (B) The full RNA of legiolysin bound to the Alpha construct. Here, the dCas9 targeting region is exposed such that dCas9 is able to bind its target.</p>");
+         $("#Content1").html("<h2>Designing a Switch</h2><p>We designed multiple RNA structures by changing the variable regions of the synthetic guide RNA. Using computational modelling from NUPACK, we designed constructs that would be unfolded by complementary binding of a target sense RNA (sRNA) (Fig. 1).</p><br><br><img src='https://static.igem.org/mediawiki/2018/4/48/T--Warwick--riboresults1AA.png' style='display: inline; width: 50%;'><img src='https://static.igem.org/mediawiki/2018/8/8b/T--Warwick--riboresults2.png' style='display: inline; width: 50%;'><br><br><p>Figure 1: Computational modelling of the secondary RNA structure of the Alpha construct (BBa_K2841503). The dCas9 targeting region is circled red. (A) Alpha construct secondary RNA structure in its normal, closed state. The targeting region is occluded by its complementary “clamp” region. (B) The full RNA of legiolysin bound to the Alpha construct. Here, the dCas9 targeting region is exposed such that dCas9 is able to bind its target.</p>");
          $("#Content2").html("<h2>Primary or Secondary Structure Design?</h2><p>Construct Alpha had a variable loop region that was built by selecting regions of the primary sequence of the target RNA and complementing these regions with our variable regions (in text format). We then tested these sequences for hairpin loop-like secondary structure using NUPACK.<br><br>Construct Gamma had a variable loop region that was built by selecting regions of the secondary structure of the target RNA and complementing these regions with our variable regions (from NUPACK-generated structures). We then tested these sequences for hairpin loop-like secondary structure using NUPACK.<br><br>We measured the fluorescence/OD600 of the two constructs, Alpha (BBa_K2841503) and Gamma (BBa_K2841501), in a 96-well plate reader (TECAN F500) to observe which method gives the best output (Fig. 2). Results were blanked against M9 minimal media and M9 minimal media with ATC (to be subtracted from data for results where we induced with ATC only). Results of fluorescence/OD600 at a time point of 18 hours (maximum OD600) shows:<br>•&nbsp;Construct Alpha shows 2.1-2.5-fold upregulation of GFP when induced<br>•&nbsp;Construct Gamma shows 1.8-fold upregulation of GFP when induced<br>•&nbsp;The Gamma construct cells did not achieve an OD600 as high as that of other cells upon induction with ATC.</p><br><br><img src='https://static.igem.org/mediawiki/2018/8/8e/T--Warwick--riboresults3.png'><br><img src='https://static.igem.org/mediawiki/2018/d/dd/T--Warwick--riboresults4.png'><br><img src='https://static.igem.org/mediawiki/2018/8/8f/T--Warwick--riboresults5.png'><br><br><p>Figure 2: Induction by ATC shows differences in fluorescence for constructs Alpha (AI + AN) and Gamma (LI + LN). Error bars show standard deviation. Number of replicas = 3. The positive control were cells constitutively expressing GFP (due to a deletion of lacI in the chromosome), the negative control were cells containing pSB1AK8 with appropriate antibiotics. (A) Fluorescence/OD600 over time. (B) OD600 over time. (C) Fluorescence over time.<br><br>A Shapiro-Wilk test for normality showed the data were not normally distributed (p = 0.000) (Fig. 3A). A Mann-Whitney U test was conducted to test for statistical significance. The data was statistically significant for both constructs, Alpha and Gamma (p = 0.000) (Fig. 3B).</p><br><br><img src='https://static.igem.org/mediawiki/2018/4/46/T--Warwick--riboresults6.png'><img src='https://static.igem.org/mediawiki/2018/7/71/T--Warwick--riboresults7.png'><br><img src='https://static.igem.org/mediawiki/2018/4/4e/T--Warwick--riboresults8.png'><br><img src='https://static.igem.org/mediawiki/2018/0/08/T--Warwick--riboresults9.png'><br><br><p>Figure 3: Statistical analysis of the raw fluorometry data for constructs Alpha and Gamma. (A) Shapiro-Wilk test for normality showed data were not normally distributed. Therefore, we needed to run a non-parametric test. (B) A Mann-Whitney U test was conducted to test for the statistical significance of our data. The data shows both constructs expressed statistically significant data (p = 0.000). (C) A Mann-Whitney U test for categories across constructs shows statistical significance, indicating that the exact sequence of our variable sRNA-binding regions gives significantly different data based on the primary sequence of these regions.<br><br>Taken together these data show that the synthetic guide RNA constructs, Alpha and Gamma, are capable of significant induction of gene regulation via their ability to guide dCas9 to its target DNA to alter gene expression.<br><br>Our data also show statistically significant differences in fluorescence/OD600 based on the construct that is used; even if targeting different regions of the same RNA (in our case, legiolysin) (Fig. 3C) (p = 0.000). This indicates that there is a significant difference in fluorescence highly dependent on the primary sequence of the variable regions of the guide RNA. It is also worth noting that the data across constructs for non-induction are also statistically significant. Thus, there is significant differences in background fluorescence and in induced fluorescence based on the primary sequence of the guide RNA.</p>");
@@ Line 174: / Line 174: @@
          $("#inorgIMG").css("width", "12%");
          $("#inorgIMG").css("background-color", "grey");
+        $("#Content1").html("<h2>Did we get our plasmids in?</h2><p>GELS:<br>To assess whether the transformations we carried were correct, we amplified the identified and ran gels to compare the lengths of the DNA present in the plasmids present in the cells. Though we did not do this for every transformation, some of the most exciting one which we ran gels of were:<br><br> * The first of our transformations was inserting JDE131 with GvpA into DH5A (E. coli). As can be seen from the gel, the bands amplified were at just under 500bp which is close to the expected length of the amplicon which would be 468bp.</p><br><img src='https://l.messenger.com/l.php?u=http%3A%2F%2F2018.igem.org%2FFile%3AT--Warwick--2018-FMX1.png&h=AT0L2PEQieLC2g1emnsiShdv2dP-E5lpl4A-F36SnIuGP72NTj-JyXzhR69Du3VpycZ06AVjP3nE7444rrahtLczJdDyFTRgw0hEX3FOCzUoMRibXiLC2ZZekQm0lFVr52UCKg'><br><p><br> * The next of our transformations was inserting ECE174 with GvpC into DH5A (E. coli). As can be seen from the gel, the bands amplified were at around 500bp which is close to the expected length of the amplicon, which only amplified the GvpC gene, which is 500bp.</p><br><img src=''><br><p>Another one our transformations was inserting JDE131 with CFP into DH5A (E. coli). As can be seen from the gel, the bands amplified were at … which is close to the expected length of the amplicon which would be 961bp.</p><br><img src='https://static.igem.org/mediawiki/2018/9/9d/T--Warwick--2018-FMX3.png'>");
+        $("#Content2").html("<h2>Plate growth shows that the plasmids did get in as they survived in antiobiotics</h2><p> * Our plasmids have antibiotic resistant genes to ensure that the cell retains the associated genes, and thus can be grown on antibiotic plates.<br> * We observed LBA Ampicillin plates with GvpA E. coli and CFP E. coli grown on them.<br> * We also observed LBA Spectinomycin with GvpA B. subtilis grown on them and LBA Chloramphenicol plates with GvpC B. subtilis grown on them.</p><br><br><h2>Sequencing the data of plasmids</h2><p>We were also able to miniprep or PCR purify the following inserts to sequence verify them: GvpA E. coli, GvpC E. coli, GvpA B. sub, CFP E. coli. The sequencing results can be seen below, and show that they were correctly inserted without significant mutations.<br><br>GvpA E. coli</p><br><img src=''><br><br><p>GvpC E. coli</p><br><img src=''><br><br><p>CFP E. coli</p><br><img src=''><br><br><p>GvpA B. subtilis</p><br><img src=''>");
+        $("#Content3").html("<h2>Did we get our plasmids to work?</h2><p>In order to characterise our promoters, we inserted a yellow fluorescent protein after both of the bacillus promoters (Hyperspank and Popp). We collected results that suggested that these promoters were successful in transcribing the fluorescent proteins and were thus functional in Bacillus subtilis. The data showed that the higher level of IPTG present, the higher level of fluorescence in the inducible promoter (Hyperspank). The data also compares the difference between the inducible (Hyperspank) and the constitutive (Popp) promoters fluorescence, showing that Popp has almost three-fold expression. The full data for our characterisation is available on our registry, as well as extensively detailed on our models page.<br><br><br>We also got addgene mega cluster into E. coli and conducted several floating experiments to see whether or not the cells floated. We saw qualitative preliminary data, that suggested gas vesicles may have been formed. This is shown in example, by the image below which shows the formation of bubbles at the top of the culture based on the level of IPTG concentration: at 0ul IPTG and 200ul IPTG there were no bubble present; this is likely because the promoters were not induced and over-worked respectively. 2ul shows the highest amount of bubbles, this is expected number at which the T7 promoter would be most efficient and, 20ul has a few bubbles, as, again, it is likely overworked.<br><br>As mentioned, this is extremely preliminary data, but was replicated and showed similar qualitative results.</p><img src=''>");
 }