Difference between revisions of "Team:Vilnius-Lithuania/Results"
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− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/b/b2/Vilnius15_gelis2.png" style="width:800px"/> |
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none">Figure 2. Resriction analysis of Cas3 protein. The wild type (WT) Cas3 protein was in a pETDuet vector and had EcoRI (E), XbaI (X) and PstI (P) restriction sites upstream of the Cas3 gene, and in the middle of Cas3 gene, all in close proximity. All five mutant plasmids have mutated successfully (except mutant plasmid #1), because after restriction with each restriction enzyme there were no restriction fragments detected. Restriction was compared to the unmutated non-digested wild type Cas3 plasmid (K).</p> |
<p class="text-justify">After a few tries we successfully made all mutagenesis reactions of both Cas3 and Cascade genes, confirming them with restriction analysis and sequencing. We also cloned them to the pSB1C3 vector and shipped them to the parts registry to provide future iGEM teams with these biobricks: | <p class="text-justify">After a few tries we successfully made all mutagenesis reactions of both Cas3 and Cascade genes, confirming them with restriction analysis and sequencing. We also cloned them to the pSB1C3 vector and shipped them to the parts registry to provide future iGEM teams with these biobricks: | ||
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<p class="text-justify">Instead, we co-expressed our biobrick parts with a plasmid, coding one of the proteins of the Cascade complex (Csy3) with a His6 sequence in E.coli BL21-DE3 strain. The proteins generated from these plasmids form the Cascade complex, which is then detected with Western blot.</p> | <p class="text-justify">Instead, we co-expressed our biobrick parts with a plasmid, coding one of the proteins of the Cascade complex (Csy3) with a His6 sequence in E.coli BL21-DE3 strain. The proteins generated from these plasmids form the Cascade complex, which is then detected with Western blot.</p> | ||
− | <p class="text-justify">Our experiment was a success, and we have obtained an expression of our Cascade constructs, compared to the negative controls, wich had no plasmids ( | + | <p class="text-justify">Our experiment was a success, and we have obtained an expression of our Cascade constructs, compared to the negative controls, wich had no plasmids (Figure 3). Cell cultures were grown until OD<sub>500</sub>~0,6, and then incubated at two different conditions. Expression of incubated cells at 16°C for 16 hours resulted in a bit stronger protein expression than those, which were incubated at 37°C for 3 hours. We wanted to see a different protein expression between the biobrick, which had a strong RBS (S), medium RBS (M) or weak RBS (W), however, the western blot did not show any differences. This may be due to the fact, that expression was too big to see subtile differences. </p> |
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+ | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none">Figure 3. Western blot of the Cascade biobricks. Expression of different Cascade biobricks was analysed in two incubation conditions after reaching a cell OD<sub>500</sub> ~0.6. Biobricks incubated at 16 °C for 16 hours showed a bit stronger expression strength rather than incubated at 37°C for 3 hours. M – molecular mass marker (Spectra broad range protein protein ladder); K – possitive control of Csy3 protein; Cascade biobricks were expressed within strong (S), medium (M) or weak (W) RBS sites. BL – BL21-DE3 strain with no transformed plasmids.</p> | ||
<p class="text-justify">Another interesting result is that Cascade with strong RBS was expressed weaker than with a medium or weak RBS site. This may have to do with experimental error or in this exact construct, the strong RBS is expressed weaker than the other two.</p> | <p class="text-justify">Another interesting result is that Cascade with strong RBS was expressed weaker than with a medium or weak RBS site. This may have to do with experimental error or in this exact construct, the strong RBS is expressed weaker than the other two.</p> | ||
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<p class="text-justify">We planned an experiment, during which we can see our system's killing profficiency. We prepared BL21-DE3 strain <em>E. coli</em> cells with our Cascade expression construct, along with a complement plasmid, carrying the Cas3 gene in a pCola-Duet vector.</p> | <p class="text-justify">We planned an experiment, during which we can see our system's killing profficiency. We prepared BL21-DE3 strain <em>E. coli</em> cells with our Cascade expression construct, along with a complement plasmid, carrying the Cas3 gene in a pCola-Duet vector.</p> | ||
− | <p class="text-justify">The whole experiment performed in order to transform the third plasmid, harbouring the crRNA region, into these bacteria. We transformed two different homogenous crRNA regions, which targeted an essential gene of <em>E. coli</em> genome: DNA pol. III δ subunit (SP1) and RNA polymerase α subunit (SP2). With these three components present in a bacteria and when IPTG is present in the medium (pColaDuet vector harbouring Cas3 needs IPTG for expression), the killing mechanism turns on. The expressed Cascade proteins assemble into a complex with the synthesized crRNA molecules, and binds to either the DNA pol. (SP1) or RNA pol. (SP2) genes. Cas3 is then recruited to | + | <p class="text-justify">The whole experiment performed in order to transform the third plasmid, harbouring the crRNA region, into these bacteria. We transformed two different homogenous crRNA regions, which targeted an essential gene of <em>E. coli</em> genome: DNA pol. III δ subunit (SP1) and RNA polymerase α subunit (SP2). With these three components present in a bacteria and when IPTG is present in the medium (pColaDuet vector harbouring Cas3 needs IPTG for expression), the killing mechanism turns on. The expressed Cascade proteins assemble into a complex with the synthesized crRNA molecules, and binds to either the DNA pol. (SP1) or RNA pol. (SP2) genes. Cas3 is then recruited to hydrolyse these genes and bacteria die. </p> |
<p class="text-justify">After counting bacterial CFU’s (colony forming units) (Figure 3), we estimated, that under conditions with no IPTG present all cells showed similar bacterial count with our control (K), which had no crRNA region. This result was expected, considering that IPTG is necessary for Cas3 expression. </p> | <p class="text-justify">After counting bacterial CFU’s (colony forming units) (Figure 3), we estimated, that under conditions with no IPTG present all cells showed similar bacterial count with our control (K), which had no crRNA region. This result was expected, considering that IPTG is necessary for Cas3 expression. </p> | ||
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− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/3/38/Vilnius15_invivo.png" style="width: 800px; " /> |
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none">Figure 4. Cell killing proficiency test. CFUs were counted of bacteria transformed with genome targeting crRNAs (SP1 or SP2), compared to no crRNA harbouring control (K). Experiment was conducted in two conditions : without IPTG and with 2.5 mM IPTG present.</p> |
<p class="text-justify">However, when IPTG is present, we can see a dramatic cell count drop of almost 100x in bacteria, harbouring our crRNA's (SP1 and SP2), compared to the control (K). According to this data, we can say, that our Cascade complex biobrick (BBa_K1773022) is expressed and actively functions in degradation of cell DNA.</p> | <p class="text-justify">However, when IPTG is present, we can see a dramatic cell count drop of almost 100x in bacteria, harbouring our crRNA's (SP1 and SP2), compared to the control (K). According to this data, we can say, that our Cascade complex biobrick (BBa_K1773022) is expressed and actively functions in degradation of cell DNA.</p> |
Revision as of 06:49, 18 September 2015