Difference between revisions of "Team:Groningen/Results"
| Line 58: | Line 58: | ||
<p class="left">Although the pSB1C3:dCas9QC is already in the pSB1C3 backbone and contains all the correct restriction sites, it does not contained the suffix. To fix this mistake we restricted the pSB1C3dCas9 with EcoRI and SpeI and ligated it into pSB1C3, which was linearized with primers G69 and G70 (see notebook) and restricted with the same enzymes. In the gel on the right lane 2 & 3 correspond to the isolated plasmid restricted with respectively EcorI and a combination of EcorI and PstI. The sizes correspond to the linearized (6,2 kb) and the separate backbone (2 kb) and dCas9 part (4,2 kb).</p> | <p class="left">Although the pSB1C3:dCas9QC is already in the pSB1C3 backbone and contains all the correct restriction sites, it does not contained the suffix. To fix this mistake we restricted the pSB1C3dCas9 with EcoRI and SpeI and ligated it into pSB1C3, which was linearized with primers G69 and G70 (see notebook) and restricted with the same enzymes. In the gel on the right lane 2 & 3 correspond to the isolated plasmid restricted with respectively EcorI and a combination of EcorI and PstI. The sizes correspond to the linearized (6,2 kb) and the separate backbone (2 kb) and dCas9 part (4,2 kb).</p> | ||
<p class="left">Since the chances of mutations in restriction-ligation cloning are negligible we did not fully sequence the part again. We did use pJet_Fwd and pJet-Rev to be absolutely certain that the correct part was cloned into the backbone. These results are shown in figure 3. </p> | <p class="left">Since the chances of mutations in restriction-ligation cloning are negligible we did not fully sequence the part again. We did use pJet_Fwd and pJet-Rev to be absolutely certain that the correct part was cloned into the backbone. These results are shown in figure 3. </p> | ||
| − | <p class="left"> This part is considered as an improvement of a previously submitted dCas9 <a href="http://parts.igem.org/Part:BBa_K1026001">BBa_K1026001</a> since our part has been made biobrick compatible | + | <p class="left"> This part is considered as an improvement of a previously submitted dCas9 <a href="http://parts.igem.org/Part:BBa_K1026001">BBa_K1026001</a> since our part has been made biobrick compatible. Also we sequence confirmed and experimentally validated our part, which was not done by the 2016 Warwick team, who also improved this part.</p> |
</li><br> | </li><br> | ||
| Line 69: | Line 69: | ||
<ul> | <ul> | ||
<li> <b>Construction</b><br> | <li> <b>Construction</b><br> | ||
| − | + | <p class="left">This part was made from the pSB1C3:dCas9QC plasmid (see dCas9) which was completely sequenced. We replaced the final part of the dCas9QC with a gBlock containing the VRER mutations. The new construct was analysed by sequencing the last part containing the gblock (figure 4). Also a restriction analysis with EcoRI and PstI was performed see the gel in dCas9 lane 4.</p> | |
| + | <p class="left">Based on the validation (see below) we cannot conclude yet whether the VRER mutations have the desired effect. If we would have mixed up the </p> | ||
| + | |||
| + | <br> insert seqeunce file VRER | ||
| + | </li><br> | ||
<li> <b>Experimental validation</b><br> | <li> <b>Experimental validation</b><br> | ||
| − | + | <p class="left"> This part was validated experimentally in the same experiment as dCas9. For this experiment we have designed four gRNA's that target GFP. Two of this gRNA's bind to sequences flanked by NGG (1&2) and two bind to a sequence flanked by NGCG (3&4). These gRNA's were inserted into the plasmid pSB3C5 containing a pLacGFP construct as well. For dCas9 and dCas9VRER expression those parts were put into a pBad vector in which they are expressed behind an Arabinose inducible promoter.</p> | |
| + | <p class="left"> In total 18 E. coli strains were produced containing pBad:dCas9, pBad:dCas9VRER or no pBad combined with pS3C5 with one of the gRNA's, pSB3C5 without a gRNA or no pSB3C5 (figure 5).</p> | ||
| + | <p class="left"> All strains were grown overnight and the next day they were dilute to an OD of 0.05 in the afternoon. At the end of the afternoon at ODof 0.4-0.6 all cultures were induced with a final concentration of 0.01% arabinose. All induced cultures were put back into the incubator (37C 220 rpm) and grown overnight. The next morning the OD of all cultures was measured and to an OD of 0.2 in LB (in final volume of 1mL). The OD and GFP (470/510) of all samples were measured in a plate reader in quadruplicate.<p> | ||
| + | <p class="left"> The data obtained from the fluorescence measurement were converted to relative fluorescence and are shown in figure 5. To convert the fluorescence to relative fluorescence we first divided all measured fluorescence values were divided by the measured OD's. Next the fluorescence/OD of the negative controls (samples without pSB1C3) was subtracted from the other samples. Next all normalized fluorescence/OD values were divided by that of the positive control (no pBad:pSB3C5 without gRNA).</p> | ||
| + | <p class="left"> | ||
| + | From the figure it can be seen that all fluorescence values are quite similar for the samples without gRNA. Further we can see a clear decrease in the relative fluorescence of dCas9 in combination with gRNA 2 and 3. For gRNA's 1 and 4 the relative fluorescence of the dCas9 is similar to that of the samples without the pBad vector. For dCas9VRER we showed CRISPR interference in combination with gRNA's 1,2, and 4. </p> | ||
| + | <p class="left">From the data we cannot conclude that the VRER mutations had any effect on the PAM preference, since dCas9VRER shows the strongest repression for gRNA 1 and 4 of which one has a NGG flanked target. Also gRNA 3 whose target is flanked by NGCG seems to be repressed by dCas9 and not by dCas9 VRER. If these results are correct this would suggest that the PAM preference has not changed for dCas9VRER, however it is quite odd that the differences between dCas9 and dCas9VRER are so big for especially gRNA 1 and gRNA4. Another explanation could be that we mixed up gRNA1 and gRNA3 in one of the steps from gBlock to double transformants. If we would switch around these data (so gRNA1-> gRNA3 and gRNA3-> gRNA1) the data would show that only dCas9VRER can be directed by towards NGCG flanked targets. Unfortunately we could not analyse the sequences of the pSB3C5 plasmids of the cultures used in this experiment before the WIKI-freeze.</p> | ||
| + | |||
| + | <br> insert figure strain table | ||
| + | </li><br> | ||
| + | |||
<li> <b>Considerations for replicating the experiments</b><br> | <li> <b>Considerations for replicating the experiments</b><br> | ||
| − | + | Besides sequencing the cultures used in the experiment, performing more experiments with biological replicates would increase the fidellity of the data. Also it would be nice to design more efficient gRNA's that bind between the -35 and -10 region of the promoter. This way we can compare the efficiency of dCas9 and dCas9VRER, since this is the optimal place to target a gRNA. | |
| − | + | </li><br> | |
| − | + | ||
</ul> | </ul> | ||
Revision as of 16:55, 1 November 2017
Results
dCas9
- Construction
For this part we started by making a biobrick compatible version of dCas9 called pSB1C3:dCas9QC. This part was also used for the construction of dCas9VRER. The full length of this part was sequenced (figure ..) and the results confirmed that the sequence was correct. In the notebook (week 18-09) a gel is shown from which it can be seen that the EcoRI site was successfully removed from our dCas9 part.
Add sequencing image and gb files
Although the pSB1C3:dCas9QC is already in the pSB1C3 backbone and contains all the correct restriction sites, it does not contained the suffix. To fix this mistake we restricted the pSB1C3dCas9 with EcoRI and SpeI and ligated it into pSB1C3, which was linearized with primers G69 and G70 (see notebook) and restricted with the same enzymes. In the gel on the right lane 2 & 3 correspond to the isolated plasmid restricted with respectively EcorI and a combination of EcorI and PstI. The sizes correspond to the linearized (6,2 kb) and the separate backbone (2 kb) and dCas9 part (4,2 kb).
Since the chances of mutations in restriction-ligation cloning are negligible we did not fully sequence the part again. We did use pJet_Fwd and pJet-Rev to be absolutely certain that the correct part was cloned into the backbone. These results are shown in figure 3.
This part is considered as an improvement of a previously submitted dCas9 BBa_K1026001 since our part has been made biobrick compatible. Also we sequence confirmed and experimentally validated our part, which was not done by the 2016 Warwick team, who also improved this part.
- Experimental validation
See dCas9VRER below
dCas9 VRER
- Construction
This part was made from the pSB1C3:dCas9QC plasmid (see dCas9) which was completely sequenced. We replaced the final part of the dCas9QC with a gBlock containing the VRER mutations. The new construct was analysed by sequencing the last part containing the gblock (figure 4). Also a restriction analysis with EcoRI and PstI was performed see the gel in dCas9 lane 4.
Based on the validation (see below) we cannot conclude yet whether the VRER mutations have the desired effect. If we would have mixed up the
insert seqeunce file VRER - Experimental validation
This part was validated experimentally in the same experiment as dCas9. For this experiment we have designed four gRNA's that target GFP. Two of this gRNA's bind to sequences flanked by NGG (1&2) and two bind to a sequence flanked by NGCG (3&4). These gRNA's were inserted into the plasmid pSB3C5 containing a pLacGFP construct as well. For dCas9 and dCas9VRER expression those parts were put into a pBad vector in which they are expressed behind an Arabinose inducible promoter.
In total 18 E. coli strains were produced containing pBad:dCas9, pBad:dCas9VRER or no pBad combined with pS3C5 with one of the gRNA's, pSB3C5 without a gRNA or no pSB3C5 (figure 5).
All strains were grown overnight and the next day they were dilute to an OD of 0.05 in the afternoon. At the end of the afternoon at ODof 0.4-0.6 all cultures were induced with a final concentration of 0.01% arabinose. All induced cultures were put back into the incubator (37C 220 rpm) and grown overnight. The next morning the OD of all cultures was measured and to an OD of 0.2 in LB (in final volume of 1mL). The OD and GFP (470/510) of all samples were measured in a plate reader in quadruplicate.
The data obtained from the fluorescence measurement were converted to relative fluorescence and are shown in figure 5. To convert the fluorescence to relative fluorescence we first divided all measured fluorescence values were divided by the measured OD's. Next the fluorescence/OD of the negative controls (samples without pSB1C3) was subtracted from the other samples. Next all normalized fluorescence/OD values were divided by that of the positive control (no pBad:pSB3C5 without gRNA).
From the figure it can be seen that all fluorescence values are quite similar for the samples without gRNA. Further we can see a clear decrease in the relative fluorescence of dCas9 in combination with gRNA 2 and 3. For gRNA's 1 and 4 the relative fluorescence of the dCas9 is similar to that of the samples without the pBad vector. For dCas9VRER we showed CRISPR interference in combination with gRNA's 1,2, and 4.
From the data we cannot conclude that the VRER mutations had any effect on the PAM preference, since dCas9VRER shows the strongest repression for gRNA 1 and 4 of which one has a NGG flanked target. Also gRNA 3 whose target is flanked by NGCG seems to be repressed by dCas9 and not by dCas9 VRER. If these results are correct this would suggest that the PAM preference has not changed for dCas9VRER, however it is quite odd that the differences between dCas9 and dCas9VRER are so big for especially gRNA 1 and gRNA4. Another explanation could be that we mixed up gRNA1 and gRNA3 in one of the steps from gBlock to double transformants. If we would switch around these data (so gRNA1-> gRNA3 and gRNA3-> gRNA1) the data would show that only dCas9VRER can be directed by towards NGCG flanked targets. Unfortunately we could not analyse the sequences of the pSB3C5 plasmids of the cultures used in this experiment before the WIKI-freeze.
insert figure strain table - Considerations for replicating the experiments
Besides sequencing the cultures used in the experiment, performing more experiments with biological replicates would increase the fidellity of the data. Also it would be nice to design more efficient gRNA's that bind between the -35 and -10 region of the promoter. This way we can compare the efficiency of dCas9 and dCas9VRER, since this is the optimal place to target a gRNA.
CRISPR arrays
- Construction
For this part we started by.... - Considerations for replicating the experiments
Before this part can be fully integrated into a system, the next parameters should be measured:... - Future plans for the project
Based on these results we suggest that the next experiments can be conducted:...
Lactis toolbox
- Construction
For this part we started by.... - Experimental validation
After creating the part within the pSB1C3 biobrick format,.... - Considerations for replicating the experiments
Before this part can be fully integrated into a system, the next parameters should be measured:... - Future plans for the project
Based on these results we suggest that the next experiments can be conducted:...
hCas9 operon
- Construction
For this part we started by.... - Considerations for replicating the experiments
Before this part can be fully integrated into a system, the next parameters should be measured:... - Future plans for the project
Based on these results we suggest that the next experiments can be conducted:...
Reporter plasmid
- Construction
For this part we started by.... - Considerations for replicating the experiments
Before this part can be fully integrated into a system, the next parameters should be measured:... - Future plans for the project
Based on these results we suggest that the next experiments can be conducted:...
You should also describe what your results mean:
- Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
- Show data, but remember all measurement and characterization data must be on part pages in the Registry.
- Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.
Project Achievements
You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.
- A list of linked bullet points of the successful results during your project
- A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.
Inspiration
See how other teams presented their results.
