Difference between revisions of "Team:EPF Lausanne/Basic Part"
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| + | <font size="100"><h3>Bikard et al. used dCas9-ω targetting the promoter PAM rich URS J23117, BBa_K1723001, in order to regulate gene expression using gRNA (single guide RNA) guided dCas9-ω. By using our own dCas9-ω system we proved that this promoter can be activated or repressed (see <a href="https://2015.igem.org/Team:EPF_Lausanne/Results">results page</a>). Now, on the model of this promoter, we created <b>a new fully synthetic promoter</b>: PAM rich URS J23117Alt promoter, BBa_K1723005. We mutated the sequence of BBa_K1723001 between and outside the -10 and -35 sequence where the RNA polymerase binds (see <a href="https://2015.igem.org/Team:EPF_Lausanne/Results">BBa_K1723005 registry page</a> for more details)</h3></font> in order to have <b>a promoter targeted by a set of sgRNAs different</b> from the sgRNAs aiming for BBa_K1723001. The creation of this part and its experimental validation (see <a href="https://2015.igem.org/Team:EPF_Lausanne/Results">results page</a>) is very promising for us as it is the proof of the MUTABILITY of the targeted promoters. We can now imagine of mutating again the promoter to obtain others promote/sgRNAs sets creating more and more different transistors-like elements in cells. | ||
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<h2>PAM rich URS J23117Alt promoter</h2> | <h2>PAM rich URS J23117Alt promoter</h2> | ||
| − | <p>This promoter was made on the same template as PAM rich URS J23117 promoter | + | <p>This promoter was made on the same template as PAM rich URS J23117 promoter, BBa_K1723001, but was mutated to be targeted by different sgRNAs. It is also fused to a PAM rich upstream regulatory region (URS). Remember that dCas9 can bind only to PAM-flanked target sites.</p> |
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Revision as of 01:28, 19 September 2015
Bikard et al. used dCas9-ω targetting the promoter PAM rich URS J23117, BBa_K1723001, in order to regulate gene expression using gRNA (single guide RNA) guided dCas9-ω. By using our own dCas9-ω system we proved that this promoter can be activated or repressed (see results page). Now, on the model of this promoter, we created a new fully synthetic promoter: PAM rich URS J23117Alt promoter, BBa_K1723005. We mutated the sequence of BBa_K1723001 between and outside the -10 and -35 sequence where the RNA polymerase binds (see BBa_K1723005 registry page for more details)
in order to have a promoter targeted by a set of sgRNAs different from the sgRNAs aiming for BBa_K1723001. The creation of this part and its experimental validation (see results page) is very promising for us as it is the proof of the MUTABILITY of the targeted promoters. We can now imagine of mutating again the promoter to obtain others promote/sgRNAs sets creating more and more different transistors-like elements in cells.
PAM rich URS J23117Alt promoter
This promoter was made on the same template as PAM rich URS J23117 promoter, BBa_K1723001, but was mutated to be targeted by different sgRNAs. It is also fused to a PAM rich upstream regulatory region (URS). Remember that dCas9 can bind only to PAM-flanked target sites.
