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<center><img src="https://static.igem.org/mediawiki/2016/1/1e/Cultures_Experiment_page.jpeg" alt="width:200px;"></center> | <center><img src="https://static.igem.org/mediawiki/2016/1/1e/Cultures_Experiment_page.jpeg" alt="width:200px;"></center> | ||
− | < | + | <h3> Testing dCas9 clamps <i>in vitro</i> </h3> |
<h5> For cultures containing strains with plasmids that had a dCas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. In order to run our plasmids containing the dCas9 clamp site, we also had to design gRNA plasmids and obtain dCas9 expression plasmids (<a href="https://www.addgene.org/48657/">DS-SPCasN-</a>). All three of these plasmids were miniprepped and then expressed using TX-TL, an in vitro, prototyping technique which mimics cell environments for transcription and translation. For our first run we did not use inducer, but for our second run we used ATc and IPTG to induce the TetR and LacI promoters on the GFP and RFP devices, respectively. In the plate reader we obtained results that measured GFP and RFP expression. (see <a href="https://2016.igem.org/Team:Alverno_CA/Results">Results</a>) Once again, because the GFP and RFP devices were placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (See our <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids) Protocol</a> for more information)</h5> | <h5> For cultures containing strains with plasmids that had a dCas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. In order to run our plasmids containing the dCas9 clamp site, we also had to design gRNA plasmids and obtain dCas9 expression plasmids (<a href="https://www.addgene.org/48657/">DS-SPCasN-</a>). All three of these plasmids were miniprepped and then expressed using TX-TL, an in vitro, prototyping technique which mimics cell environments for transcription and translation. For our first run we did not use inducer, but for our second run we used ATc and IPTG to induce the TetR and LacI promoters on the GFP and RFP devices, respectively. In the plate reader we obtained results that measured GFP and RFP expression. (see <a href="https://2016.igem.org/Team:Alverno_CA/Results">Results</a>) Once again, because the GFP and RFP devices were placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (See our <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids) Protocol</a> for more information)</h5> | ||
Revision as of 23:08, 19 October 2016