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| | <div class="tabcontents"> | | <div class="tabcontents"> |
| | <div class="anchor" id="view0"> | | <div class="anchor" id="view0"> |
| - | <h2>CRISPRi</h2> | + | <img src="https://static.igem.org/mediawiki/2014/e/e2/CRISPRi_1.png"></a><br> |
| - | <strong>June 6, 2014</strong><br>
| + | <img src="https://static.igem.org/mediawiki/2014/5/53/CRISPRi_2.png"></a><br> |
| - | Digested pSB1C3 backbone with EcoRI and PstI and gel purified<br>
| + | <img src="https://static.igem.org/mediawiki/2014/d/d6/CRISPR_3.png"></a><br> |
| - | <strong>June 9, 2014</strong><br>
| + | <img src="https://static.igem.org/mediawiki/2014/f/f9/CRISPR_4.png"></a><br> |
| - | Received synthesized sgRNA 1 and sgRNA 2 constructs from BioBasic and transformed the DNA into E. coli DH5α <br>
| + | <img src="https://static.igem.org/mediawiki/2014/1/1d/CRISPR_5.png"></a><br> |
| - | <strong>June 11, 2014</strong><br>
| + | <img src="https://static.igem.org/mediawiki/2014/5/5c/CRISPRi_6.png"></a><br> |
| - | Inoculated sgRNA 1 and 2 colonies from the transformation into over night liquid culture for frozen stocks and miniprep<br>
| + | <img src="https://static.igem.org/mediawiki/2014/8/82/CIRSPRi_7.png"></a><br> |
| - | <strong>June 18, 2014</strong><br>
| + | <img src="https://static.igem.org/mediawiki/2014/0/0b/CRISPRi_8.png"></a><br> |
| - | Digested sgRNA 1 and 2 constructs with EcoRI and PstI, then gel purified the inserts for cloning into pSB1A3
| + | <img src="https://static.igem.org/mediawiki/2014/3/37/CRISPRi_9.png"></a><br> |
| - | Also digested sgRNA 1 with EcoRI and SpeI, and sgRNA 2 with XbaI and PstI for modified 3A assembly into pSB1A3<br>
| + | <p> |
| - | <strong>June 19, 2014</strong><br>
| + | |
| - | Digested pSB1A3 linearized backbone with EcoRI and PstI, then column purified. <br>Ligated pSB1A3 with the sgRNA 1 and 2 (E/P) inserts individually and with both sgRNA 1 (E/S) and sgRNA 2 (X/P) together <br>Ligated pSB1C3 backbone with sgRNA 1 (E/P) and sgRNA 2 (E/P) individually for BioBricks<br>
| + | |
| - | <strong>June 20, 2014</strong><br>
| + | |
| - | Transformed all ligations into E. coli DH5α<br>
| + | |
| - | <strong>June 21, 2014</strong><br>
| + | |
| - | Streak purified 4 colonies from each ligation<br>
| + | |
| - | <strong>June 22, 2014</strong><br>
| + | |
| - | Inoculated 4 colonies of each ligation into overnight liquid culture for miniprep (patched colonies onto agar plates before inoculating<br>
| + | |
| - | <strong>June 23, 2014</strong><br>
| + | |
| - | Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts:
| + | |
| - | -sgRNA 1 in pSB1A3: 1 successful<br>
| + | |
| - | -sgRNA 2 in pSB1A3: 3 successful<br>
| + | |
| - | -sgRNA 1 and sgRNA 2 in pSB1A3: 0 successful<br>
| + | |
| - | -sgRNA 1 in pSB1C3: 1 successful<br>
| + | |
| - | -sgRNA 2 in pSB1C3: 2 successful<br>
| + | |
| - | <strong>June 24, 2014</strong><br>
| + | |
| - | Streak purified 4 more colonies from the sgRNA 1 and sgRNA 2 in pSB1A3 ligation to screen for successful clones<br>
| + | |
| - | <strong>June 25, 2014</strong><br>
| + | |
| - | Inoculated the 4 clones of sgRNA 1 and sgRNA 2 in pSB1A3 for miniprep (patched colonies onto agar plates before inoculating)<br>
| + | |
| - | <strong>June 26, 2014</strong><br>
| + | |
| - | Miniprepped and did a test digest to screen for clones with the correct insert size: 0 successful<br>
| + | |
| - | Decided to wait for the sgRNA 1 in pSB1A3 to be sequence confirmed and then clone in sgRNA 2 instead of doing modified 3A assembly<br>
| + | |
| - | <strong>July 3, 2014</strong><br>
| + | |
| - | Inoculated successful clones from patches into over night liquid culture to miniprep and send for sequencing<br>
| + | |
| - | <strong>July 4, 2014</strong><br>
| + | |
| - | Miniprepped and sent clones for sequencing<br>
| + | |
| - | <strong>July 9, 2014</strong><br>
| + | |
| - | Received synthesized dCas9 cassette from BioBasic and transformed the DNA into E. coli DH5α<br>
| + | |
| - | <strong>July 10, 2014</strong><br>
| + | |
| - | Digested the dCas9 construct with EcoRI and PstI then gel purified the insert<br>
| + | |
| - | Digested more pSB1A3 and pSB1C3 with EcoRI and PstI and gel purified the backbone.<br>
| + | |
| - | Ligated the dCas9 cassette (E/P) into pSB1A3 (E/P) and pSB1C3 (E/P)<br>
| + | |
| - | <strong>July 11, 2014</strong><br>
| + | |
| - | Transformed all ligations into E. coli DH5α<br>
| + | |
| - | <strong>July 12, 2014</strong><br>
| + | |
| - | Streak purified 8 colonies from each ligation<br>
| + | |
| - | <strong>July 13, 2014</strong><br>
| + | |
| - | Inoculated the colonies into over night liquid cultures for miniprep (patched colonies onto agar plates before inoculating)<br>
| + | |
| - | <strong>July 14, 2014</strong><br>
| + | |
| - | Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts:<br>
| + | |
| - | - dCas9 cassette in pSB1A3: 0 successful<br>
| + | |
| - | - dCas9 cassette in pSB1C3 : 2 successful<br>
| + | |
| - | Ran digests of sgRNA for gel extraction<br>
| + | |
| - | <strong>July 15, 2014</strong><br>
| + | |
| - | Streak purified 8 colonies from dCas9 pSB1A3<br>
| + | |
| - | Gel extracted sgRNA<br>
| + | |
| - | Restriction digest and gel extraction of dsRed-ermM-oriV, ermM-oriV, pSB1C3, pSB1A3<br>
| + | |
| - | <strong>July 16, 2014</strong><br>
| + | |
| - | Inoculated and patched 8 colonies of dCas9 pSB1A3<br>
| + | |
| - | Miniprepped sequence-confirmed sgRNA strains, pSB1C3, pSB1A3, ermM-oriV, dsRed-ermM-oriV<br>
| + | |
| - | Digested, column purified, and ligated<br>
| + | |
| - | <strong>July 17, 2014</strong><br>
| + | |
| - | Miniprep and restriction digest of 8 colonies of dCas9 pSB1A3: 0 successful<br>
| + | |
| - | Transformed ligations of sgRNA, pSB1C3, pSB1A3, and ermM-oriV<br>
| + | |
| - | <strong>July 18, 2014</strong><br>
| + | |
| - | Streak purified 4 colonies for each ligation<br>
| + | |
| - | <strong>July 19, 2014</strong><br>
| + | |
| - | Miniprepped and digested:<br>
| + | |
| - | sgRNA, ermM-oriV, sgRNA-ermM-oriV, dCas9-ermM-oriV<br>
| + | |
| - | sgRNA1+sgRNA2+pSB1A3: 2 successful<br>
| + | |
| - | sgRNA1+pSB1C3: 4 successful<br>
| + | |
| - | ermM-oriV-pSB1A3: 1 successful<br>
| + | |
| - | sgRNA1-ermM-oriV-pSB1A3: 0 successful<br>
| + | |
| - | sgRNA2-ermM-oriV-pSB1A3: 1 successful<br>
| + | |
| - | dCas9-ermM-oriV-pSB1A3: 2 successful<br>
| + | |
| - | <strong>July 22, 2014</strong><br>
| + | |
| - | ermM-oriV PCR<br>
| + | |
| - | <strong>July 24, 2014</strong><br>
| + | |
| - | ermM-oriV restriction digest and ligation in pSB1A3<br>
| + | |
| - | Miniprep and digest: spc construct-pUC57, dCas9 cassette<br>
| + | |
| - | dsRed-ermM-oriV in pUC57 digest and gel extraction<br>
| + | |
| - | <strong>July 25, 2014</strong><br>dsRed-ermM-oriV digest and transformation into DC10B<br>
| + | |
| - | Transformation: ermM-oriV-pSB1A3 4 colonies<br>
| + | |
| - | <strong>July 27, 2014</strong><br>Miniprep and digest of:<br>
| + | |
| - | ermM-oriV-pSB1A3: 3 successful<br>
| + | |
| - | dsRed-ermM-oriV-pUC57-DC10B: 3 successful<br>
| + | |
| - | <strong>July 28, 2014</strong><br>
| + | |
| - | Gel extracted dCas9-ermM-oriV<br>
| + | |
| - | <strong>July 29, 2014</strong><br>
| + | |
| - | Ligation of dCas9-ermM-oriV with sgRNA<br>
| + | |
| - | <strong>July 30, 2014</strong><br>
| + | |
| - | Restriction digest, column purification and ligation of dCas9-ermM-oriV and sgRNA<br>
| + | |
| - | <strong>July 31, 2014</strong><br>
| + | |
| - | Transformed and inoculated dCas9-ermM-oriV to DH5α and pSB1A3<br>
| + | |
| - | <strong>August 1, 2014</strong><br>
| + | |
| - | Miniprepped and picked colonies from transformation, then grown in LB + Amp<br>
| + | |
| - | <strong>August 2, 2014</strong><br>
| + | |
| - | Miniprepped and digested:<br>
| + | |
| - | dCas9-ermM-oriV+sgRNA1: 1 successful<br>
| + | |
| - | dCas9-ermM-oriV+sgRNA2: 1 successful<br>
| + | |
| - | dCas9-ermM-oriV+sgRNA1+sgRNA2: 2 successful<br>
| + | |
| - | dCas9-ermM-oriV+pSB1A3: 1 successful<br>
| + | |
| - | <strong>August 5, 2014</strong><br>
| + | |
| - | PCR of ermM-oriV<br>
| + | |
| - | <strong>August 6, 2014</strong><br>
| + | |
| - | PCR ermM and dsRed: 0 successful<br>
| + | |
| - | Ligation and digest for ermM-oriV, sgRNA and pSB1A3<br>
| + | |
| - | ermM-oriV column purified<br>
| + | |
| - | <strong>August 8, 2014</strong><br>
| + | |
| - | Transformation of ermM-oriV+pSB1A3 into DH5α<br>
| + | |
| - | Many colonies, of which 6 were picked<br>
| + | |
| - | PCR ermM and dsRed<br>
| + | |
| - | <strong>August 9, 2014</strong><br>Miniprep and digest of ermM-oriV+pSB1A3: 0 successful<br>
| + | |
| - | <strong>August 11, 2014</strong><br>ermM and dsRed column purified: 0 successful<br>
| + | |
| - | <strong>August 12, 2014</strong><br>
| + | |
| - | Transformed:<br>
| + | |
| - | DsRed-ermM-oriV into DH5α<br>
| + | |
| - | DsRed CDS into pSB1C3<br>
| + | |
| - | ermM CDS into pSB1C3<br>
| + | |
| - | Colony PCR of:<br>
| + | |
| - | ermM-oriV+pSB1A3<br>
| + | |
| - | dCas9-ermM-oriV+pSB1A3<br>
| + | |
| - | <strong>August 13, 2014</strong><br>
| + | |
| - | Miniprepped, made stocks and picked colonies:<br>
| + | |
| - | sgRNA1-dCas9-ermM-oriV<br>
| + | |
| - | sgRNA2-dCas9-ermM-oriV<br>
| + | |
| - | sgRNA1-sgRNA2-dCas9-ermM-oriV<br>
| + | |
| - | Inoculated:<br>
| + | |
| - | YFP construct<br>
| + | |
| - | aadD construct-pUC57<br>
| + | |
| - | spc construct-pUC57<br>
| + | |
| - | dsRed-ermM-oriV-pUC57 in DH5α<br>
| + | |
| - | ermM-oriV-pSB1A3<br>
| + | |
| - | dCas9-ermM-oriV-pSB1A3<br>
| + | |
| - | <strong>August 14, 2014</strong><br>Restriction digest of inoculated samples: 4 successful<br>
| + | |
| - | <strong>August 19, 2014</strong><br>Digested dsRed-ermM-oriV<br>
| + | |
| - | <strong>September 4, 2014</strong><br>
| + | |
| - | Transformed J23101 and GFP into DH5α and plated on kanamycin plates<br>
| + | |
| - | Streak purified DsRed-ermM-oriV and sgRNA from stock<br>
| + | |
| - | <strong>September 5, 2014</strong><br>
| + | |
| - | Testing erythromycin resistance in e. coli <br>
| + | |
| - | DsRed-ermM-oriV<sub><i>S. aureus</i></sub> in pUC57, DC10B<br>
| + | |
| - | sgRNA in pUC57, DH5α (negative control)<br>
| + | |
| - | negative control had growth, so erythromycin stock was remade<br>
| + | |
| - | PCR of dsRed<br>
| + | |
| - | <strong>September 6, 2014</strong><br>Inoculated and miniprepped J23101, GFP<br>
| + | |
| - | <strong>September 8, 2014</strong><br>PCR of xylose promoter: no products<br>
| + | |
| - | oriV – PCR, column purification, and digest with PstI and EcoRI<br>
| + | |
| - | <strong>September 9, 2014</strong><br>Ligation of oriV<br>
| + | |
| - | Colony PCR for ermM-oriV+pSB1A3 and dCas9-ermM-oriV+pSB1A3: no amplification seen<br>
| + | |
| - | Gel extraction of DsRed-ermM-oriV<br>
| + | |
| - | <strong>September 10, 2014</strong><br>
| + | |
| - | Colony PCR testing KOD and Taq<br>
| + | |
| - | PCR of xylose promoter<br>
| + | |
| - | <strong>September 11, 2014</strong><br>
| + | |
| - | Gel extraction of xylose promoter<br>
| + | |
| - | Inoculated oriV to LB with chloramphenicol <br>
| + | |
| - | <strong>September 12, 2014</strong><br>
| + | |
| - | Digested xylose promoter with EcoRI and SpeI<br>
| + | |
| - | Digested oriV pSK41 with EcoRI and PstI<br>
| + | |
| - | Restriction digest and ligation of ermM-oriV with pSB1A3:
| + | |
| - | 0 successful<br>
| + | |
| - | <strong>September 13, 2014</strong><br>
| + | |
| - | Heat shock transformation of ermM-oriV + pSB1A3 into DH5α and gel extracted<br>
| + | |
| - | <strong>September 14, 2014</strong><br>Restriction digest, gel extraction, and ligation of ermM-oriV-pSB1A3 in DH5α<br>
| + | |
| - | Inoculation and 10 colonies picked<br>
| + | |
| - | PCR of dsRed-oriV-ermM<br>
| + | |
| - | <strong>September 15, 2014</strong><br>
| + | |
| - | Gel extraction of PCR<br>
| + | |
| - | Miniprep of 10 colonies<br>
| + | |
| - | PCR, diagnostic digest of miniprepped ermM-oriV-pSB1A3:
| + | |
| - | 2 successful<br>
| + | |
| - | <strong>September 16, 2014</strong><br>
| + | |
| - | Digest of oriV: 0 successful<br>
| + | |
| - | Streak purified:<br>
| + | |
| - | pSB1C3+RFP<br>
| + | |
| - | J23101-GFP<br>
| + | |
| - | sgRNA<br>
| + | |
| - | dsRed-ermM-oriV<br>
| + | |
| - | Inoculated and patched 12 oriV-pSB1C3 colonies<br>
| + | |
| - | <strong>September 17, 2014</strong><br>
| + | |
| - | Xylose promoter-GFP miniprep and digest: 0 successful <br>
| + | |
| - | ermM-oriV-pSB1A3 digest: 0 successful<br>
| + | |
| - | Restriction digest of sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV cut with EcoRI and PstI:
| + | |
| - | 1 successful<br>
| + | |
| - | <strong>September 18, 2014</strong><br>Miniprep and restriction digest of oriV: 7 successful<br>
| + | |
| - | <strong>September 19, 2014</strong><br>
| + | |
| - | Xylose promoter and dsRed PCR: successful<br>
| + | |
| - | Miniprep of ermM-oriV and sgRNA1-dCas9-ermM-oriV<br>
| + | |
| - | <strong>September 22, 2014</strong><br>
| + | |
| - | Gel extraction and PCR of xylose promoter, dsRed, ermM<br>
| + | |
| - | Restriction digest of ermM-oriV<br>
| + | |
| - | PCR of pSB1C3 and dsRed-ermM-oriV<br>
| + | |
| - | <strong>September 23, 2014</strong><br>
| + | |
| - | Miniprep of xylose promoter, oriV<br>
| + | |
| - | Restriction digest of xylose promoter: 0 successful<br>
| + | |
| - | Gel extraction of pSB1C3 and dsRed-ermM-oriV<br>
| + | |
| - | Restriction digest of pSB1C3 shuttle vector, ermM-oriV shuttle vector, sarA promoter, xylose promoter, dsRed cassette, ermM cassette<br>
| + | |
| - | <strong>September X, 2014</strong><br>TEXT<br>
| + | |
| - | <strong>September X, 2014</strong><br>TEXT<br>
| + | |
| - | </div>
| + | |
| - | <div class="anchor" id="view1">
| + | |
| - | <h2>RNAi</h2>
| + | |
| - | <p>
| + | |
| - | content 1 <br>
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| - | content 1 <br>
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| - | content 1 <br>
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| - | </p>
| + | |
| | </div> | | </div> |
| | <div class="anchor" id="view2"> | | <div class="anchor" id="view2"> |
| - | <h2>YFP</h2> | + | <img src="https://static.igem.org/mediawiki/2014/1/1d/YFP_1.png"></a><br> |
| - | <p>
| + | <img src="https://static.igem.org/mediawiki/2014/5/59/YFP_2.png"></a><br> |
| - | content 2 <br>
| + | <img src="https://static.igem.org/mediawiki/2014/0/00/YFP_3.png"></a><br> |
| - | content 2 <br>
| + | <img src="https://static.igem.org/mediawiki/2014/1/18/YFP_4.png"></a><br> |
| - | content 2 <br>
| + | <img src="https://static.igem.org/mediawiki/2014/9/96/YFP_5.png"></a><br> |
| - | content 2 <br>
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| - | content 2 <br>
| + | |
| - | </p>
| + | |
| | </div> | | </div> |
| | <div class="anchor" id="view3"> | | <div class="anchor" id="view3"> |
| - | <h2>CONJUGATION</h2> | + | <img src="https://static.igem.org/mediawiki/2014/2/28/Conjugation_1.png"></a><br> |
| - | <p>
| + | <img src="https://static.igem.org/mediawiki/2014/0/0a/Conjugation_2.png"></a><br> |
| - | content 3 <br>
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| - | </p>
| + | |
| | </div> | | </div> |
| | <div class="anchor" id="view4"> | | <div class="anchor" id="view4"> |
| - | <h2>OTHER</h2> | + | <img src="https://static.igem.org/mediawiki/2014/8/8e/Other_1.png"></a><br> |
| - | asdfasdfasdfasdfasgasdgasdg
| + | <img src="https://static.igem.org/mediawiki/2014/7/7d/Other_2.png"></a><br> |
| - | asgasdgad
| + | |
| - | sgadsga
| + | |
| - | sgsagsa
| + | |
| - | gasg<br>
| + | |
| - | asdfasdf
| + | |
| - | adfsf
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
"
