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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 X, 2014</strong><br>TEXT<br>
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- | <strong>September X, 2014</strong><br>TEXT<br>
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- | <strong>September X, 2014</strong><br>TEXT<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>
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- | <p>
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- | content 1 <br>
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- | </p>
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| </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>
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- | </p>
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| </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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- | content 3 <br>
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- | </p>
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| </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
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- | sgadsga
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- | sgsagsa
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- | gasg<br>
| + | |
- | asdfasdf
| + | |
- | adfsf
| + | |
| </div> | | </div> |
| </div> | | </div> |