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| Line 31: |
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| | <img src="https://static.igem.org/mediawiki/2014/3/37/CRISPRi_9.png"></a><br> | | <img src="https://static.igem.org/mediawiki/2014/3/37/CRISPRi_9.png"></a><br> |
| | <p> | | <p> |
| - | <strong>June 6, 2014</strong><br>
| |
| - | Digested pSB1C3 backbone with EcoRI and PstI and gel purified<br>
| |
| - | <strong>June 9, 2014</strong><br>
| |
| - | Received synthesized sgRNA 1 and sgRNA 2 constructs from BioBasic and transformed the DNA into <i>E. coli</i> DH5α <br>
| |
| - | <strong>June 11, 2014</strong><br>
| |
| - | Inoculated sgRNA 1 and 2 colonies from the transformation into over night liquid culture for frozen stocks and miniprep<br>
| |
| - | <strong>June 18, 2014</strong><br>
| |
| - | Digested sgRNA 1 and 2 constructs with EcoRI and PstI, then gel purified the inserts for cloning into pSB1A3
| |
| - | Also digested sgRNA 1 with EcoRI and SpeI, and sgRNA 2 with XbaI and PstI for modified 3A assembly into pSB1A3<br>
| |
| - | <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 <i>E. coli</i> 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 <i>E. coli</i> 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 <i>E. coli</i> 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 <i>E. coli</i> <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>
| |
| - | Heat inactivated sarA promoter<br>
| |
| - | Column purified all the digests<br>
| |
| - | <strong>September 24, 2014</strong><br>
| |
| - | GFP miniprep and digest with EcoRI-HF and XBaI<br>
| |
| - | Ligations of digests<br>
| |
| - | Inoculation of J23101-GFP, dsRed-ermM-oriV, sgRNA to check fluorescence<br>
| |
| - | Transformation into DH5α: shuttle vector, xylose promoter-pSB1C3, dsRed, ermM, sarA promoter, xylose promoter, shuttle vector control, pSB1C3 control, GFP control<br>
| |
| - | <strong>September 27, 2014</strong><br>
| |
| - | PCR of ermM-oriV and pSB1A3<br>
| |
| - | Restriction digest:<br>
| |
| - | xylose promoter-GFP with 5 successful<br>
| |
| - | sarA promoter-GFP with 5 successful<br>
| |
| - | shuttle vector with RFP with 1 successful<br>
| |
| - | xylose promoter with 1 successful<br>
| |
| - | GFP (negative control)<br>
| |
| - | Inoculated and patched ermM-oriV-pSB1A3<br>
| |
| - | Inoculated oriV, xylose promoter-GFP, sarA promoter-GFP, shuttle vector-RFP, sgRNA1, sgRNA2 <br>
| |
| - | <strong>September 27, 2014</strong><br>
| |
| - | PCR of pSB3K3, ermM-oriV with prefix/suffix, ermM-oriV with BamHI/BglII, pSB1A3 with BamHI/BglII<br>
| |
| - | Miniprepped oriV, xylose promoter-GFP, sarA promoter-GFP, shuttle vector-RFP, xylose promoter<br>
| |
| - | <strong>October 1, 2014</strong><br>Inoculated xylose promoter-GFP, sarA promoter-GFP, J23101-GFP, DH5α<br>
| |
| - | Miniprep of dsRed-ermM-oriV and RFP<br>
| |
| - | <strong>October 2, 2014</strong><br>
| |
| - | Diluted cultures 1/100 and incubated for 3 hours and read with plate reader for fluorescence and again after half an hour for xylose promter-GFP, xylose promoter-GFP with xylose, sarA promoter-GFP, J23101-GFP, DH5α<br>
| |
| - | Fluorescence seen in J23101-GFP, sarA promoter and in xylose promoter-GFP with xylose<br>
| |
| - | <strong>October 3, 2014</strong><br>
| |
| - | Digest of dsRed-ermM-oriV<br>
| |
| - | <strong>October 5, 2014</strong><br>
| |
| - | Streak purified DH5α, J23101-GFP, GFP, xylose promoter-GFP, sarA promoter-GFP<br>
| |
| - | <strong>October 9, 2014</strong><br>
| |
| - | Inoculated sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-ermM-oriV, dCas9-ermM-oriV<br>
| |
| - | Inoculated J23101-GFP, GFP, DH5α, xylose promoter-GFP, sarA promoter-GFP, dsRed-ermM-oriV<sub><i>S.epidermidis</i></sub>, dsRed-ermM-oriVDC10B, <i>S.epidermidis</i>, YFP cassette, YFP-F, hfq cassette, oriV, RFP, xylose promoter, sarA promoter<br>
| |
| - | Digest of dCas9-ermM-oriV, sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV<br>
| |
| - | <strong>October 10, 2014</strong><br>
| |
| - | Gel extraction of dCas9-ermM-oriV, sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV<br>
| |
| - | Ligation of dCas9-ermM-oriV in pSB1A3<br>
| |
| - | Transformation of dCas9-ermM-oriV-pSB1A3 into DH5α and DC10B<br>
| |
| - | Measure absorbance spectra for dsRed-ermM-oriV<br>
| |
| - | <strong>October 13, 2014</strong><br>
| |
| - | Ligation of dCas9-ermM-oriV and pSB1A3<br>
| |
| - | <strong>October 14, 2014</strong><br>
| |
| - | Digest for CRISPR assay <br>
| |
| - | co-transformation successful<br>
| |
| - | Promoter characterization:<br>
| |
| - | 3 colonies each :xylose promoter-GFP, sarA promoter-GFP, J23101-GFP, GFP, DH5α, xylose promoter-GFP with xylose<br>
| |
| - | Inoculated from overnight cultures into 5ml LB in 1/100 dilution with antibiotics<br>
| |
| - | Incubated for 3 hours and read absorbance and fluorescence on plate reader<br>
| |
| - | Xylose promoter unsuccessful – use fresh xylose next time<br>
| |
| - | Reinoculated cultures for next day<br>
| |
| - | <strong>October 15, 2014</strong><br>
| |
| - | CRISPR assay<br>
| |
| - | Inoculated overnight cultures to fresh LB + antibiotic + xylose in triplicate at 1/100 dilution<br>
| |
| - | Grew until 0.5-0.6 OD and read fluorescence with plate reader unsuccessful<br>
| |
| - | </div>
| |
| - | <div class="anchor" id="view1">
| |
| - | <img src="https://static.igem.org/mediawiki/2014/e/ea/RNAi_1.png"></a><br>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/3/32/RNAi_2.png"></a><br>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/a/a5/RNAi_3.png"></a><br>
| |
| - | <p>
| |
| - | <strong>June 10, 2014</strong><br>
| |
| - | Received pJB38 integrative plasmid, pKAN, pSPC, pYFP-F, pYFP-R, pRFP-F, and pRFP-R from Dr. Bose, Dr. Bayles, and Dr. Endres from the University of Nebraska, Nebraska, U.S.A in E. coli DH5α. Inoculated colonies into over night liquid cultures for frozen stocks and miniprep<br>
| |
| - | <strong>June 16, 2014</strong><br>
| |
| - | Received synthesized YFP construct (1st half of integration insert) from BioBasic and transformed the DNA into E. coli DH5α<br>
| |
| - | <strong>June 18, 2014</strong><br>
| |
| - | Inoculated 4 colonies of the YFP construct from the transformation into over night liquid culture for frozen stocks and miniprep<br>
| |
| - | <strong>June 19, 2014</strong><br>
| |
| - | Minipreped the 4 cultures and did a diagnostic digest before making frozen stocks<br>
| |
| - | <strong>June 20, 2014</strong><br>
| |
| - | Received the synthesized 2nd half of the integration insert containing the aadD gene from BioBasic. Transformed DNA into E. coli DH5α<br>
| |
| - | <strong>June 21, 2014</strong><br>
| |
| - | Streak purified 4 colonies of aadD containing construct<br>
| |
| - | <strong>June 22, 2014</strong><br>
| |
| - | Inoculated the 4 colonies of aadD containing construct into over night liquid cultures for miniprep and frozen stocks<br>
| |
| - | <strong>June 23, 2014</strong><br>
| |
| - | Minipreped the 4 cultures and did a diagnostic digest before making frozen stocks
| |
| - | Digested the following for modified 3A assembly:<br>
| |
| - | - YFP construct with EcoRI and SpeI<br>
| |
| - | - aadD containing construct with XbaI and SalI<br>
| |
| - | - pJB38 backbone with EcoRI and SalI<br>
| |
| - | Gel purified the inserts and ligated all the three parts together<br>
| |
| | </div> | | </div> |
| | <div class="anchor" id="view2"> | | <div class="anchor" id="view2"> |
"
