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Team:Hong Kong HKUST/wetlab/notebook
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· Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600<br> | · Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600<br> | ||
· Transformation of BBa_B0034 and BBa_E0040 into DH10B<br> | · Transformation of BBa_B0034 and BBa_E0040 into DH10B<br> | ||
| - | + | ||
| - | + | ||
<br><b>June 10</b> <br> | <br><b>June 10</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Found reference promoter J23101 in BBa_I20260 from part registry<br> | · Found reference promoter J23101 in BBa_I20260 from part registry<br> | ||
| - | · Looked for | + | · Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br> |
· Bacterial inoculation of BBa_B0034 and BBa_E0040<br> | · Bacterial inoculation of BBa_B0034 and BBa_E0040<br> | ||
| - | + | ||
<br><b>June 11</b> <br> | <br><b>June 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Looked for | + | · Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br> |
· Studied the Characteristic, function and mechnism of how promoters work<br> | · Studied the Characteristic, function and mechnism of how promoters work<br> | ||
· Miniprep of BBa_B0034 and BBa_E0040<br> | · Miniprep of BBa_B0034 and BBa_E0040<br> | ||
| - | + | ||
<br><b>June 12</b> <br> | <br><b>June 12</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Looked for | + | · Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br> |
| - | · research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization<br> | + | · research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of p~<i>comE</i> to use for characterization<br> |
· explain of scar<br> | · explain of scar<br> | ||
| - | + | ||
<br><b>June 13</b> <br> | <br><b>June 13</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Looked for | + | · Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br> |
| - | · research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization<br> | + | · research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of p~<i>comE</i> to use for characterization<br> |
| - | + | ||
| - | + | ||
</div> | </div> | ||
</div> | </div> | ||
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<br><b>June 16</b> <br> | <br><b>June 16</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for | + | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br> |
| - | · Found a possible | + | · Found a possible P<sub>combox</sub><br> |
· Transformation of BBa_J04450 and BBa_I20260 into DH10B<br> | · Transformation of BBa_J04450 and BBa_I20260 into DH10B<br> | ||
· Bacterial inoculation of BBa_E0240<br> | · Bacterial inoculation of BBa_E0240<br> | ||
| Line 142: | Line 141: | ||
<br><b>June 17</b> <br> | <br><b>June 17</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for | + | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br> |
| - | · Found a possible | + | · Found a possible P<sub>combox</sub><br> |
| - | · Searched for other possible | + | · Searched for other possible P<sub>combox</sub> sequences<br> |
· Miniprep of BBa_E0240<br> | · Miniprep of BBa_E0240<br> | ||
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br> | · Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br> | ||
| Line 150: | Line 149: | ||
<br><b>June 18</b> <br> | <br><b>June 18</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Designed primers for the | + | · Designed primers for the P<sub>combox</sub> sequences<br> |
| - | · Received a | + | · Received a P<sub>combox</sub> sequence from Wellcome Trust Sanger Institute<br> |
· Miniprep of BBa_J04450(CHL) and BBa_I20260<br> | · Miniprep of BBa_J04450(CHL) and BBa_I20260<br> | ||
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br> | · Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br> | ||
| Line 165: | Line 164: | ||
· <br> | · <br> | ||
<br><b>June 20</b> <br> | <br><b>June 20</b> <br> | ||
| - | · Amended primers of | + | · Amended primers of P<sub>combox</sub> sequences<br> |
· Miniprep of BBa_J04450(CHL)<br> | · Miniprep of BBa_J04450(CHL)<br> | ||
· Digestion of BBa_J04450(CHL)<br> | · Digestion of BBa_J04450(CHL)<br> | ||
· Gel electrophoresis of digested product<br> | · Gel electrophoresis of digested product<br> | ||
| - | · Designed primers for cloning <i>comD</i> and <i>comE</i><br> | + | · Designed primers for cloning <i>comD</i> and <i><i>comE</i></i><br> |
| Line 180: | Line 179: | ||
Wet lab<br> | Wet lab<br> | ||
· Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br> | · Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br> | ||
| - | · Primer design for PCR of comX-myc and comW-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br> | + | · Primer design for PCR of comX-myc and <i>comW</i>-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br> |
| - | · Designed plasmid construct for | + | · Designed plasmid construct for P<sub>combox</sub><br> |
· Bacterial inoculation of BBa_J04450(CHL)<br> | · Bacterial inoculation of BBa_J04450(CHL)<br> | ||
| - | + | ||
| - | + | ||
| - | + | ||
· <br> | · <br> | ||
<br><b>June 24</b> <br> | <br><b>June 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Inoculation of BBa_B0015<br> | · Inoculation of BBa_B0015<br> | ||
| - | · Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp | + | · Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br> |
| - | · Designed forward and reverse oligos of 67bp | + | · Designed forward and reverse oligos of 67bp P<sub>combox</sub><br> |
· Transformation of pSB1C3-BBa_I13504<br> | · Transformation of pSB1C3-BBa_I13504<br> | ||
· Miniprep of BBa_J04450(CHL)<br> | · Miniprep of BBa_J04450(CHL)<br> | ||
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· Transformation of BBa_J04450(KAN)<br> | · Transformation of BBa_J04450(KAN)<br> | ||
· Bacterial inoculation of BBa_J04450(CHL)<br> | · Bacterial inoculation of BBa_J04450(CHL)<br> | ||
| - | |||
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Gel check of purified BBa_B0015 insert<br> | · Gel check of purified BBa_B0015 insert<br> | ||
· Transformation of ligated pSB1C3-K880005.B0015<br> | · Transformation of ligated pSB1C3-K880005.B0015<br> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
· <br> | · <br> | ||
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<br> <b> July 2</b> <br> | <br> <b> July 2</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Arrival of primers order for comX-myc and comW-FLAG<br> | + | · Arrival of primers order for comX-myc and <i>comW</i>-FLAG<br> |
| - | · Dilution of primers and design of experiment for both comX-myc and comW-FLAG<br> | + | · Dilution of primers and design of experiment for both comX-myc and <i>comW</i>-FLAG<br> |
· Inoculation of ligated pSB1C3-K880005.B0015<br> | · Inoculation of ligated pSB1C3-K880005.B0015<br> | ||
· Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br> | · Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br> | ||
· Oligo annealing for PcomX<br> | · Oligo annealing for PcomX<br> | ||
· Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)<br> | · Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)<br> | ||
| - | · PCR for cloning <i>comD</i> and <i>comE</i><br> | + | · PCR for cloning <i>comD</i> and <i><i>comE</i></i><br> |
| Line 291: | Line 281: | ||
· Gel check for comX-myc PCR product<br> | · Gel check for comX-myc PCR product<br> | ||
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br> | · Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br> | ||
| - | · Received and stored the new common reverse primers for | + | · Received and stored the new common reverse primers for P<sub>combox</sub> and sense oligo and anti-sense oligo of 67bp P<sub>combox</sub><br> |
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Gel purification of digested BBa_E0240 and BBa_J04450<br> | · Gel purification of digested BBa_E0240 and BBa_J04450<br> | ||
| Line 300: | Line 290: | ||
· E0240:Plasmid extraction ; Digestion; Gel electrophoresis; Gel extraction ; Gel purification<br> | · E0240:Plasmid extraction ; Digestion; Gel electrophoresis; Gel extraction ; Gel purification<br> | ||
· B0015: Plasmid extraction ; Nanodrop<br> | · B0015: Plasmid extraction ; Nanodrop<br> | ||
| - | · | + | · <i>comE<sup>D58E</sup></i>: PCR<br> |
· <br> | · <br> | ||
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· PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | · PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | ||
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · | + | · <i>comE<sup>D58E</sup></i> :Phusion PCR<br> |
· E0240: Gel purification<br> | · E0240: Gel purification<br> | ||
| - | |||
| - | |||
| - | |||
| - | |||
· <br> | · <br> | ||
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· Digestion of comX-myc with XbaI and PstI<br> | · Digestion of comX-myc with XbaI and PstI<br> | ||
· Gel check of digested comX-myc, digestion clean up<br> | · Gel check of digested comX-myc, digestion clean up<br> | ||
| - | · PCR of comW-FLAG using Vent DNA Polymerase<br> | + | · PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br> |
| - | · Gel check for comW-FLAG PCR product, PCR clean up<br> | + | · Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br> |
· Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br> | · Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br> | ||
· Gel purification of digested BBa_E0240 and BBa_J04450<br> | · Gel purification of digested BBa_E0240 and BBa_J04450<br> | ||
| - | · Submitted primers for Gibson assembly of pSB3K3- | + | · Submitted primers for Gibson assembly of pSB3K3-100bpP<sub>combox</sub><br> |
· Inoculation of pSB3K3-BBa_J04450<br> | · Inoculation of pSB3K3-BBa_J04450<br> | ||
· E0240 - Bacterial inoculation<br> | · E0240 - Bacterial inoculation<br> | ||
· pSB1C3-PcomX: Bacterial inoculation<br> | · pSB1C3-PcomX: Bacterial inoculation<br> | ||
· pSB1C3- PcomCDE: Bacterial inoculation, Streaking<br> | · pSB1C3- PcomCDE: Bacterial inoculation, Streaking<br> | ||
| - | · | + | · <i>comE<sup>D58E</sup></i> :PCR cleanup; Digestion; Ligation with pSB1C3; Transformation<br> |
· <br> | · <br> | ||
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· pSB1C3-PcomX: Plasmid extraction<br> | · pSB1C3-PcomX: Plasmid extraction<br> | ||
· pSB1C3- PcomCDE: Plasmid extraction<br> | · pSB1C3- PcomCDE: Plasmid extraction<br> | ||
| - | · | + | · <i>comE<sup>D58E</sup></i>: Bacterial inoculation; Ligation; Transformation<br> |
· <br> | · <br> | ||
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· Gel purification of digested BBa_E0240<br> | · Gel purification of digested BBa_E0240<br> | ||
· Gel check for BBa_E0240 insert and pSB3K3 backbone<br> | · Gel check for BBa_E0240 insert and pSB3K3 backbone<br> | ||
| - | · PCR of 100bp | + | · PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br> |
· Inoculation of pCEPcin<br> | · Inoculation of pCEPcin<br> | ||
· E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis<br> | · E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis<br> | ||
· J04450 (KAN): Gel purification<br> | · J04450 (KAN): Gel purification<br> | ||
| - | · | + | · <i>comE<sup>D58E</sup></i>:Plasmid extraction; NanoDrop; Gel electrophoresis (not <i>comE</i>); Bacterial inoculation<br> |
| - | + | ||
| - | + | ||
| - | + | ||
<br> <b> July 10</b> <br> | <br> <b> July 10</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Another PCR of comW-FLAG using Vent DNA Polymerase<br> | + | · Another PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br> |
| - | · Gel check for comW-FLAG PCR product, PCR clean up<br> | + | · Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br> |
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | · Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | ||
· Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br> | · Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br> | ||
· Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI<br> | · Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI<br> | ||
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · PCR of 100bp | + | · PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br> |
· Ethanol precipitation of BBa_E0240 insert<br> | · Ethanol precipitation of BBa_E0240 insert<br> | ||
· Ligate E0240-PcomX & PcomCDE <br> | · Ligate E0240-PcomX & PcomCDE <br> | ||
· Extract J04450 (KAN)<br> | · Extract J04450 (KAN)<br> | ||
| - | · Restrict check & run mutagenesis | + | · Restrict check & run mutagenesis <i>comE<sup>D58E</sup></i><br> |
| - | + | ||
| - | + | ||
<br> <b> July 11</b> <br> | <br> <b> July 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion of comW-FLAG with XbaI and PstI, digestion clean up<br> | + | · Digestion of <i>comW</i>-FLAG with XbaI and PstI, digestion clean up<br> |
· Gel check for the PCR product obtained on July 10<br> | · Gel check for the PCR product obtained on July 10<br> | ||
· Digestion of pCEPcin at NcoI and XhoI<br> | · Digestion of pCEPcin at NcoI and XhoI<br> | ||
· Ethanol precipitation of pCEPcin<br> | · Ethanol precipitation of pCEPcin<br> | ||
· Extract J04450 (KAN)<br> | · Extract J04450 (KAN)<br> | ||
| - | · Ligate pSB1C3 with | + | · Ligate pSB1C3 with <i>comE<sup>D58E</sup></i> <br> |
· TransformPcomCDE<br> | · TransformPcomCDE<br> | ||
· <br> | · <br> | ||
| Line 402: | Line 384: | ||
<br> <b> July 14</b> <br> | <br> <b> July 14</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Ligation of comW-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI<br> | + | · Ligation of <i>comW</i>-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI<br> |
· Transformation of ligation products<br> | · Transformation of ligation products<br> | ||
· Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br> | · Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br> | ||
· Digestion of pCEPcin at NcoI and XhoI<br> | · Digestion of pCEPcin at NcoI and XhoI<br> | ||
| - | · PCR of 100bp | + | · PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br> |
· Gel purification of digested BBa_J04450<br> | · Gel purification of digested BBa_J04450<br> | ||
· Inoculation of pCEPcin<br> | · Inoculation of pCEPcin<br> | ||
| - | · PCR of | + | · PCR of <i>comE<sup>D58E</sup></i> with only 1 primer → check whether the primers are working or not<br> |
· Ligation of pSB1C3-PcomX<br> | · Ligation of pSB1C3-PcomX<br> | ||
· Inoculation of pSB1C3-PcomCDE<br> | · Inoculation of pSB1C3-PcomCDE<br> | ||
· Digest B0015<br> | · Digest B0015<br> | ||
| - | · Digest | + | · Digest <i>comE<sup>D58E</sup></i><br> |
· Ligation of PCR product <i>comE</i> to pSB1C3<br> | · Ligation of PCR product <i>comE</i> to pSB1C3<br> | ||
| Line 424: | Line 406: | ||
· Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase<br> | · Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase<br> | ||
· Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br> | · Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br> | ||
| - | · Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.<i>comW</i>-FLAG<br> |
· Ethanol precipitation of pCEPcin<br> | · Ethanol precipitation of pCEPcin<br> | ||
· Gel check of the PCR product obtained on July 14<br> | · Gel check of the PCR product obtained on July 14<br> | ||
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp | + | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br> |
· Plasmid extraction of pSB1C3-PcomCDE<br> | · Plasmid extraction of pSB1C3-PcomCDE<br> | ||
· Bacterial inoculation (5 for PcomCDE, 4 for PcomX)<br> | · Bacterial inoculation (5 for PcomCDE, 4 for PcomX)<br> | ||
· Bacterial inoculation of B0015 (6 tubes)<br> | · Bacterial inoculation of B0015 (6 tubes)<br> | ||
| - | · Extract gel B0015 & | + | · Extract gel B0015 & <i>comE<sup>D58E</sup></i><br> |
· K880005 dephosphorylation; Digestion<br> | · K880005 dephosphorylation; Digestion<br> | ||
· <br> | · <br> | ||
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<br> <b> July 16</b> <br> | <br> <b> July 16</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of ligated pSB1C3-K880005.comW-FLAG<br> | + | · Digestion check of ligated pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Colony PCR of pSB1C3-K880005.comW-FLAG<br> | + | · Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Gel check of pSB1C3-K880005.comW-FLAG PCR product (no PCR product present)<br> | + | · Gel check of pSB1C3-K880005.<i>comW</i>-FLAG PCR product (no PCR product present)<br> |
| - | · PCR of comW using Vent DNA Polymerase (no PCR product present)<br> | + | · PCR of <i>comW</i> using Vent DNA Polymerase (no PCR product present)<br> |
· Digestion of comX on XbaI and PstI<br> | · Digestion of comX on XbaI and PstI<br> | ||
· Gel check of the PCR product obtained on July 15<br> | · Gel check of the PCR product obtained on July 15<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
· Digestion of pCEPcin2 at XhoI and SalI<br> | · Digestion of pCEPcin2 at XhoI and SalI<br> | ||
| - | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp | + | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br> |
· Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br> | · Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br> | ||
· Plasmid extraction of Promoters and B0015<br> | · Plasmid extraction of Promoters and B0015<br> | ||
· Colony PCR<br> | · Colony PCR<br> | ||
| - | · Extract gel B0015 & | + | · Extract gel B0015 & <i>comE<sup>D58E</sup></i><br> |
| - | · Mutagenesis attempts for <i>comE</i><br> | + | · Mutagenesis attempts for <i><i>comE</i></i><br> |
<br> <b> July 17</b> <br> | <br> <b> July 17</b> <br> | ||
| Line 457: | Line 439: | ||
· PCR of comX-myc and comX using Vent DNA Polymerase<br> | · PCR of comX-myc and comX using Vent DNA Polymerase<br> | ||
· Gel check of comX-myc and comX PCR product, PCR clean-up<br> | · Gel check of comX-myc and comX PCR product, PCR clean-up<br> | ||
| - | · PCR of comW using Vent DNA Polymerase<br> | + | · PCR of <i>comW</i> using Vent DNA Polymerase<br> |
| - | · Gel extraction and gel purification of comW PCR product<br> | + | · Gel extraction and gel purification of <i>comW</i> PCR product<br> |
· Dephosporylation of BBa_K880005<br> | · Dephosporylation of BBa_K880005<br> | ||
| - | · Gel check of the PCR product obtained on July 16, all types of | + | · Gel check of the PCR product obtained on July 16, all types of P<sub>combox</sub> shown positive result<br> |
| - | · PCR clean-up of | + | · PCR clean-up of P<sub>combox</sub><br> |
· PCR product ligation with pSB1C3<br> | · PCR product ligation with pSB1C3<br> | ||
| - | · Bacterial inoculation of B0015, | + | · Bacterial inoculation of B0015, <i>comE<sup>D58E</sup></i><br> |
| - | · Extract gel B0015 & | + | · Extract gel B0015 & <i>comE<sup>D58E</sup></i> - Digestion<br> |
· Overnight Ligation<br> | · Overnight Ligation<br> | ||
· <br> | · <br> | ||
| Line 472: | Line 454: | ||
· Gel check of comX PCR product, PCR clean-up<br> | · Gel check of comX PCR product, PCR clean-up<br> | ||
· Digestion of comX PCR product on XbaI and PstI, digestion clean-up<br> | · Digestion of comX PCR product on XbaI and PstI, digestion clean-up<br> | ||
| - | · Digestion of comX-myc and comW PCR products, digestion clean-up<br> | + | · Digestion of comX-myc and <i>comW</i> PCR products, digestion clean-up<br> |
· Gel extraction and gel purification of digested comX and comX-myc<br> | · Gel extraction and gel purification of digested comX and comX-myc<br> | ||
· Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)<br> | · Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)<br> | ||
· Digestion of pCEPcin2 (at BamHI and NcoI)<br> | · Digestion of pCEPcin2 (at BamHI and NcoI)<br> | ||
| - | · PCR of 100bp | + | · PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br> |
| - | · Gel check of the purified | + | · Gel check of the purified P<sub>combox</sub> obtained July 17<br> |
| - | · Gel check of the 100bp | + | · Gel check of the 100bp P<sub>combox</sub> (PCR product)<br> |
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
| - | · Ligation of | + | · Ligation of <i>comE<sup>D58E</sup></i><br> |
| - | · Gel extract | + | · Gel extract <i>comE<sup>D58E</sup></i><br> |
| - | + | · BBa_B0015 Gel purification<br> | |
| - | · <br> | + | |
· <br> | · <br> | ||
| Line 497: | Line 478: | ||
· Gel purification of comX and comX-myc<br> | · Gel purification of comX and comX-myc<br> | ||
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | · Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | ||
| - | · Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | + | · Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> |
· Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio<br> | · Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio<br> | ||
| - | · Ligation of comW into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | + | · Ligation of <i>comW</i> into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> |
| - | · Colony PCR of pSB1C3-K880005.comW-FLAG using Taq DNA Polymerase<br> | + | · Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG using Taq DNA Polymerase<br> |
| - | · PCR of 100bp and 150bp | + | · PCR of 100bp and 150bp P<sub>combox</sub> using Phusion polymerase<br> |
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
· Colony PCR of Promoters<br> | · Colony PCR of Promoters<br> | ||
| - | |||
| - | |||
| - | |||
<br> <b> July 22</b> <br> | <br> <b> July 22</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Colony PCR of comX, comW, comX-myc, comW-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br> | + | · Colony PCR of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br> |
| - | · Inoculation of comX, comW, comX-myc, comW-FLAG<br> | + | · Inoculation of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG<br> |
· Gel check of the PCR product obtained on July 21<br> | · Gel check of the PCR product obtained on July 21<br> | ||
| - | · Oligo annealing of 67bp | + | · Oligo annealing of 67bp P<sub>combox</sub><br> |
· Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br> | ||
| - | · PCR of 100bp,150bp and 160bp | + | · PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br> |
· Gel electrophoresis of colony PCR results<br> | · Gel electrophoresis of colony PCR results<br> | ||
| - | · Mutagenesis of | + | · Mutagenesis of <i>comE<sup>D58E</sup></i><br> |
· <br> | · <br> | ||
<br> <b> July 23</b> <br> | <br> <b> July 23</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comX, comW, comX-myc, comW-FLAG colony PCR products on XbaI and SpeI<br> | + | · Digestion check of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG colony PCR products on XbaI and SpeI<br> |
| - | · Inoculation of comW-FLAG<br> | + | · Inoculation of <i>comW</i>-FLAG<br> |
· Gel check of PCR product obtained on July 22<br> | · Gel check of PCR product obtained on July 22<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
| - | · PCR of 100bp,150bp and 160bp | + | · PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br> |
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
· Gel electrophoresis of colony PCR results<br> | · Gel electrophoresis of colony PCR results<br> | ||
| - | · Mutagenesis of | + | · Mutagenesis of <i>comE<sup>D58E</sup></i><br> |
| - | + | ||
· <br> | · <br> | ||
<br> <b> July 24</b> <br> | <br> <b> July 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and SpeI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and SpeI-HF<br> |
| - | · Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone<br> | + | · Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone<br> |
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone<br> | · Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone<br> | ||
· PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br> | · PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br> | ||
| Line 546: | Line 523: | ||
· Received pCEPcin2<br> | · Received pCEPcin2<br> | ||
· Gel electrophoresis of colony PCR and mutagenesis results<br> | · Gel electrophoresis of colony PCR and mutagenesis results<br> | ||
| - | |||
| - | |||
| - | |||
| - | |||
· <br> | · <br> | ||
| Line 560: | Line 533: | ||
· Gel check on K880005<br> | · Gel check on K880005<br> | ||
· Transformation of ligated product (promoters+pSB1C3)<br> | · Transformation of ligated product (promoters+pSB1C3)<br> | ||
| - | |||
| - | |||
· <br> | · <br> | ||
</div> | </div> | ||
| Line 572: | Line 543: | ||
Wet lab<br> | Wet lab<br> | ||
· Colony PCR of comX-myc using Taq DNA Polymerase<br> | · Colony PCR of comX-myc using Taq DNA Polymerase<br> | ||
| - | · Colony PCR of | + | · Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br> |
| - | · Inoculation of comX-myc and comW-FLAG<br> | + | · Inoculation of comX-myc and <i>comW</i>-FLAG<br> |
· Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br> | · Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br> | ||
· Colony PCR of promoters-pSB1C3<br> | · Colony PCR of promoters-pSB1C3<br> | ||
| - | · Bacterial inoculation of K880005, | + | · Bacterial inoculation of K880005, <i>comE<sup>D58E</sup></i> <br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| Line 582: | Line 553: | ||
<br> <b> July 29</b> <br> | <br> <b> July 29</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF<br> | + | · Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br> |
| - | · Transformation of comX-myc and comW-FLAG<br> | + | · Transformation of comX-myc and <i>comW</i>-FLAG<br> |
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Digestion of pCEPcin2 at EcoRI and PstI<br> | · Digestion of pCEPcin2 at EcoRI and PstI<br> | ||
· Inoculation of pSB3K3-BBa_J04450<br> | · Inoculation of pSB3K3-BBa_J04450<br> | ||
| - | · PCR of 100bp,150bp and 160bp | + | · PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Vent polymerase<br> |
· Restrict check K880005 <br> | · Restrict check K880005 <br> | ||
· Promoters colony PCR <br> | · Promoters colony PCR <br> | ||
| Line 596: | Line 567: | ||
<br> <b> July 30</b> <br> | <br> <b> July 30</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comX-myc and comW-FLAG<br> | + | · Digestion check of comX-myc and <i>comW</i>-FLAG<br> |
| - | · Inoculation of comX-myc and comW-FLAG <br> | + | · Inoculation of comX-myc and <i>comW</i>-FLAG <br> |
| - | · Streaking plates of comX-myc and comW-FLAG<br> | + | · Streaking plates of comX-myc and <i>comW</i>-FLAG<br> |
| - | · PCR clean-up of 100bp,150bp and 160bp | + | · PCR clean-up of 100bp,150bp and 160bp P<sub>combox</sub><br> |
· Digestion of pCEPcin2 at EcoRI and PstI<br> | · Digestion of pCEPcin2 at EcoRI and PstI<br> | ||
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Restrict check K880005 <br> | · Restrict check K880005 <br> | ||
| + | |||
<br> <b> July 31</b> <br> | <br> <b> July 31</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF<br> | + | · Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br> |
| - | · Redigest comW-FLAG on XmnI and NcoI<br> | + | · Redigest <i>comW</i>-FLAG on XmnI and NcoI<br> |
| - | · PCR sequencing for comW and comX<br> | + | · PCR sequencing for <i>comW</i> and comX<br> |
| - | · Ethanol precipitation for comW and comX<br> | + | · Ethanol precipitation for <i>comW</i> and comX<br> |
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· K880005 dephosphorylation & ligation<br> | · K880005 dephosphorylation & ligation<br> | ||
| Line 653: | Line 625: | ||
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> | · Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> | ||
· Transformation of pSB1C3-B0015.comX-myc ligation product<br> | · Transformation of pSB1C3-B0015.comX-myc ligation product<br> | ||
| - | · Sequencing of comX-myc and comW-FLAG<br> | + | · Sequencing of comX-myc and <i>comW</i>-FLAG<br> |
· Inoculation of comX-myc<br> | · Inoculation of comX-myc<br> | ||
| - | · Digestion of 100bp, 150bp, 160bp and 180bp | + | · Digestion of 100bp, 150bp, 160bp and 180bp P<sub>combox</sub> at XbaI and PstI<br> |
| - | · Ligation of 100bp and 160bp | + | · Ligation of 100bp and 160bp P<sub>combox</sub> with pSB1C3<br> |
· Digestion of pCEPcin2 at EcoRI and PstI<br> | · Digestion of pCEPcin2 at EcoRI and PstI<br> | ||
· PcomCDE Precipitation<br> | · PcomCDE Precipitation<br> | ||
| Line 671: | Line 643: | ||
· Inoculation of comX-myc<br> | · Inoculation of comX-myc<br> | ||
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · Ligation of 150bp, 180bp and 249bp | + | · Ligation of 150bp, 180bp and 249bp P<sub>combox</sub> with pSB1C3<br> |
| - | · Transformation of pSB1C3-150bp | + | · Transformation of pSB1C3-150bp P<sub>combox</sub> , pSB1C3-180bp P<sub>combox</sub> and pSB1C3-249bp P<sub>combox</sub><br> |
· Gel purification of digested pSB1C3 backbone<br> | · Gel purification of digested pSB1C3 backbone<br> | ||
· Inoculation of pCEPcin2<br> | · Inoculation of pCEPcin2<br> | ||
· Send to sequencing: PcomCDE and PcomX<br> | · Send to sequencing: PcomCDE and PcomX<br> | ||
· Bacterial inoculation of <i>comE<sup>D58E</sup></i> <br> | · Bacterial inoculation of <i>comE<sup>D58E</sup></i> <br> | ||
| - | · Colony PCR to check pSB1C3-<i>comE</i> and pSB1C3-<i>comE<sup>D58E</sup></i><br> | + | · Colony PCR to check pSB1C3-<i><i>comE</i></i> and pSB1C3-<i><i>comE</i><sup>D58E</sup></i><br> |
| Line 684: | Line 656: | ||
· Digestion check of comX-myc on XbaI and PstI<br> | · Digestion check of comX-myc on XbaI and PstI<br> | ||
· Gel check of comX-myc PCR product (no PCR product present)<br> | · Gel check of comX-myc PCR product (no PCR product present)<br> | ||
| - | · Sequencing of comW-FLAG using VF2 and VR<br> | + | · Sequencing of <i>comW</i>-FLAG using VF2 and VR<br> |
· Digestion of comX-myc on StyI<br> | · Digestion of comX-myc on StyI<br> | ||
· Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI<br> | · Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI<br> | ||
· Gel purification of digested pSB1C3 backbone<br> | · Gel purification of digested pSB1C3 backbone<br> | ||
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · Phosphorylation of 67bp | + | · Phosphorylation of 67bp P<sub>combox</sub> oligos<br> |
| - | · Oligo annealing for 67bp | + | · Oligo annealing for 67bp P<sub>combox</sub><br> |
| - | + | · Align sequence with expected: P<sub>comCDE</sub> and P<sub>comX</sub><br> | |
| - | · Align sequence with expected: | + | |
| - | + | ||
| - | + | ||
| - | + | ||
<br><b>Aug 7</b> <br> | <br><b>Aug 7</b> <br> | ||
| Line 701: | Line 670: | ||
· Digestion of comX-myc on StyI<br> | · Digestion of comX-myc on StyI<br> | ||
· Digestion check of comX on SphI and NcoI<br> | · Digestion check of comX on SphI and NcoI<br> | ||
| - | · Digestion check of comW on XmnI and NcoI<br> | + | · Digestion check of <i>comW</i> on XmnI and NcoI<br> |
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp | + | · Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br> |
· Gel purification of digested pSB1C3 backbone<br> | · Gel purification of digested pSB1C3 backbone<br> | ||
· Precipitate after sequencing reaction PcomCDE<br> | · Precipitate after sequencing reaction PcomCDE<br> | ||
· Make glycerol stock for PcomX<br> | · Make glycerol stock for PcomX<br> | ||
| - | · Run colony PCR for | + | · Run colony PCR for <i>comE<sup>D58E</sup></i><br> |
· <br> | · <br> | ||
<br><b>Aug 8</b> <br> | <br><b>Aug 8</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion of comW-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification<br> | + | · Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification<br> |
| - | · Digestion of comW-FLAG on SpeI and PstI-HF, digestion clean-up<br> | + | · Digestion of <i>comW</i>-FLAG on SpeI and PstI-HF, digestion clean-up<br> |
· Sequencing of comX-myc succeeded but mutation at EcoRI site<br> | · Sequencing of comX-myc succeeded but mutation at EcoRI site<br> | ||
· Digestion check of comX-myc at EcoRI-HF and PstI-HF<br> | · Digestion check of comX-myc at EcoRI-HF and PstI-HF<br> | ||
| - | · Ligation of comX and comW into dephosporylated pSB1C3-K880005<br> | + | · Ligation of comX and <i>comW</i> into dephosporylated pSB1C3-K880005<br> |
| - | · Transformation of comX and comW ligation product<br> | + | · Transformation of comX and <i>comW</i> ligation product<br> |
· Digestion of comX-myc at EcoRI-HF and SpeI-HF<br> | · Digestion of comX-myc at EcoRI-HF and SpeI-HF<br> | ||
· Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br> | · Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br> | ||
| - | · Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp | + | · Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> with pSB1C3 backbone<br> |
· Ligate with E0240 PcomX <br> | · Ligate with E0240 PcomX <br> | ||
| - | · Colony PCR for comE mutant<br> | + | · Colony PCR for <i>comE</i> mutant<br> |
· Check sequence PcomCDE<br> | · Check sequence PcomCDE<br> | ||
· <br> | · <br> | ||
| Line 732: | Line 701: | ||
<br><b>Aug 11</b> <br> | <br><b>Aug 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF<br> | + | · Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br> |
· Gel purification of comX-myc digested on EcoRI-HF and SpeI<br> | · Gel purification of comX-myc digested on EcoRI-HF and SpeI<br> | ||
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> | · Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> | ||
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 180bp and 249bp) at XbaI and PstI<br> |
| - | · Colony PCR of pSB1C3- | + | · Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp)<br> |
· Gel purification of digested pSB1C3 backbone<br> | · Gel purification of digested pSB1C3 backbone<br> | ||
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br> | ||
· Extract pSB1C3 from J04450<br> | · Extract pSB1C3 from J04450<br> | ||
· Redo ligation part 1<br> | · Redo ligation part 1<br> | ||
| - | · Gibson assembly of <i>comE<sup>D58E</sup></i><br> | + | · Gibson assembly of <i><i>comE</i><sup>D58E</sup></i><br> |
· Ligation of PCR product <i>comD</i> to pSB1C3<br> | · Ligation of PCR product <i>comD</i> to pSB1C3<br> | ||
<br><b>Aug 12</b> <br> | <br><b>Aug 12</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF<br> | + | · Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF<br> |
| - | · Dilution of comW-FLAG<br> | + | · Dilution of <i>comW</i>-FLAG<br> |
· Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up<br> | · Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up<br> | ||
· Re-ligation of pSB1C3-B0015.comX-myc<br> | · Re-ligation of pSB1C3-B0015.comX-myc<br> | ||
| Line 753: | Line 722: | ||
· Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc<br> | · Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc<br> | ||
· Digestion of pCEPcin2 at EcoRI and EcoRV<br> | · Digestion of pCEPcin2 at EcoRI and EcoRV<br> | ||
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br> |
| - | · Ligate K880005 and comE(D58E)<br> | + | · Ligate K880005 and <i>comE</i>(D58E)<br> |
· Inoculate for glycerol stock (PcomX)<br> | · Inoculate for glycerol stock (PcomX)<br> | ||
· <br> | · <br> | ||
| Line 760: | Line 729: | ||
Wet lab<br> | Wet lab<br> | ||
· Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | · Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | ||
| - | · Inoculation of pSB1C3- | + | · Inoculation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br> |
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br> |
| - | · Colony PCR of pSB1C3- | + | · Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br> |
· Check new dNTP<br> | · Check new dNTP<br> | ||
· Restrict check of ligation product part 1<br> | · Restrict check of ligation product part 1<br> | ||
| Line 773: | Line 742: | ||
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | ||
· Transformation of ligated product pSB1C3-K880005.comX-myc.B0015<br> | · Transformation of ligated product pSB1C3-K880005.comX-myc.B0015<br> | ||
| - | · Dilution of comW-FLAG<br> | + | · Dilution of <i>comW</i>-FLAG<br> |
| - | · Digestion of comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
| - | · Ligation of comW-FLAG into pSB1C3-B0015 backbone<br> | + | · Ligation of <i>comW</i>-FLAG into pSB1C3-B0015 backbone<br> |
| - | · Re-digestion of comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Re-digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
| - | · Gel check of the digested pSB1C3- | + | · Gel check of the digested pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13<br> |
| - | · Colony PCR of Colony PCR of pSB1C3- | + | · Colony PCR of Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br> |
· glycerol stock for PcomX<br> | · glycerol stock for PcomX<br> | ||
· colony PCR ligation product part 1<br> | · colony PCR ligation product part 1<br> | ||
· colony PCR gibson assembly<br> | · colony PCR gibson assembly<br> | ||
· Run gel for PCR products<br> | · Run gel for PCR products<br> | ||
| - | · Inoculation of | + | · Inoculation of <i>comE<sup>D58E</sup></i><br> |
· <br> | · <br> | ||
<br><b>Aug 15</b> <br> | <br><b>Aug 15</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br> | · Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br> | ||
| - | · Colony PCR of comW-FLAG using Taq DNA Polymerase<br> | + | · Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br> |
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI<br> | · Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI<br> | ||
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF<br> | · Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF<br> | ||
| Line 796: | Line 765: | ||
· Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product<br> | · Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product<br> | ||
· Ligation of comX-myc into pSB1C3-B0015 backbone<br> | · Ligation of comX-myc into pSB1C3-B0015 backbone<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br> |
· Preparation for submittion of samples to be sequenced<br> | · Preparation for submittion of samples to be sequenced<br> | ||
| - | · Inoculation of pSB1C3- | + | · Inoculation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br> |
· colony PCR for ligation product part 1<br> | · colony PCR for ligation product part 1<br> | ||
· colony PCR for ligation product part 2<br> | · colony PCR for ligation product part 2<br> | ||
· colony PCR for gibson assembly<br> | · colony PCR for gibson assembly<br> | ||
· Run gel for PCR products<br> | · Run gel for PCR products<br> | ||
| - | · comE-D58E plasmid extraction<br> | + | · <i>comE</i>-D58E plasmid extraction<br> |
· <br> | · <br> | ||
| Line 815: | Line 784: | ||
Wet lab<br> | Wet lab<br> | ||
· Transformation of ligated pSB1C3-K880005.comX-myc<br> | · Transformation of ligated pSB1C3-K880005.comX-myc<br> | ||
| - | · Ligation of comW-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone<br> | + | · Ligation of <i>comW</i>-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone<br> |
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on SpeI-HF and PstI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on SpeI-HF and PstI-HF<br> |
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | · Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | ||
· Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015<br> | · Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015<br> | ||
| Line 823: | Line 792: | ||
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | ||
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br> | · Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br> | ||
| - | · Sent out pSB1C3- | + | · Sent out pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp) samples for commercial sequencing<br> |
| - | · Digestion of pSB1C3- | + | · Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at SpeI and PstI<br> |
| - | · Gel purification of digested pSB1C3- | + | · Gel purification of digested pSB1C3-P<sub>combox</sub> (150bp, 160bp, 180bp and 249bp)<br> |
· Inoculation of pSB1C3-BBa_E0240<br> | · Inoculation of pSB1C3-BBa_E0240<br> | ||
· Gel extraction and purification of E0240<br> | · Gel extraction and purification of E0240<br> | ||
· Bacterial inoculation of PcomX (PcomCDE(3))<br> | · Bacterial inoculation of PcomX (PcomCDE(3))<br> | ||
| - | · Gel extraction and purification of | + | · Gel extraction and purification of <i>comE</i> mutant<br> |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
· <br> | · <br> | ||
| + | |||
<br><b>Aug 19</b> <br> | <br><b>Aug 19</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| Line 841: | Line 806: | ||
· Digestion check of comX-myc on XbaI and PstI-HF<br> | · Digestion check of comX-myc on XbaI and PstI-HF<br> | ||
· Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> | · Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br> |
| - | · Gel purification of digested pSB1C3- | + | · Gel purification of digested pSB1C3-P<sub>combox</sub> (100bp)<br> |
| - | · Ligation of pSB1C3- | + | · Ligation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) with BBa_E0240 insert<br> |
| - | · Transformation of pSB1C3- | + | · Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br> |
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br> | ||
· Gel purification of E0240 insert<br> | · Gel purification of E0240 insert<br> | ||
| Line 850: | Line 815: | ||
· Make aliquotes for oligo - PcomCDE<br> | · Make aliquotes for oligo - PcomCDE<br> | ||
· PcomCDE Oligo annealing<br> | · PcomCDE Oligo annealing<br> | ||
| - | · Ligation of comE mutant insert with K880005<br> | + | · Ligation of <i>comE</i> mutant insert with K880005<br> |
| - | · Ligation of comE insert with K880005<br> | + | · Ligation of <i>comE</i> insert with K880005<br> |
· Gel electrophoresis of Gibson assembly mastermix checking<br> | · Gel electrophoresis of Gibson assembly mastermix checking<br> | ||
· Plasmid extraction of J04450(CHL), PcomX(PcomCDE(3)) <br> | · Plasmid extraction of J04450(CHL), PcomX(PcomCDE(3)) <br> | ||
| - | · Bacterial inoculation of comE WT, J04450 (CHL)<br> | + | · Bacterial inoculation of <i>comE</i> WT, J04450 (CHL)<br> |
· <br> | · <br> | ||
<br><b>Aug 20</b> <br> | <br><b>Aug 20</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> |
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | ||
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF<br> | ||
· Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI<br> | · Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI<br> | ||
| - | · Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br> |
· Gel purification of digested pSB1C3-BBa_E0240<br> | · Gel purification of digested pSB1C3-BBa_E0240<br> | ||
| - | · Digestion of pSB1C3-180bp | + | · Digestion of pSB1C3-180bp P<sub>combox</sub> at SpeI and PstI<br> |
| - | · Digestion clean-up of pSB1C3-180bp | + | · Digestion clean-up of pSB1C3-180bp P<sub>combox</sub><br> |
| - | · Ligation of pSB1C3-180bp | + | · Ligation of pSB1C3-180bp P<sub>combox</sub> with BBa_E0240 insert<br> |
| - | · Transformation of pSB1C3- | + | · Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br> |
| - | · Colony PCR for ligated pSB1C3- | + | · Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br> |
| - | · Inoculation of pSB1C3- | + | · Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br> |
· Ligation of E0240 insert with PcomX<br> | · Ligation of E0240 insert with PcomX<br> | ||
· Transformation [E0240-PcomCDE exp+ -ve, pSB1C3-PcomCDE exp+ -ve]<br> | · Transformation [E0240-PcomCDE exp+ -ve, pSB1C3-PcomCDE exp+ -ve]<br> | ||
| - | · Ligation of | + | · Ligation of <i>comE<sup>D58E</sup></i> insert with K880005<br> |
| - | · Gel extract comE WT<br> | + | · Gel extract <i>comE</i> WT<br> |
| - | · PCR check for | + | · PCR check for <i>comE<sup>D58E</sup></i>-myc<br> |
· Digest J04450(CHL) Gel extraction<br> | · Digest J04450(CHL) Gel extraction<br> | ||
| - | · Plasmid extraction of comE WT, J04450 (CHL)<br> | + | · Plasmid extraction of <i>comE</i> WT, J04450 (CHL)<br> |
· <br> | · <br> | ||
<br><b>Aug 21</b> <br> | <br><b>Aug 21</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br> | ||
| - | · Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> | + | · Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br> |
· Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase<br> | · Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase<br> | ||
| - | · Digestion of comW-FLAG on NotI-HF<br> | + | · Digestion of <i>comW</i>-FLAG on NotI-HF<br> |
| - | · Digestion of comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
| - | · Inoculation of pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Transformation pSB1C3-K880005.comW-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015<br> | + | · Transformation pSB1C3-K880005.<i>comW</i>-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015<br> |
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) at NcoI<br> |
| - | · Colony PCR for ligated pSB1C3- | + | · Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br> |
| - | · Inoculation of pSB1C3- | + | · Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br> |
| - | · Received sequencing result of pSB1C3- | + | · Received sequencing result of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp)<br> |
· Ligate PcomX with E0240 insert<br> | · Ligate PcomX with E0240 insert<br> | ||
· PCR, Restrict check Part I (PcomCDE)<br> | · PCR, Restrict check Part I (PcomCDE)<br> | ||
· O/N Ligation w/ PcomCDE<br> | · O/N Ligation w/ PcomCDE<br> | ||
| - | · Extract comE WT-pSB1C3<br> | + | · Extract <i>comE</i> WT-pSB1C3<br> |
| - | · PCR and restrict check | + | · PCR and restrict check <i>comE<sup>D58E</sup></i>-myc<br> |
· Glycerol stock for PcomX <br> | · Glycerol stock for PcomX <br> | ||
· <br> | · <br> | ||
<br><b>Aug 22</b> <br> | <br><b>Aug 22</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion check of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
· Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF<br> | · Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF<br> | ||
· Inoculation pSB1C3-K880005.comX-myc.B0015<br> | · Inoculation pSB1C3-K880005.comX-myc.B0015<br> | ||
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br> |
| - | · PCR of pSB1C3- | + | · PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) using Vent polymerase <br> |
| - | · Sent out pSB1C3- | + | · Sent out pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) for commercial sequencing<br> |
· Transformation of newly ligated (E0240)<br> | · Transformation of newly ligated (E0240)<br> | ||
· Bacterial inoculation of checked colony (E0240)<br> | · Bacterial inoculation of checked colony (E0240)<br> | ||
· O/N ligation w/ pSB1C3<br> | · O/N ligation w/ pSB1C3<br> | ||
| - | · Ligation comE WT-pSB1C3<br> | + | · Ligation <i>comE</i> WT-pSB1C3<br> |
| - | · Ligate | + | · Ligate <i>comE<sup>D58E</sup></i>-myc w/ K880005<br> |
· <br> | · <br> | ||
| Line 923: | Line 888: | ||
<br><b>Aug 25</b> <br> | <br><b>Aug 25</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI | + | · Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI |
Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF | Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF | ||
| - | Transformation of pSB1C3-K880005.comW-FLAG | + | Transformation of pSB1C3-K880005.<i>comW</i>-FLAG |
Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, gel extraction and gel purification | Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, gel extraction and gel purification | ||
Inoculation of pSB1C3-K880005.comX-myc.B0015 for glycerol stock<br> | Inoculation of pSB1C3-K880005.comX-myc.B0015 for glycerol stock<br> | ||
| - | · Received sequencing result of pSB1C3- | + | · Received sequencing result of pSB1C3-P<sub>combox</sub> (160bp and 249bp)<br> |
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br> |
| - | · Digestion of pSB1C3- | + | · Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 160bp, 180bp and 249bp) at XbaI and PstI<br> |
| - | · Gel purification of digested | + | · Gel purification of digested P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) insert<br> |
· Ligation of PcomX-E0240<br> | · Ligation of PcomX-E0240<br> | ||
· Digestion of PcomCDE-E0240<br> | · Digestion of PcomCDE-E0240<br> | ||
| - | · Colony PCR pSB1C3-PcomCDE, K880005-comE WT, K880005- | + | · Colony PCR pSB1C3-PcomCDE, K880005-<i>comE</i> WT, K880005-<i>comE<sup>D58E</sup></i>-myc<br> |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
· <br> | · <br> | ||
<br><b>Aug 26</b> <br> | <br><b>Aug 26</b> <br> | ||
| Line 945: | Line 906: | ||
· Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF<br> | · Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF<br> | ||
· Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification<br> | · Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br> |
| - | · Transformation of pSB1C3-K880005.comW-FLAG<br> | + | · Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Ligation of comX, 100bp | + | · Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br> |
· Gel pSB1C3-PcomCDE<br> | · Gel pSB1C3-PcomCDE<br> | ||
· PcomX-E0240 Colony PCR check<br> | · PcomX-E0240 Colony PCR check<br> | ||
· PcomCDE glycerol stock <br> | · PcomCDE glycerol stock <br> | ||
| - | · Restrict check K880005-comE WT<br> | + | · Restrict check K880005-<i>comE</i> WT<br> |
| - | · Ligation w/ comE WT<br> | + | · Ligation w/ <i>comE</i> WT<br> |
| - | · Restrict check K880005- | + | · Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br> |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
· <br> | · <br> | ||
<br><b>Aug 27</b> <br> | <br><b>Aug 27</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br> |
| - | · Sequencing of pSB1C3-K880005.comW-FLAG.B0015<br> | + | · Sequencing of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br> |
| - | · Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone <br> | + | · Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone <br> |
| - | · Transformation of pSB1C3-K880005.comW-FLAG<br> | + | · Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Colony PCR of pSB1C3- | + | · Colony PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br> |
| - | · Ligation of comX, 100bp | + | · Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br> |
| - | · Inoculation of pSB1C3- | + | · Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 180bp and 249bp), pSB1C3-P<sub>combox</sub> (160bp)<br> |
· pSB1C3-PcomCDE<br> | · pSB1C3-PcomCDE<br> | ||
· PcomCDE sequencing <br> | · PcomCDE sequencing <br> | ||
| - | · PCR check K880005-comE WT<br> | + | · PCR check K880005-<i>comE</i> WT<br> |
| - | · PCR check K880005- | + | · PCR check K880005-<i>comE<sup>D58E</sup></i>-myc<br> |
| - | + | · Ligate K880005-<i>comE</i>-myc<br> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | · < | + | |
| - | + | ||
· <br> | · <br> | ||
<br><b>Aug 28</b> <br> | <br><b>Aug 28</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification<br> |
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> |
· Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone<br> | · Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone<br> | + | · Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone<br> |
| - | · Restriction check of pSB1C3- | + | · Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br> |
| - | · Colony PCR of pSB3K3-comX-100bp | + | · Colony PCR of pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br> |
· pSB1C3-PcomCDE overnight ligation<br> | · pSB1C3-PcomCDE overnight ligation<br> | ||
· PcomX-E0240 ligation<br> | · PcomX-E0240 ligation<br> | ||
· Gel extract I20260 and E0240<br> | · Gel extract I20260 and E0240<br> | ||
| - | · Glycerol stock for K880005-comE WT<br> | + | · Glycerol stock for K880005-<i>comE</i> WT<br> |
| - | · Colony PCR for K880005- | + | · Colony PCR for K880005-<i>comE<sup>D58E</sup></i>-myc <br> |
| - | · Ligate K880005-comE-myc <br> | + | · Ligate K880005-<i>comE</i>-myc <br> |
| - | · PCR | + | · PCR <i>comE<sup>D58E</sup></i>-myc<br> |
· Ligation of Part 1 and Part 2<br> | · Ligation of Part 1 and Part 2<br> | ||
| Line 1,002: | Line 955: | ||
<br><b>Aug 29</b> <br> | <br><b>Aug 29</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br> |
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br> |
| - | · Sent out pSB1C3-160bp | + | · Sent out pSB1C3-160bp P<sub>combox</sub> for commercial sequencing<br> |
| - | · Digestion of pSB1C3-160bp | + | · Digestion of pSB1C3-160bp P<sub>combox</sub> at SpeI and PstI<br> |
| - | · Colony PCR for pSB3K3-comX-100bp | + | · Colony PCR for pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br> |
| - | · Ligation of pSB1C3-160bp | + | · Ligation of pSB1C3-160bp P<sub>combox</sub> with BBa_E0240 insert<br> |
· Transform pSB1C3-PcomCDE <br> | · Transform pSB1C3-PcomCDE <br> | ||
· Transform PcomX-E0240 <br> | · Transform PcomX-E0240 <br> | ||
· Ligate I20260 and E0240 <br> | · Ligate I20260 and E0240 <br> | ||
| - | · Ligate K880005-comE WT <br> | + | · Ligate K880005-<i>comE</i> WT <br> |
| - | · Ligate K880005-comE-myc<br> | + | · Ligate K880005-<i>comE</i>-myc<br> |
| - | · Ligate K880005- | + | · Ligate K880005-<i>comE<sup>D58E</sup></i>-myc <br> |
| - | · Gel check | + | · Gel check <i>comE<sup>D58E</sup></i>-myc <br> |
· <br> | · <br> | ||
| Line 1,043: | Line 996: | ||
<br><b>Sept 1</b> <br> | <br><b>Sept 1</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br> |
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI<br> | ||
· Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br> | · Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br> | ||
| - | · Received the sequencing result of pSB1C3-160bp | + | · Received the sequencing result of pSB1C3-160bp P<sub>combox</sub><br> |
| - | · Colony PCR: pSB1C3-PcomCDE, PcomX-E0240, I20260+Part2, E0240+Part2, E0240+Part2, K880005-comE WT, K990005-comE-myc, K990005- | + | · Colony PCR: pSB1C3-PcomCDE, PcomX-E0240, I20260+Part2, E0240+Part2, E0240+Part2, K880005-<i>comE</i> WT, K990005-<i>comE</i>-myc, K990005-<i>comE<sup>D58E</sup></i>-myc<br> |
· <br> | · <br> | ||
| Line 1,054: | Line 1,007: | ||
· Ligation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP cut on EcoRI-HF and PstI-HF into pSB3K3 cut on EcoRI-HF and PstI-HF<br> | · Ligation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP cut on EcoRI-HF and PstI-HF into pSB3K3 cut on EcoRI-HF and PstI-HF<br> | ||
· Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br> | · Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br> | ||
| - | · Restriction check of pSB1C3-160bp | + | · Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240 at XmnI and NcoI<br> |
· Whole construct gel check <br> | · Whole construct gel check <br> | ||
· Restrict check PcomX-E0240<br> | · Restrict check PcomX-E0240<br> | ||
· Restrict check Part 1 + Part 2<br> | · Restrict check Part 1 + Part 2<br> | ||
· Restrict check I20260 and E0240 <br> | · Restrict check I20260 and E0240 <br> | ||
| - | · Restrict check K880005-comE WT<br> | + | · Restrict check K880005-<i>comE</i> WT<br> |
| - | · Restrict check K880005-comE-myc<br> | + | · Restrict check K880005-<i>comE</i>-myc<br> |
| - | · Restrict check K880005- | + | · Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br> |
· <br> | · <br> | ||
| Line 1,067: | Line 1,020: | ||
Wet lab<br> | Wet lab<br> | ||
· Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br> | · Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br> |
| - | · Restriction check of pSB1C3-160bp | + | · Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br> |
· Restrict check I20260-Part 2<br> | · Restrict check I20260-Part 2<br> | ||
· Restrict check E0240-Part 2<br> | · Restrict check E0240-Part 2<br> | ||
| Line 1,078: | Line 1,031: | ||
<br><b>Sept 4</b> <br> | <br><b>Sept 4</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br> |
· Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br> | · Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br> | ||
· Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF<br> | · Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF<br> | ||
· Transformation of pSB3K3-Combox.GFP ligation product<br> | · Transformation of pSB3K3-Combox.GFP ligation product<br> | ||
| - | · Digestion of pSB1C3-160bp | + | · Digestion of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br> |
| - | · Ligation of 160bp | + | · Ligation of 160bp P<sub>combox</sub>-BBa_E0240,comX and pSB3K3<br> |
· Restrict check I20260-Part 2, E0240-Part 2, I20260-Part 2, whole construct, -ve of whole construct ligation<br> | · Restrict check I20260-Part 2, E0240-Part 2, I20260-Part 2, whole construct, -ve of whole construct ligation<br> | ||
· Restrict check PcomCDE-E0240<br> | · Restrict check PcomCDE-E0240<br> | ||
· Restrict check I20260, E0240, Whole construct 3,4,5<br> | · Restrict check I20260, E0240, Whole construct 3,4,5<br> | ||
· Restrict check and colony PCR for pSB1C3-PcomCDE<br> | · Restrict check and colony PCR for pSB1C3-PcomCDE<br> | ||
| - | · Restrict check K880005+ | + | · Restrict check K880005+<i>comE<sup>D58E</sup></i>-N-myc<br> |
| - | · Restrict check K880005+comE-C-myc<br> | + | · Restrict check K880005+<i>comE</i>-C-myc<br> |
· <br> | · <br> | ||
| Line 1,110: | Line 1,063: | ||
· Restrict check and plasmid PCR for PcomCDE-E0240<br> | · Restrict check and plasmid PCR for PcomCDE-E0240<br> | ||
· Obtain sequencing results for PcomCDE-pSB1C3, whole construct (PcomX)<br> | · Obtain sequencing results for PcomCDE-pSB1C3, whole construct (PcomX)<br> | ||
| - | · K880005-comE/ | + | · K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc digest<br> |
· Ligation of Part 1 and 2 (also I20260, E0240)<br> | · Ligation of Part 1 and 2 (also I20260, E0240)<br> | ||
· Transformation<br> | · Transformation<br> | ||
| Line 1,127: | Line 1,080: | ||
· Obtain sequencing results for PcomCDE-pSB1C3<br> | · Obtain sequencing results for PcomCDE-pSB1C3<br> | ||
· Whole construct (PcomX)<br> | · Whole construct (PcomX)<br> | ||
| - | · K880005-comE/ | + | · K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc send to sequencing<br> |
· Ligation and transformation of Part 1 and 2 (also I20260, E0240)<br> | · Ligation and transformation of Part 1 and 2 (also I20260, E0240)<br> | ||
· <br> | · <br> | ||
| Line 1,133: | Line 1,086: | ||
<br><b>Sept 11</b> <br> | <br><b>Sept 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Restriction check of pSB3K3-comX-160bp | + | · Restriction check of pSB3K3-comX-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br> |
· Obtain sequencing results PcomCDE-pSB1C3<br> | · Obtain sequencing results PcomCDE-pSB1C3<br> | ||
· Gel electrophoresis of colony PCR product from 10/9 PcomCDE-E0240<br> | · Gel electrophoresis of colony PCR product from 10/9 PcomCDE-E0240<br> | ||
| Line 1,140: | Line 1,093: | ||
· Gel extraction and purification of E0240 and I20260<br> | · Gel extraction and purification of E0240 and I20260<br> | ||
· Gel extraction and purification of PcomCDE-E0240<br> | · Gel extraction and purification of PcomCDE-E0240<br> | ||
| - | · Streak plate for K880005-comE-C-myc<br> | + | · Streak plate for K880005-<i>comE</i>-C-myc<br> |
· <br> | · <br> | ||
| Line 1,146: | Line 1,099: | ||
<br><b>Sept 12</b> <br> | <br><b>Sept 12</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Restriction check of pSB3K3-160bp | + | · Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br> |
| - | · Transformation of pSB3K3-160bp | + | · Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br> |
· Make glycerol stock for PcomCDE-pSB1C3<br> | · Make glycerol stock for PcomCDE-pSB1C3<br> | ||
· Gel electrophoresis of colony PCR product of PcomCDE-E0240 from 10/9; Bacterial inoculation of checked products (for sequencing); Streak plate<br> | · Gel electrophoresis of colony PCR product of PcomCDE-E0240 from 10/9; Bacterial inoculation of checked products (for sequencing); Streak plate<br> | ||
· Check colony and fluorescence for whole construct (PcomX)<br> | · Check colony and fluorescence for whole construct (PcomX)<br> | ||
| - | · Streak plate for K880005-comE-C-myc<br> | + | · Streak plate for K880005-<i>comE</i>-C-myc<br> |
· <br> | · <br> | ||
</div> | </div> | ||
| Line 1,160: | Line 1,113: | ||
<br><b>Sept 15</b> <br> | <br><b>Sept 15</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br> |
| - | · PCR check and inoculation of colonies of I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005- | + | · PCR check and inoculation of colonies of I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
· Restrict check, Gel extraction and Gel purification of pSB3K3 J04450 (CHL)<br> | · Restrict check, Gel extraction and Gel purification of pSB3K3 J04450 (CHL)<br> | ||
· <br> | · <br> | ||
| Line 1,168: | Line 1,121: | ||
<br><b>Sept 16</b> <br> | <br><b>Sept 16</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI<br> | + | · Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI<br> |
· <br> | · <br> | ||
<br><b>Sept 17</b> <br> | <br><b>Sept 17</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion of pSB1C3-K880005.comW-FLAG on XbaI and PstI<br> | + | · Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI<br> |
| - | · Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> | + | · Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> |
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br> | ||
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI<br> | ||
· Digestion check of Combox + GFP on XmnI and PstI<br> | · Digestion check of Combox + GFP on XmnI and PstI<br> | ||
| - | · Restriction check of pSB3K3-160bp | + | · Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at NotI and PstI<br> |
| - | · PCR check and inoculation for I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005- | + | · PCR check and inoculation for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
· Restrict check pSB3K3<br> | · Restrict check pSB3K3<br> | ||
· Gel extraction and Gel purification of J04450 (CHL) insert X,S<br> | · Gel extraction and Gel purification of J04450 (CHL) insert X,S<br> | ||
| - | · Sequencing reaction for K880005- | + | · Sequencing reaction for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
· <br> | · <br> | ||
| Line 1,188: | Line 1,141: | ||
Wet lab<br> | Wet lab<br> | ||
· Ligation of comX-myc into pSB1C3 cut on XbaI and PstI<br> | · Ligation of comX-myc into pSB1C3 cut on XbaI and PstI<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br> |
| - | · Colony PCR and inoculation of correct colonies for I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005- | + | · Colony PCR and inoculation of correct colonies for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
· Gel extraction and gel purification for J04450 (CHL), J04450 (KAN)<br> | · Gel extraction and gel purification for J04450 (CHL), J04450 (KAN)<br> | ||
| - | · Sequencing reaction and Restrict check for K880005- | + | · Sequencing reaction and Restrict check for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
· <br> | · <br> | ||
<br><b>Sept 19</b> <br> | <br><b>Sept 19</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI<br> | + | · Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI<br> |
| - | · Colony PCR, inoculation for I20260/E0240-K880005- | + | · Colony PCR, inoculation for I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
| - | · Miniprep for I20260/E0240-K880005-comE-C-myc<br> | + | · Miniprep for I20260/E0240-K880005-<i>comE</i>-C-myc<br> |
· Gel extraction and gel purification J04450 (CHL), J04450 (KAN) XS<br> | · Gel extraction and gel purification J04450 (CHL), J04450 (KAN) XS<br> | ||
| - | · Sequencing reaction for K880005-comE-C-myc<br> | + | · Sequencing reaction for K880005-<i>comE</i>-C-myc<br> |
· <br> | · <br> | ||
| Line 1,212: | Line 1,165: | ||
Wet lab<br> | Wet lab<br> | ||
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br> | · Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br> | ||
| - | · Inoculation of pSB1C3-K880005.comW-FLAG.B0015<br> | + | · Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br> |
| - | · Ligation of pSB3K3 backbone with 160bp | + | · Ligation of pSB3K3 backbone with 160bp P<sub>combox</sub>-BBa_E0240<br> |
| - | · Gel extraction and gel purification of I20260/E0240-K880005- | + | · Gel extraction and gel purification of I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc EP<br> |
| - | · Colony PCR and inoculation of I20260/E0240-K880005-comE-C-myc <br> | + | · Colony PCR and inoculation of I20260/E0240-K880005-<i>comE</i>-C-myc <br> |
· Gel extraction and gel purification J04450 (CHL), J04450 (KAN)<br> | · Gel extraction and gel purification J04450 (CHL), J04450 (KAN)<br> | ||
· Oligo annealing and overnight ligation w/ pSB1C3for PcomCDE<br> | · Oligo annealing and overnight ligation w/ pSB1C3for PcomCDE<br> | ||
| Line 1,222: | Line 1,175: | ||
<br><b>Sept 23</b> <br> | <br><b>Sept 23</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Digestion of pSB1C3-K880005.comW-FLAG.B0015 of EcoRI-HF and SpeI-HF<br> | + | · Digestion of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 of EcoRI-HF and SpeI-HF<br> |
· Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | · Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br> | ||
· Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> | · Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br> | ||
| - | · Transformation of pSB3K3-160bp | + | · Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 ligated on Sept 22<br> |
| - | · Gel check and redo liagtion for I20260-K880005- | + | · Gel check and redo liagtion for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br> |
| - | · Gel extraction and purification for I20260-K880005- | + | · Gel extraction and purification for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br> |
| - | · Ethanol precipitation for sequencing, restrict check K880005- | + | · Ethanol precipitation for sequencing, restrict check K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
· Dephosphorylation and gel extraction and gel purification of J04450 (CHL) (X&S)<br> | · Dephosphorylation and gel extraction and gel purification of J04450 (CHL) (X&S)<br> | ||
· Gel extraction and Gel purification, dephosphorylation of J04450 (KAN) (E&P)<br> | · Gel extraction and Gel purification, dephosphorylation of J04450 (KAN) (E&P)<br> | ||
| Line 1,237: | Line 1,190: | ||
Wet lab<br> | Wet lab<br> | ||
· Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification<br> | · Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification<br> | ||
| - | · Inoculation of bacterial colonies carrying pSB3K3-160bp | + | · Inoculation of bacterial colonies carrying pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br> |
| - | · Restrict check, gel extraction and purification, Ligation w/ pSB3K3 I20260-K880005- | + | · Restrict check, gel extraction and purification, Ligation w/ pSB3K3 I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-· myc, E0240-K880005-<i>comE</i>-C-myc <br> |
| - | · Obtain sequencing results for K880005- | + | · Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
· Transform pSB1C3-PcomCDE<br> | · Transform pSB1C3-PcomCDE<br> | ||
· <br> | · <br> | ||
| Line 1,245: | Line 1,198: | ||
<br><b>Sept 25</b> <br> | <br><b>Sept 25</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Restrcition digestion of ligated pSB3K3- | + | · Restrcition digestion of ligated pSB3K3-160P<sub>combox</sub>-BBa_E0240<br> |
| - | · Restrict check, gel extraction and purification, Ligation w/ pSB3K3, I20260-K880005- | + | · Restrict check, gel extraction and purification, Ligation w/ pSB3K3, I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br> |
| - | · Obtain sequencing results for K880005- | + | · Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
· Colony PCR w/ ‘NEW FPcomCDE’ and VR, inoculation of pSB1C3-PcomCDE<br> | · Colony PCR w/ ‘NEW FPcomCDE’ and VR, inoculation of pSB1C3-PcomCDE<br> | ||
| Line 1,254: | Line 1,207: | ||
<br><b>Sept 26</b> <br> | <br><b>Sept 26</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · Transformation of pSB3K3- | + | · Transformation of pSB3K3-160P<sub>combox</sub>-BBa_E0240 sample1<br> |
· Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240<br> | · Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240<br> | ||
| - | · Gel extraction and purificationI and ligation20260-K880005- | + | · Gel extraction and purificationI and ligation20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br> |
| - | · Restrict check K880005- | + | · Restrict check K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
| - | · Inoculation for sequencing K880005- | + | · Inoculation for sequencing K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br> |
· Transformation of pSB1C3-PcomCDE<br> | · Transformation of pSB1C3-PcomCDE<br> | ||
| Line 1,374: | Line 1,327: | ||
<br> <b> July 7 </b> <br> | <br> <b> July 7 </b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Oligos | + | . Oligos phosphorylation <br> |
. ligation of TAs and CRs <br> | . ligation of TAs and CRs <br> | ||
. Colony PCR <br> | . Colony PCR <br> | ||
. Digest Receptor Backbone pSB1C3 <br> | . Digest Receptor Backbone pSB1C3 <br> | ||
. Agar-LB plates <br> | . Agar-LB plates <br> | ||
| - | . Inoculation for | + | . Inoculation for transformed Biobricks <br> |
. Primer Design for TA construct Gibson Assembly <br> | . Primer Design for TA construct Gibson Assembly <br> | ||
. Gel check for the colony PCR <br> | . Gel check for the colony PCR <br> | ||
| Line 1,389: | Line 1,342: | ||
. Transformation of ligated TAs and CRs <br> | . Transformation of ligated TAs and CRs <br> | ||
. Miniprep previous TAs CRs constructs <br> | . Miniprep previous TAs CRs constructs <br> | ||
| - | . | + | . restriction check previous TAs CRs constructs <br> |
. Colony PCR for CRs and TAs <br> | . Colony PCR for CRs and TAs <br> | ||
. Re-ligation of TAs and CRs <br> | . Re-ligation of TAs and CRs <br> | ||
| Line 1,407: | Line 1,360: | ||
. Sequencing preparation of P<sub>bad</sub> <br> | . Sequencing preparation of P<sub>bad</sub> <br> | ||
. Miniprep previous TAs CRs constructs <br> | . Miniprep previous TAs CRs constructs <br> | ||
| - | . | + | . restriction check previous TAs CRs constructs <br> |
. Colony PCR <br> | . Colony PCR <br> | ||
. Inoculation for glycerol stocks <br> | . Inoculation for glycerol stocks <br> | ||
| - | . Digest Receptor Backbone , digestion of BBa_ | + | . Digest Receptor Backbone , digestion of BBa_ BBa_E0240 with EcoR1-HF and Xbal and gel purification <br> |
<br> <b> July 11</b> <br> | <br> <b> July 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
. P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <br> | . P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <br> | ||
| - | . Ligation of P<sub>bad</sub> and | + | . Ligation of P<sub>bad</sub> and BBa_E0240 <br> |
| - | . | + | . Overlapping PCR design <br> |
. Gibson assembly master mix preparation <br> | . Gibson assembly master mix preparation <br> | ||
. Glycerol stock <br> | . Glycerol stock <br> | ||
| - | . Competent cells preparations for | + | . Competent cells preparations for DH5alpha and DH10B <br> |
. Run gel for yesterday's restriction check <br> | . Run gel for yesterday's restriction check <br> | ||
| - | . Miniprep | + | . Miniprep BBa_E0240, mRFP, BBa_B0015 <br> |
. Colony PCR <br> | . Colony PCR <br> | ||
. One full set re-ligation (short time) <br> | . One full set re-ligation (short time) <br> | ||
| - | . Miniprep | + | . Miniprep BBa_I13401 and BBa_K592009 <br> |
</div> | </div> | ||
| Line 1,435: | Line 1,388: | ||
Wet lab<br> | Wet lab<br> | ||
. Gibson assembly master mix preparation <br> | . Gibson assembly master mix preparation <br> | ||
| - | . | + | . PCR <br> |
| - | . Ligation of P<sub>bad</sub> an | + | . Ligation of P<sub>bad</sub> an BBa_E0240 <br> |
| - | . Competent cells preparations for | + | . Competent cells preparations for DH5alpha and DH10B <br> |
| - | . Prepare receptor backbone for oligo anealing, pSB1C3- | + | . Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI) <br> |
. One full set of re-ligation (short time) <br> | . One full set of re-ligation (short time) <br> | ||
. One full set of re-ligation (O/N) <br> | . One full set of re-ligation (O/N) <br> | ||
| Line 1,447: | Line 1,400: | ||
Wet lab<br> | Wet lab<br> | ||
. Inoculation for P<sub>bad</sub> -GFP-TT <br> | . Inoculation for P<sub>bad</sub> -GFP-TT <br> | ||
| - | . (2nd time) Colony PCR | + | . (2nd time) Colony PCR using VF2 and VR <br> |
. Design primer for P<sub>bad</sub> promoter sequencing <br> | . Design primer for P<sub>bad</sub> promoter sequencing <br> | ||
. Gibson assembly master mix preparation <br> | . Gibson assembly master mix preparation <br> | ||
. Preparations for P<sub>bad</sub> re-characterization <br> | . Preparations for P<sub>bad</sub> re-characterization <br> | ||
| - | . Competent cells preparations for | + | . Competent cells preparations for DH5alpha and DH10B <br> |
. One full set re-ligation (O/N) <br> | . One full set re-ligation (O/N) <br> | ||
. Prepare receptor backbone for oligo anealing <br> | . Prepare receptor backbone for oligo anealing <br> | ||
| Line 1,460: | Line 1,413: | ||
. Gibson assembly master mix preparation <br> | . Gibson assembly master mix preparation <br> | ||
. Preparations for P<sub>bad</sub> re-characterization <br> | . Preparations for P<sub>bad</sub> re-characterization <br> | ||
| - | . Competent cells preparations for | + | . Competent cells preparations for DH5alpha and DH10B <br> |
. Prepare receptor backbone for oligo anealing <br> | . Prepare receptor backbone for oligo anealing <br> | ||
| - | . | + | . inoculate successful oligo annealing colonies <br> |
. Oligo annealing products C. PCR? <br> | . Oligo annealing products C. PCR? <br> | ||
| Line 1,470: | Line 1,423: | ||
. P<sub>bad</sub> re-characterization <br> | . P<sub>bad</sub> re-characterization <br> | ||
. Miniprep CRs TAs <br> | . Miniprep CRs TAs <br> | ||
| - | . | + | . Restriction check CRs,TAs <br> |
. Oligo annealing products C. PCR <br> | . Oligo annealing products C. PCR <br> | ||
. Prepare receptor backbone for oligo anealing <br> | . Prepare receptor backbone for oligo anealing <br> | ||
| Line 1,493: | Line 1,446: | ||
. M9 mininaml medium preparation <br> | . M9 mininaml medium preparation <br> | ||
. Colony PCR <br> | . Colony PCR <br> | ||
| - | . plate streaking for P<sub>bad</sub>- | + | . plate streaking for P<sub>bad</sub>-BBa_E0240 <br> |
. inoculation of TAs and CRs <br> | . inoculation of TAs and CRs <br> | ||
| Line 1,511: | Line 1,464: | ||
. Gibson assembly for amilCP <br> | . Gibson assembly for amilCP <br> | ||
. Miniprep CRs TAs <br> | . Miniprep CRs TAs <br> | ||
| - | . | + | . restriction check pSB1C3-TAs/CRs constructs <br> |
. Restrict GFP.dTT for CRs <br> | . Restrict GFP.dTT for CRs <br> | ||
. Restrict P<sub>bad</sub> for TAs <br> | . Restrict P<sub>bad</sub> for TAs <br> | ||
| - | . Restrict | + | . Restrict BBa_B0015 EcoR1 and XbaI <br> |
| - | . | + | . BBa_J04450 backbone extraction <br> |
. Colony PCR <br> | . Colony PCR <br> | ||
. inoculation of TAs and CRs <br> | . inoculation of TAs and CRs <br> | ||
| - | . prepare P<sub>bad</sub>L(+) A. concentration gradients for | + | . prepare P<sub>bad</sub> L(+) A. concentration gradients for characterization <br> |
. Restrict CRs <br> | . Restrict CRs <br> | ||
. Restrict TAs-Version1 <br> | . Restrict TAs-Version1 <br> | ||
| Line 1,533: | Line 1,486: | ||
. Colony PCR <br> | . Colony PCR <br> | ||
. Miniprep CRs TAs <br> | . Miniprep CRs TAs <br> | ||
| - | . | + | . restriction check pSB1C3-TAs/CRs constructs <br> |
| - | . Restrict | + | . Restrict BBa_B0015-Version2 <br> |
| Line 1,540: | Line 1,493: | ||
Wet lab<br> | Wet lab<br> | ||
. Inoculation candidate colony or construct <br> | . Inoculation candidate colony or construct <br> | ||
| - | . Miniprep and | + | . Miniprep and restriction check inoculations <br> |
. Gibson assembly check <br> | . Gibson assembly check <br> | ||
. Gibson PCR <br> | . Gibson PCR <br> | ||
. Teach inoue competent cells to main project <br> | . Teach inoue competent cells to main project <br> | ||
| - | . Miniprep of | + | . Miniprep of BBa_B0015, P<sub>bad</sub>-BBa_E0240 <br> |
| - | . glycerol stock (P<sub>bad</sub>- | + | . glycerol stock (P<sub>bad</sub>-BBa_E0240) <br> |
| - | . | + | . Re-ligation of oligo annealing O/N <br> |
<br> <b> July 24</b> <br> | <br> <b> July 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
. miniprep candidate colony or construct <br> | . miniprep candidate colony or construct <br> | ||
| - | . | + | . restriction check pSB1C3-TAs/CRs constructs <br> |
| - | . Re-Re-ligation | + | . Re-Re-ligation transformation <br> |
| - | . Re-Re-ligation | + | . Re-Re-ligation transformation V.X <br> |
. Gibson assembly check <br> | . Gibson assembly check <br> | ||
| Line 1,568: | Line 1,521: | ||
<br> <b> July 28 </b> <br> | <br> <b> July 28 </b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . oligo | + | . oligo phosphorylation <br> |
. oligo annealing <br> | . oligo annealing <br> | ||
. ligation of annealed oligos O/N <br> | . ligation of annealed oligos O/N <br> | ||
. gel for C. PCR <br> | . gel for C. PCR <br> | ||
. receptor backbone for annealed oligos <br> | . receptor backbone for annealed oligos <br> | ||
| - | . | + | . inoculate last ligation <br> |
. Check past locks/keys status <br> | . Check past locks/keys status <br> | ||
. P<sub>bad</sub> sequencing <br> | . P<sub>bad</sub> sequencing <br> | ||
| - | . transformation of some of the existing riboregulator | + | . transformation of some of the existing riboregulator Biobricks <br> |
. Benchling Check <br> | . Benchling Check <br> | ||
| Line 1,582: | Line 1,535: | ||
<br> <b> July 29 </b> <br> | <br> <b> July 29 </b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Miniprep and | + | . Miniprep and restriction check inoculations <br> |
| - | . | + | . restriction check pSB1C3-TAs/CRs constructs <br> |
. Inoculation of CR and TA from distribution kit <br> | . Inoculation of CR and TA from distribution kit <br> | ||
| - | . prepare | + | . prepare BBa_I20260 in pSB1C3 <br> |
. prepare Side project Buffer <br> | . prepare Side project Buffer <br> | ||
. prepare side project gel red 200:1 <br> | . prepare side project gel red 200:1 <br> | ||
. Check digested GFP <br> | . Check digested GFP <br> | ||
| - | . Check digested | + | . Check digested BBa_B0015 <br> |
. ligate oligos into GFP <br> | . ligate oligos into GFP <br> | ||
| - | . ligate oligos | + | . ligate oligos into BBa_B0015 <br> |
. prepare M9 for characterization <br> | . prepare M9 for characterization <br> | ||
. transform our P<sub>bad</sub> into DH5alpha <br> | . transform our P<sub>bad</sub> into DH5alpha <br> | ||
| Line 1,600: | Line 1,553: | ||
<br> <b> July 30</b> <br> | <br> <b> July 30</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . transformation of CLON001-010 in pSB1C3 ( | + | . transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401) <br> |
| - | . C. PCR for yesterday | + | . C. PCR for yesterday transformations (CLON001-010) <br> |
. If construct after gel check inoculation, restriction, ligation <br> | . If construct after gel check inoculation, restriction, ligation <br> | ||
| - | . prepare | + | . prepare BBa_I20260 in pSB1C3 <br> |
. transform our P<sub>bad</sub>into DH5alpha <br> | . transform our P<sub>bad</sub>into DH5alpha <br> | ||
. prepare M9 for characterization <br> | . prepare M9 for characterization <br> | ||
| Line 1,612: | Line 1,565: | ||
. Try Gibson assembly mastermix <br> | . Try Gibson assembly mastermix <br> | ||
. Digest Biobrick TA and CR <br> | . Digest Biobrick TA and CR <br> | ||
| - | . PCR clean up | + | . PCR clean up for existing CR and TA Biobricks <br> |
. run gel for yesterday's colony PCR <br> | . run gel for yesterday's colony PCR <br> | ||
. check dNTPs <br> | . check dNTPs <br> | ||
| - | . Sequencing preparation for | + | . Sequencing preparation for possible construct <br> |
. colony PCR for 5th <br> | . colony PCR for 5th <br> | ||
. inoculate candidates <br> | . inoculate candidates <br> | ||
| - | . prepare | + | . prepare BBa_I20260 in pSB1C3 <br> |
| Line 1,652: | Line 1,605: | ||
. miniprep CLON005 <br> | . miniprep CLON005 <br> | ||
. Digest 005 Spe1 and Pst1 <br> | . Digest 005 Spe1 and Pst1 <br> | ||
| - | . ligate CLON 005 with | + | . ligate CLON 005 with BBa_B0015 <br> |
| - | . prepare backbone : | + | . prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// BBa_J23102 X, P <br> |
. Ligate P<sub>bad</sub> with Key1 dTT <br> | . Ligate P<sub>bad</sub> with Key1 dTT <br> | ||
| - | . digest | + | . digest P<sub>tet</sub> lock 1 with S and P <br> |
<br><b>Aug 2</b> <br> | <br><b>Aug 2</b> <br> | ||
| Line 1,674: | Line 1,627: | ||
. Restrict P<sub>bad</sub> for TAs <br> | . Restrict P<sub>bad</sub> for TAs <br> | ||
. Restrict GFP.dTT for CRs <br> | . Restrict GFP.dTT for CRs <br> | ||
| - | . Restrict | + | . Restrict BBa_B0015 <br> |
. Ligate P<sub>bad</sub>.TA <br> | . Ligate P<sub>bad</sub>.TA <br> | ||
| - | . | + | . ligate CR.GFP.dTT <br> |
. Ligate P<sub>bad</sub>.dTT <br> | . Ligate P<sub>bad</sub>.dTT <br> | ||
. Transform TAs, CRs <br> | . Transform TAs, CRs <br> | ||
. inoculate 001-010 <br> | . inoculate 001-010 <br> | ||
. Sequence 001-010 <br> | . Sequence 001-010 <br> | ||
| - | . inoculate | + | . inoculate BBa_B0015 <br> |
. inoculate constitutive promoter <br> | . inoculate constitutive promoter <br> | ||
| - | . | + | . inoculate P<sub>bad</sub> with key 1 dTT <br> |
. Competent cells preparations DH10B <br> | . Competent cells preparations DH10B <br> | ||
| - | . Prepare for functional assay of | + | . Prepare for functional assay of P<sub>bad</sub> promoter <br> |
. Prepare M9 minimal Medium <br> | . Prepare M9 minimal Medium <br> | ||
. make a second list of oligos to be sent out <br> | . make a second list of oligos to be sent out <br> | ||
. Test out GA mastermix efficiency <br> | . Test out GA mastermix efficiency <br> | ||
| - | . Ligate TAs oligo with | + | . Ligate TAs oligo with BBa_B0015 <br> |
. inoue Buffer <br> | . inoue Buffer <br> | ||
. Sequence 001-010 <br> | . Sequence 001-010 <br> | ||
| Line 1,697: | Line 1,650: | ||
. Colony PCR for TA.dTTs <br> | . Colony PCR for TA.dTTs <br> | ||
. inoculate TA.dTT <br> | . inoculate TA.dTT <br> | ||
| - | . ligation of | + | . ligation of P<sub>bad</sub> and key3 <br> |
. Restrict TAs <br> | . Restrict TAs <br> | ||
. restrict CRs <br> | . restrict CRs <br> | ||
| - | . Restrict | + | . Restrict P<sub>bad</sub> for TAs <br> |
. Restrict GFP.dTT for CRs <br> | . Restrict GFP.dTT for CRs <br> | ||
| - | . Ligate | + | . Ligate P<sub>bad</sub>.TA <br> |
| - | . | + | . ligate CR.GFP.dTT <br> |
| - | . Ligate | + | . Ligate P<sub>bad</sub>.dTT <br> |
. Transform TAs, CRs <br> | . Transform TAs, CRs <br> | ||
. Prepare M9 medium <br> | . Prepare M9 medium <br> | ||
| - | . Prepare | + | . Prepare inoculations for P<sub>bad</sub> characterization <br> |
| - | . Sequecing | + | . Sequecing preparation <br> |
| - | . Digest | + | . Digest P<sub>tet</sub> lock 1 with S and P <br> |
. Glycerol stock for 001-010 <br> | . Glycerol stock for 001-010 <br> | ||
. Inoue competent cells <br> | . Inoue competent cells <br> | ||
| - | . "Mini Prep pSB1C3- | + | . "Mini Prep pSB1C3-PCR P<sub>bad</sub><br> |
<br><b>Aug 6</b> <br> | <br><b>Aug 6</b> <br> | ||
| Line 1,721: | Line 1,674: | ||
. Restrict TAs <br> | . Restrict TAs <br> | ||
. Restrict CRs <br> | . Restrict CRs <br> | ||
| - | . Restrict | + | . Restrict BBa_B0015 for TAs <br> |
. Colony PCR for TA.dTTs <br> | . Colony PCR for TA.dTTs <br> | ||
. Oligo annealing for TA.dTT <br> | . Oligo annealing for TA.dTT <br> | ||
. Inoue competent cells <br> | . Inoue competent cells <br> | ||
. Sequencing results <br> | . Sequencing results <br> | ||
| - | . Ligate | + | . Ligate P<sub>bad</sub> with Key1 dTT <br> |
| - | . Ligate | + | . Ligate P<sub>tet</sub> Lock 1 (GFP dTT) <br> |
| - | . Send PCR | + | . Send PCR P<sub>bad</sub> for sequencing <br> |
| - | . Assay | + | . Assay P<sub>bad</sub> promoter <br> |
<br><b>Aug 7</b> <br> | <br><b>Aug 7</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
. Gel check for C. PCR <br> | . Gel check for C. PCR <br> | ||
| - | . | + | . inoculate possible TA.dTT <br> |
. Ligation of TA and dTT (4TAs) <br> | . Ligation of TA and dTT (4TAs) <br> | ||
| - | . Ligation of | + | . Ligation of P<sub>bad</sub> and key dTT <br> |
. Gel check for TAs CRs <br> | . Gel check for TAs CRs <br> | ||
. competent cells <br> | . competent cells <br> | ||
| - | . miniprep | + | . miniprep P<sub>bad</sub>-dTT <br> |
| - | . miniprep | + | . miniprep P<sub>bad</sub>-key3 <br> |
| - | . Colony PCR for | + | . Colony PCR for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br> |
| - | . Restriction of | + | . Restriction of P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br> |
| - | . prepare backbone and or insert if any // and | + | . prepare backbone and or insert if any // and restriction check <br> |
| - | . Preparation for | + | . Preparation for P<sub>bad</sub> promoter characterization <br> |
| - | . Colony PCR for | + | . Colony PCR for P<sub>bad</sub> with Key 1 dTT <br> |
| - | . Colony PCR for | + | . Colony PCR for P<sub>tet</sub> Lock 1 GFP dTT <br> |
| - | . | + | . inoculation of ligated DNA <br> |
<br><b>Aug 8</b> <br> | <br><b>Aug 8</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . digestion of | + | . digestion of P<sub>bad</sub>-key3 <br> |
| - | . Restriction check for | + | . Restriction check for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3-DTT <br> |
| - | . ligation of | + | . ligation of P<sub>bad</sub>-key3-dTT <br> |
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br> | . Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br> | ||
| - | . Miniprep | + | . Miniprep P<sub>bad</sub> Key 1 dTT <br> |
| - | . Miniprep | + | . Miniprep P<sub>tet</sub> Lock 1 GFP dTT <br> |
. Restriction Check for the key 1 and lock 1 mentioned above <br> | . Restriction Check for the key 1 and lock 1 mentioned above <br> | ||
. Prepare digestion of key 1 and lock 1 for ligation <br> | . Prepare digestion of key 1 and lock 1 for ligation <br> | ||
| Line 1,762: | Line 1,715: | ||
. Restriction check CLON007(J), CLON 010(I) and CLON (G) <br> | . Restriction check CLON007(J), CLON 010(I) and CLON (G) <br> | ||
. digestion of CLON007(J), CLON 010(I) and CLON (G) <br> | . digestion of CLON007(J), CLON 010(I) and CLON (G) <br> | ||
| - | . ligation of CLON007(J), CLON 010(I) and CLON (G) with | + | . ligation of CLON007(J), CLON 010(I) and CLON (G) with P<sub>bad</sub> <br> |
. C. PCR for CLON 022-025 TAs.dTT <br> | . C. PCR for CLON 022-025 TAs.dTT <br> | ||
| - | . ligation of | + | . ligation of P<sub>bad</sub> and TAs <br> |
. ligation of GFP and CRs <br> | . ligation of GFP and CRs <br> | ||
. Inoue competent cells <br> | . Inoue competent cells <br> | ||
| - | . | + | . P<sub>bad</sub> promoter characterization <br> |
<br><b>Aug 9</b> <br> | <br><b>Aug 9</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . transformation , all in | + | . transformation , all in CHL, overnight ligation 16C <br> |
. Colony PCR gel check <br> | . Colony PCR gel check <br> | ||
. Check competent cells <br> | . Check competent cells <br> | ||
| Line 1,792: | Line 1,745: | ||
. Gel check for C. PCR TAs.dTT <br> | . Gel check for C. PCR TAs.dTT <br> | ||
. Colony PCR for CR.GFP.dTTs <br> | . Colony PCR for CR.GFP.dTTs <br> | ||
| - | . Colony PCR for | + | . Colony PCR for P<sub>bad</sub>.TAs <br> |
. Inoculate CR.GFP.dTTs <br> | . Inoculate CR.GFP.dTTs <br> | ||
| - | . Inoculate | + | . Inoculate P<sub>bad</sub>.TAs <br> |
. Restrict TA.dTTs <br> | . Restrict TA.dTTs <br> | ||
| - | . Ligate | + | . Ligate P<sub>bad</sub>.TA.dTTs <br> |
| - | . Transform | + | . Transform P<sub>bad</sub>.TA.dTTs <br> |
| - | . Restriction check for | + | . Restriction check for P<sub>tet</sub> Lock 1 GFP dTT and P<sub>bad</sub> Key 1 dTT <br> |
. Transform 2014 Kit plate 4 well GF <br> | . Transform 2014 Kit plate 4 well GF <br> | ||
. Transformation of restricted checked DNA <br> | . Transformation of restricted checked DNA <br> | ||
| - | . | + | . inoculate BBa_J13002 <br> |
| - | . | + | . inoculate more P<sub>bad</sub> dTTs <br> |
. sequencing PCR for MCR, MTA <br> | . sequencing PCR for MCR, MTA <br> | ||
. transformation for backbone with KAN resistant <br> | . transformation for backbone with KAN resistant <br> | ||
. inoculation B0034 from glycerol stock B0034 <br> | . inoculation B0034 from glycerol stock B0034 <br> | ||
. restriction check <br> | . restriction check <br> | ||
| - | . Ligation of | + | . Ligation of P<sub>bad</sub>-key3 into BBa_B0015-pSB1AK3 plasmid <br> |
| - | . inoculation of | + | . inoculation of P<sub>bad</sub>-key3 <br> |
<br><b>Aug 12</b> <br> | <br><b>Aug 12</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . transformation | + | . transformation P<sub>bad</sub> -TA <br> |
. Gel for TA.dTTs <br> | . Gel for TA.dTTs <br> | ||
. GEl for C. PCR for CR.GFP.dTT <br> | . GEl for C. PCR for CR.GFP.dTT <br> | ||
| - | . Digestion of PCR | + | . Digestion of PCR P<sub>bad</sub> <br> |
<br><b>Aug 13</b> <br> | <br><b>Aug 13</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
. miniprep CR.GFP.dTTs <br> | . miniprep CR.GFP.dTTs <br> | ||
| - | . miniprep | + | . miniprep P<sub>bad</sub> <br> |
| - | . | + | . restriction check CR.GFP.dTTs <br> |
| - | . | + | . inoculate TA.dTTs <br> |
. Gel extraction-purification of constitutive promoter <br> | . Gel extraction-purification of constitutive promoter <br> | ||
| - | . Ligate CR.GFP.dTTs into | + | . Ligate CR.GFP.dTTs into constitutive promoter <br> |
| - | . transform, | + | . transform, P<sub>bad</sub>.Key3 <br> |
| - | . transform | + | . transform constitutive promoter.CR.GFP.dTTs <br> |
. Gibson Assembly mastermix <br> | . Gibson Assembly mastermix <br> | ||
| - | . Digest | + | . Digest P<sub>bad</sub> Key dTT and P<sub>tet</sub> Lock 1 GFP dTT for 3A assembly <br> |
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br> | . Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br> | ||
. 3A assembly <br> | . 3A assembly <br> | ||
| - | . Plan for | + | . Plan for P<sub>bad</sub> characterization <br> |
. Prepare stock arabinose solution M9 medium <br> | . Prepare stock arabinose solution M9 medium <br> | ||
| Line 1,839: | Line 1,792: | ||
. check dNTPs <br> | . check dNTPs <br> | ||
. miniprep TA.dTTs <br> | . miniprep TA.dTTs <br> | ||
| - | . | + | . restriction check TA.dTTs <br> |
| - | . miniprep Lock3. | + | . miniprep Lock3.GFP.dTT <br> |
| - | . colony PCR for | + | . colony PCR for P<sub>bad</sub>-key3-dTT <br> |
. Gel extraction-purification of constitutive promoter <br> | . Gel extraction-purification of constitutive promoter <br> | ||
| - | . Ligate CR.GFP.dTTs into | + | . Ligate CR.GFP.dTTs into constitutive promoter <br> |
| - | . ligate TA.dTT with | + | . ligate TA.dTT with P<sub>bad</sub> <br> |
| - | . transform ligate | + | . transform ligate P<sub>bad</sub>.TA.dTTs <br> |
| - | . transform | + | . transform constitutive promoter.CR.GFP.dTTs <br> |
<br><b>Aug 15</b> <br> | <br><b>Aug 15</b> <br> | ||
| Line 1,852: | Line 1,805: | ||
. Gibson Assembly mastermix <br> | . Gibson Assembly mastermix <br> | ||
. purify CR.GFP.dTTs <br> | . purify CR.GFP.dTTs <br> | ||
| - | . Ligate CR.GFP.dTTs into | + | . Ligate CR.GFP.dTTs into constitutive promoter <br> |
. Digestion of TA-dTT and PCR clean-up <br> | . Digestion of TA-dTT and PCR clean-up <br> | ||
. Restriction Check for TA-dTT <br> | . Restriction Check for TA-dTT <br> | ||
| - | . transform ligate | + | . transform ligate P<sub>bad</sub>.TA.dTTs <br> |
| - | . transform | + | . transform constitutive promoter.CR.GFP.dTTs <br> |
. make 10x DNA loading Dye <br> | . make 10x DNA loading Dye <br> | ||
. Order new TA and CR <br> | . Order new TA and CR <br> | ||
| Line 1,875: | Line 1,828: | ||
<br><b>Aug 18</b> <br> | <br><b>Aug 18</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Restriction check for | + | . Restriction check for P<sub>bad</sub>.TA.dTTs <br> |
. Restriction check for constitutive promoter.CR.GFP.dTT <br> | . Restriction check for constitutive promoter.CR.GFP.dTT <br> | ||
. Restriction check for constitutive promoter.RBS.GFP.dTT <br> | . Restriction check for constitutive promoter.RBS.GFP.dTT <br> | ||
| Line 1,882: | Line 1,835: | ||
. PCR check for constitutive promoter.CR.GFP.dTT <br> | . PCR check for constitutive promoter.CR.GFP.dTT <br> | ||
. PCR check for constitutive promoter.RBS.GFP.dTT <br> | . PCR check for constitutive promoter.RBS.GFP.dTT <br> | ||
| - | . PCR check for | + | . PCR check for P<sub>bad</sub>.TA.dTT <br> |
| - | . | + | . restriction check TA.dTTs <br> |
. Prepare functional assay for RBS, CRs <br> | . Prepare functional assay for RBS, CRs <br> | ||
. Inoculate candidates <br> | . Inoculate candidates <br> | ||
. Prepare pSB3K3 <br> | . Prepare pSB3K3 <br> | ||
. Prepare pSB1C3 for oligo ligation <br> | . Prepare pSB1C3 for oligo ligation <br> | ||
| - | . Prepare | + | . Prepare BBa_I13401 for oligo ligation <br> |
| - | . Prepare | + | . Prepare BBa_B0015 for oligo ligation <br> |
. Inoue competent cells <br> | . Inoue competent cells <br> | ||
| - | . Prepare for | + | . Prepare for P<sub>bad</sub> characterization <br> |
| - | . Take over the tank B and C | + | . Take over the tank B and C around 1pm-5;30 <br> |
<br><b>Aug 19</b> <br> | <br><b>Aug 19</b> <br> | ||
| Line 1,901: | Line 1,854: | ||
. miniprep possible candidates <br> | . miniprep possible candidates <br> | ||
. Restrict TA.dTT <br> | . Restrict TA.dTT <br> | ||
| - | . pSB1C3- | + | . pSB1C3-BBa_I13401 for oligo ligation <br> |
| - | . | + | . BBa_B0015 for oligo ligation <br> |
| - | . pSB1C3- | + | . pSB1C3-BBa_J04450 for oligo ligation <br> |
. Inoue competent cells <br> | . Inoue competent cells <br> | ||
| - | . | + | . P<sub>bad</sub>-key3-dTT restriction check <br> |
<br><b>Aug 20</b> <br> | <br><b>Aug 20</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Gel check for | + | . Gel check for P<sub>bad</sub>.TA.dTTs <br> |
| - | . purification for | + | . purification for BBa_B0015, BBa_I13401 <br> |
| - | . pSB1C3- | + | . pSB1C3-BBa_J04450 for oligo ligation <br> |
. Gel check for PCR products (19/08) <br> | . Gel check for PCR products (19/08) <br> | ||
| - | . PCR check for | + | . PCR check for BBa_J23102, LOCK 3 GFP DTT <br> |
| - | . | + | . Restriction check for key1 DTT <br> |
<br><b>Aug 21</b> <br> | <br><b>Aug 21</b> <br> | ||
| Line 1,921: | Line 1,874: | ||
. Ligate UST lock <br> | . Ligate UST lock <br> | ||
. restriction check for key1.dTT <br> | . restriction check for key1.dTT <br> | ||
| - | . ligate | + | . ligate P<sub>bad</sub>.key1.dTT <br> |
. ligate lock 3.GFP.dTT with C. promoter <br> | . ligate lock 3.GFP.dTT with C. promoter <br> | ||
. Colony PCR for c. promoter.MCR.GFP.dTT <br> | . Colony PCR for c. promoter.MCR.GFP.dTT <br> | ||
| Line 1,935: | Line 1,888: | ||
. Colony PCR for pSB1C3-HKUSTlock1 <br> | . Colony PCR for pSB1C3-HKUSTlock1 <br> | ||
. Colony PCR for HKUSTlock1.GFP.dTT <br> | . Colony PCR for HKUSTlock1.GFP.dTT <br> | ||
| - | . miniprep | + | . miniprep pSB3K3-J04550 for pSB3K3 Backbone <br> |
. restrict pSB3K3 for 3 pieces assemblage <br> | . restrict pSB3K3 for 3 pieces assemblage <br> | ||
. ligate TA-CR into pSB3K3 <br> | . ligate TA-CR into pSB3K3 <br> | ||
. Transform TA-CR <br> | . Transform TA-CR <br> | ||
| - | . colony PCR for | + | . colony PCR for P<sub>bad</sub>-key3-dTT <br> |
| - | . inoculate | + | . inoculate P<sub>bad</sub>.key1.dTT <br> |
. gel check for C. PCR on 21/08 <br> | . gel check for C. PCR on 21/08 <br> | ||
. extraction of pSB3K3 <br> | . extraction of pSB3K3 <br> | ||
| Line 1,950: | Line 1,903: | ||
Wet lab<br> | Wet lab<br> | ||
. Gel check for TAs <br> | . Gel check for TAs <br> | ||
| - | . colony PCR for UST lock in | + | . colony PCR for UST lock in BBa_I13401 and pSB1C3 <br> |
. transform ust key <br> | . transform ust key <br> | ||
. Check competent cells <br> | . Check competent cells <br> | ||
| Line 1,958: | Line 1,911: | ||
<br><b>Aug 24</b> <br> | <br><b>Aug 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Colony PCR for | + | . Colony PCR for P<sub>bad</sub>.Key1.dTT <br> |
| - | . Gel check for | + | . Gel check for P<sub>bad</sub>.Key1.dTT <br> |
| Line 1,970: | Line 1,923: | ||
<br><b>Aug 25</b> <br> | <br><b>Aug 25</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Colony PCR for | + | . Colony PCR for P<sub>bad</sub>.Key1.dTT <br> |
. PCR check for CR.GFP.dTT <br> | . PCR check for CR.GFP.dTT <br> | ||
. PCR check for Cons. prom.CR.GFP.dTT <br> | . PCR check for Cons. prom.CR.GFP.dTT <br> | ||
| Line 1,977: | Line 1,930: | ||
. transform ust lock1 <br> | . transform ust lock1 <br> | ||
. transform ust key <br> | . transform ust key <br> | ||
| - | . ligation again key 1 into | + | . ligation again key 1 into BBa_B0015 <br> |
| - | + | ||
| Line 1,987: | Line 1,939: | ||
. Colony PCR for "un-named key" <br> | . Colony PCR for "un-named key" <br> | ||
. inoculate ones which SEEMS like it contains key <br> | . inoculate ones which SEEMS like it contains key <br> | ||
| - | . colony | + | . colony PCR for Key1.DTT <br> |
. Gel check for O/NPCR products <br> | . Gel check for O/NPCR products <br> | ||
| - | . | + | . PCR check for CR.GFP.dTTs <br> |
. Inoculate all possible candidates <br> | . Inoculate all possible candidates <br> | ||
| - | . | + | . Prepare BBa_I13401 for CRs <br> |
| - | . Streak | + | . Streak possible c.p.CR/RBS.GFP.dTT <br> |
. make SOC <br> | . make SOC <br> | ||
. single cut check for TAs <br> | . single cut check for TAs <br> | ||
| Line 2,002: | Line 1,954: | ||
Wet lab<br> | Wet lab<br> | ||
. miniprerp candidates <br> | . miniprerp candidates <br> | ||
| - | . | + | . PCR check for candidates <br> |
| - | . gel check for o/n | + | . gel check for o/n PCR <br> |
| - | . | + | . restriction check for ust key 1 <br> |
| - | . | + | . restriction check for key 1 <br> |
| - | . | + | . P<sub>bad</sub> for ust key 1 <br> |
| - | . extract ust lock 1 for | + | . extract ust lock 1 for BBa_J23102 <br> |
. ligate ust lock and key <br> | . ligate ust lock and key <br> | ||
. transform ust lock and key <br> | . transform ust lock and key <br> | ||
| Line 2,017: | Line 1,969: | ||
. plasmid PCR for HKUSt lock and key <br> | . plasmid PCR for HKUSt lock and key <br> | ||
. overnight digestion of B0034 for control construct <br> | . overnight digestion of B0034 for control construct <br> | ||
| - | . Digestion of HKUST key- | + | . Digestion of HKUST key-BBa_B0015 <br> |
| Line 2,025: | Line 1,977: | ||
. miniprep locks <br> | . miniprep locks <br> | ||
. glycerol stocks for locks <br> | . glycerol stocks for locks <br> | ||
| - | . minipreps for | + | . minipreps for BBa_B0015 and B0034 <br> |
| - | . ligation of | + | . ligation of P<sub>bad</sub>-Hkust key-BBa_B0015 <br> |
. digestion of HKUST lock <br> | . digestion of HKUST lock <br> | ||
. ligation of HKUST lock with GFP <br> | . ligation of HKUST lock with GFP <br> | ||
| Line 2,034: | Line 1,986: | ||
. inoculation of key and lock <br> | . inoculation of key and lock <br> | ||
. miniprep <br> | . miniprep <br> | ||
| - | . Key one | + | . Key one restriction check <br> |
| - | . key one restriction for inserting | + | . key one restriction for inserting P<sub>bad</sub> <br> |
| - | . Restrict and | + | . Restrict and extract lock3c rbs and ligate into c.promoter <br> |
. ligate lock 3c.GFP.dTT with C. promoter <br> | . ligate lock 3c.GFP.dTT with C. promoter <br> | ||
| - | . transform c. | + | . transform c. promoter lock 3c rbs.GFP.DTT <br> |
. Extract mcr/mta <br> | . Extract mcr/mta <br> | ||
| - | . ligate MCR and MTA into | + | . ligate MCR and MTA into pSB3K3 <br> |
. transform MCR/MTA <br> | . transform MCR/MTA <br> | ||
. Glycerol stock for locks <br> | . Glycerol stock for locks <br> | ||
| - | . | + | . BBa_E0240 ligate with C. Promoter <br> |
. TB <br> | . TB <br> | ||
| - | . parallel ligation and | + | . parallel ligation and anealing for key 1 <br> |
. Sequencing preparations <br> | . Sequencing preparations <br> | ||
| Line 2,056: | Line 2,008: | ||
. competent cells <br> | . competent cells <br> | ||
. Extract mcr/mta <br> | . Extract mcr/mta <br> | ||
| - | . ligate MCR and MTA into | + | . ligate MCR and MTA into pSB3K3 <br> |
. transform MCR/MTA <br> | . transform MCR/MTA <br> | ||
. Glycerol stock for locks <br> | . Glycerol stock for locks <br> | ||
| - | . PCR check for | + | . PCR check for Biobrick key1 <br> |
| - | + | ||
| Line 2,098: | Line 2,049: | ||
<br><b>Sept 1</b> <br> | <br><b>Sept 1</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . | + | . restriction check P<sub>bad</sub>- Lost your key-DTT <br> |
| - | . | + | . restriction check P<sub>bad</sub>-Lost your key-DTT <br> |
. colony PCR for CR.TAs (orthogonality) <br> | . colony PCR for CR.TAs (orthogonality) <br> | ||
| - | . colony PCR for Key1, | + | . colony PCR for Key1,DTT <br> |
. ligate lock3c.GFP.dTT into c.p plasmid <br> | . ligate lock3c.GFP.dTT into c.p plasmid <br> | ||
| - | . transform lock3. | + | . transform lock3.GFP.DTT <br> |
. Colony PCR for CR.TAs (orthogonality) <br> | . Colony PCR for CR.TAs (orthogonality) <br> | ||
| - | . Colony PCR for sherLock-GFP- | + | . Colony PCR for sherLock-GFP-DTT <br> |
| - | . | + | . restriction check for Lost Your Key in pSB1C3 <br> |
| - | . transform | + | . transform P<sub>bad</sub>.lost your key.DTT <br> |
. inoculate orthogonality candidates <br> | . inoculate orthogonality candidates <br> | ||
| - | . | + | . Prepare midi-prep for pSB3K3 <br> |
| Line 2,116: | Line 2,067: | ||
<br><b>Sept 2</b> <br> | <br><b>Sept 2</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Gel check for O/N colony | + | . Gel check for O/N colony PCR for key1 and pSB3K3 <br> |
. massive prep <br> | . massive prep <br> | ||
. PCR check sense reverse <br> | . PCR check sense reverse <br> | ||
| - | . colony | + | . colony PCR for orthogonality <br> |
| - | . colony | + | . colony PCR for cons. pro. lock3crbs.GFP.DTT <br> |
| - | . migrate cpromoter | + | . migrate cpromoter BBa_E0240 to pSB3K3 with mta key <br> |
| - | . inoculation of B0034-GFP- | + | . inoculation of B0034-GFP-DTT <br> |
| - | . inoculation of Sherlock-GFP- | + | . inoculation of Sherlock-GFP-DTT <br> |
. sequencing for sherlock in pSB1C3 <br> | . sequencing for sherlock in pSB1C3 <br> | ||
| - | . digestion of B0034-GFP- | + | . digestion of B0034-GFP-DTT <br> |
| - | . digestion of sherlock-GFP- | + | . digestion of sherlock-GFP-DTT <br> |
| Line 2,133: | Line 2,084: | ||
<br><b>Sept 3</b> <br> | <br><b>Sept 3</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . digestion of | + | . digestion of P<sub>bad</sub> MTA dTT <br> |
| - | . digestion of | + | . digestion of P<sub>bad</sub> dTT <br> |
| - | . digestion of | + | . digestion of BBa_J23102 MCR GFP dTT <br> |
| - | . digestion of | + | . digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br> |
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br> | . digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br> | ||
| - | . Ligation of | + | . Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br> |
| - | . Ligation of | + | . Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br> |
| - | . Ligation of | + | . Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> dTT <br> |
| - | . Plasmid PCR for SherLock-GFP- | + | . Plasmid PCR for SherLock-GFP-DTT <br> |
| - | . ligation of B0034-GFP- | + | . ligation of B0034-GFP-DTT <br> |
| - | . ligation of SherLock-GFP- | + | . ligation of SherLock-GFP-DTT <br> |
. sequencing <br> | . sequencing <br> | ||
. check plate for arabinose induced mcr mta while singing <br> | . check plate for arabinose induced mcr mta while singing <br> | ||
. extraction of key and lock <br> | . extraction of key and lock <br> | ||
. restriction check for key with lock <br> | . restriction check for key with lock <br> | ||
| - | . | + | . extraction of key1 DTT <br> |
| - | . ligation of key1 | + | . ligation of key1 DTT with P<sub>bad</sub> <br> |
. Gel check for O/N colonyPCR <br> | . Gel check for O/N colonyPCR <br> | ||
| - | . | + | . PCR check <br> |
. Check 031 <br> | . Check 031 <br> | ||
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br> | . ligate lock3c RBS.GFP.dTT into c.p plasmid <br> | ||
. transform lock3c RBS.GFP.dTT <br> | . transform lock3c RBS.GFP.dTT <br> | ||
| - | . Midi prep preparations | + | . Midi-prep preparations <br> |
| - | + | ||
| Line 2,164: | Line 2,114: | ||
Wet lab<br> | Wet lab<br> | ||
. Gel purification of digested insert and backbone <br> | . Gel purification of digested insert and backbone <br> | ||
| - | . Ligation of | + | . Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br> |
| - | . Ligation of | + | . Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br> |
| - | . digestion of | + | . digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br> |
. Transformation of ligated projects mentioned above <br> | . Transformation of ligated projects mentioned above <br> | ||
| - | . Colony PCR for sherLock-GFP- | + | . Colony PCR for sherLock-GFP-DTT <br> |
| - | . Colony PCR for B0034-GFP- | + | . Colony PCR for B0034-GFP-DTT <br> |
. Inoculation of successful colonies <br> | . Inoculation of successful colonies <br> | ||
. Gel check for O/N Colony PCR <br> | . Gel check for O/N Colony PCR <br> | ||
| - | . Gel check for O/N | + | . Gel check for O/N PCR <br> |
. Gel check for Erics restriction check <br> | . Gel check for Erics restriction check <br> | ||
. Inoculation of successful colonies <br> | . Inoculation of successful colonies <br> | ||
| Line 2,182: | Line 2,132: | ||
<br><b>Sept 5</b> <br> | <br><b>Sept 5</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . miniprep/glycerol | + | . miniprep/ glycerol stock <br> |
| - | . Transform | + | . Transform P<sub>bad</sub> dTT <br> |
| - | . Transform ligated produce of | + | . Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br> |
| - | + | ||
| Line 2,198: | Line 2,147: | ||
<br><b>Sept 8</b> <br> | <br><b>Sept 8</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | + | · <br> | |
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| Line 2,213: | Line 2,168: | ||
<br><b>Sept 10</b> <br> | <br><b>Sept 10</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . colony | + | . colony PCR <br> |
. ligations for final constructs <br> | . ligations for final constructs <br> | ||
. inoculations <br> | . inoculations <br> | ||
| - | . | + | . P<sub>bad</sub>.lost your key.DTT <br> |
. sherlock <br> | . sherlock <br> | ||
| - | . gel check for colony | + | . gel check for colony PCR <br> |
| - | . colony | + | . colony PCR <br> |
| Line 2,233: | Line 2,188: | ||
<br><b>Sept 12</b> <br> | <br><b>Sept 12</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . colony | + | . colony PCR <br> |
| - | . restrict key1. | + | . restrict key1.DTT for P<sub>bad</sub> insertion <br> |
| - | . ligate | + | . ligate P<sub>bad</sub> with key1.DTT <br> |
. transform key1.dTT <br> | . transform key1.dTT <br> | ||
. extract pSB3K3 <br> | . extract pSB3K3 <br> | ||
| Line 2,245: | Line 2,200: | ||
. inoculation <br> | . inoculation <br> | ||
. Digestion of CLON 036 <br> | . Digestion of CLON 036 <br> | ||
| - | . colony | + | . colony PCR mcr mta <br> |
| Line 2,264: | Line 2,219: | ||
<br><b>Sept 16</b> <br> | <br><b>Sept 16</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . mini prep | + | . mini prep P<sub>bad</sub>.key1.DTT <br> |
| - | . | + | . restriction check P<sub>bad</sub>.key1.DTT <br> |
| - | . gel check for O/N c. | + | . gel check for O/N c.PCR <br> |
. extract P<sub>bad</sub>.key1 <br> | . extract P<sub>bad</sub>.key1 <br> | ||
. inoculate every CR.TA construct <br> | . inoculate every CR.TA construct <br> | ||
| Line 2,280: | Line 2,235: | ||
Wet lab<br> | Wet lab<br> | ||
. purify P<sub>bad</sub>.key1 <br> | . purify P<sub>bad</sub>.key1 <br> | ||
| - | . ligate P<sub>bad</sub>key1 | + | . ligate P<sub>bad</sub>key1 DTT with locks <br> |
| - | . transform locks.P<sub>bad</sub>.key1. | + | . transform locks.P<sub>bad</sub>.key1.DTT <br> |
. 2xYT (agar) AMP <br> | . 2xYT (agar) AMP <br> | ||
. transform all <br> | . transform all <br> | ||
| Line 2,292: | Line 2,247: | ||
. Spread a plate <br> | . Spread a plate <br> | ||
. miniprep for 3 inoculations <br> | . miniprep for 3 inoculations <br> | ||
| - | . Sense oligo and VR PCR for HKUST Lock- | + | . Sense oligo and VR PCR for HKUST Lock-DTT construct <br> |
. Overnight digestion of plasmid which gives a band around 1300bp <br> | . Overnight digestion of plasmid which gives a band around 1300bp <br> | ||
. EtoHpp for sequencing <br> | . EtoHpp for sequencing <br> | ||
| - | . Miniprep | + | . Miniprep constructs for submission <br> |
| - | . | + | . inoculation <br> |
. transform 031, 034 <br> | . transform 031, 034 <br> | ||
. Check "Diagonal constructs" and controls <br> | . Check "Diagonal constructs" and controls <br> | ||
| - | . migrate CR | + | . migrate CR constructs into pSB3K3 <br> |
| Line 2,308: | Line 2,263: | ||
. miniprep lock.key candidate <br> | . miniprep lock.key candidate <br> | ||
. ligate problematic cr.key <br> | . ligate problematic cr.key <br> | ||
| - | . | + | . restriction check sherlock <br> |
| - | . | + | . gel extraction<br> |
| Line 2,323: | Line 2,278: | ||
<br><b>Sept 21</b> <br> | <br><b>Sept 21</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . miniprep | + | . miniprep matching CR.TA <br> |
. Restriction check by EcoR1 and Pst1 <br> | . Restriction check by EcoR1 and Pst1 <br> | ||
| Line 2,337: | Line 2,292: | ||
Wet lab<br> | Wet lab<br> | ||
. Run Gel check for matching set of CR.TA <br> | . Run Gel check for matching set of CR.TA <br> | ||
| - | . ligate | + | . ligate problematic sets <br> |
| - | . transform | + | . transform problematic sets <br> |
. prepare DNA for submission <br> | . prepare DNA for submission <br> | ||
| - | . Gel | + | . Gel purification <br> |
| - | . Ligation lock- | + | . Ligation lock-DTT with constitutive promoter <br> |
. transformation <br> | . transformation <br> | ||
| - | . pSB3K3- CRs ligation <br> | + | . pSB3K3-CRs ligation <br> |
| Line 2,350: | Line 2,305: | ||
<br><b>Sept 23</b> <br> | <br><b>Sept 23</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . pSB3K3- CRs transformation <br> | + | . pSB3K3-CRs transformation <br> |
. Gel extractions <br> | . Gel extractions <br> | ||
. Gel purifications <br> | . Gel purifications <br> | ||
| Line 2,356: | Line 2,311: | ||
. Agar L (+) plates <br> | . Agar L (+) plates <br> | ||
. Send DNA to BGI for sequencing <br> | . Send DNA to BGI for sequencing <br> | ||
| - | . C. PCR | + | . C. PCR <br> |
. Inoculate possible candidates <br> | . Inoculate possible candidates <br> | ||
. ligate problematic sets <br> | . ligate problematic sets <br> | ||
| Line 2,367: | Line 2,322: | ||
<br><b>Sept 24</b> <br> | <br><b>Sept 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . Competent cells preparations | + | . Competent cells preparations <br> |
. minipreps <br> | . minipreps <br> | ||
. colony PCR <br> | . colony PCR <br> | ||
| - | . miniprep for | + | . miniprep for Biobrick submission <br> |
| - | . send | + | . send plasmid for sequencing <br> |
| Line 2,402: | Line 2,357: | ||
. C. PCR for H series... <br> | . C. PCR for H series... <br> | ||
. inoculate possible candidates <br> | . inoculate possible candidates <br> | ||
| - | . check self ligated | + | . check self ligated pSB3K3 <br> |
. Gel check for possible candidates AseI <br> | . Gel check for possible candidates AseI <br> | ||
. Gel check for possible candidates PvuII <br> | . Gel check for possible candidates PvuII <br> | ||
. Ligated H6 H7 <br> | . Ligated H6 H7 <br> | ||
. transformed H6 H7 <br> | . transformed H6 H7 <br> | ||
| - | . O/N | + | . O/N inoculation of MCR/MTA for characterization <br> |
| Line 2,414: | Line 2,369: | ||
Wet lab<br> | Wet lab<br> | ||
. Extract pSB1C3 <br> | . Extract pSB1C3 <br> | ||
| - | . | + | . Extract 036 <br> |
. miniprep "H" series candidates <br> | . miniprep "H" series candidates <br> | ||
. Repeat CHL restrictions <br> | . Repeat CHL restrictions <br> | ||
| - | . Repeat CHL | + | . Repeat CHL ligations <br> |
. Repeat CHL transformations <br> | . Repeat CHL transformations <br> | ||
. Gel check for "7" series using NcoI <br> | . Gel check for "7" series using NcoI <br> | ||
| - | . | + | . restriction check AseI <br> |
| - | . | + | . restriction check NcoI <br> |
. Fluorescence measurement of MCR/MTA for characterization. <br> | . Fluorescence measurement of MCR/MTA for characterization. <br> | ||
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br> | . Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br> | ||
| Line 2,436: | Line 2,391: | ||
<br><b>Sept 29</b> <br> | <br><b>Sept 29</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . | + | . restriction check AseI <br> |
| - | . | + | . restriction check NcoI <br> |
| - | . mini prep 6 + MCR/MTA (thanks from | + | . mini prep 6 + MCR/MTA (thanks from Doyle) <br> |
. Restriction check for MCR/MTA in pSB3K3. <br> | . Restriction check for MCR/MTA in pSB3K3. <br> | ||
. restrict 036 <br> | . restrict 036 <br> | ||
| Line 2,444: | Line 2,399: | ||
. ligate monkey with sherlock <br> | . ligate monkey with sherlock <br> | ||
. Gel check for NcoI and AseI <br> | . Gel check for NcoI and AseI <br> | ||
| - | . Get pSB1C3 | + | . Get pSB1C3 backbone EcoRI and PstI <br> |
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br> | . Fluorescence measurement of MCR/MTA for characterization (second trial) <br> | ||
| - | . | + | . inoculate Lock and Keys for crude functional assay. <br> |
| Line 2,454: | Line 2,409: | ||
Wet lab<br> | Wet lab<br> | ||
. miniprep 11 tubes in shaker c <br> | . miniprep 11 tubes in shaker c <br> | ||
| - | . | + | . restriction check H" series with NcoI <br> |
. Restrict inoculated colonies wiht AseI <br> | . Restrict inoculated colonies wiht AseI <br> | ||
. Restrict inoculated colonies wiht NcoI <br> | . Restrict inoculated colonies wiht NcoI <br> | ||
. Gel purifications <br> | . Gel purifications <br> | ||
. ligation of H series and some from other series... <br> | . ligation of H series and some from other series... <br> | ||
| - | . | + | . transformation of 16 confirmed constructs <br> |
<br><b>Oct 1</b> <br> | <br><b>Oct 1</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | . digestion of key3,HKUSTkey | + | . digestion of key3,HKUSTkey<br> |
. miniprep- final constructs <br> | . miniprep- final constructs <br> | ||
| Line 2,499: | Line 2,454: | ||
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Latest revision as of 00:38, 18 October 2014
Notebook
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Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately. PneumosensorRiboregulator |
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