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Team:Hong Kong HKUST/wetlab/notebook
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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> | · <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> | ||
| Line 117: | Line 117: | ||
<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> | · <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> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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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> | ||
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<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 150: | ||
<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 165: | ||
· <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 180: | ||
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> | ||
| Line 190: | Line 190: | ||
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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<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 291: | ||
· 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 300: | ||
· 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> | · <br> | ||
· <br> | · <br> | ||
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<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> | · <br> | ||
· <br> | · <br> | ||
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<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> | ||
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<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 424: | ||
· 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 457: | ||
· 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 472: | ||
· 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> | · <br> | ||
| Line 497: | Line 497: | ||
· 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> | ||
| Line 511: | Line 511: | ||
<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> | · <br> | ||
| Line 537: | Line 537: | ||
<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 572: | Line 572: | ||
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 582: | ||
<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 596: | ||
<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> | ||
| Line 605: | Line 605: | ||
<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 653: | ||
· 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 671: | ||
· 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 684: | ||
· 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 698: | ||
· 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 729: | ||
<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 750: | ||
· 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 757: | ||
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 770: | ||
· 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 793: | ||
· 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 812: | ||
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 820: | ||
· 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 comE mutant<br> | + | · Gel extraction and purification of <i>comE</i> mutant<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| Line 841: | Line 838: | ||
· 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 847: | ||
· 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 920: | ||
<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> | · <br> | ||
| Line 945: | Line 942: | ||
· 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> | · <br> | ||
| Line 962: | Line 959: | ||
<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-comE-myc<br> | + | · Ligate K880005-<i>comE</i>-myc<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| Line 983: | Line 980: | ||
<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 999: | ||
<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 1,040: | ||
<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 1,051: | ||
· 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 1,064: | ||
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 1,075: | ||
<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 1,107: | ||
· 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 1,124: | ||
· 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 1,130: | ||
<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 1,137: | ||
· 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 1,143: | ||
<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 1,157: | ||
<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 1,165: | ||
<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 1,185: | ||
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 1,209: | ||
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 1,219: | ||
<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 1,234: | ||
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 1,242: | ||
<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 1,251: | ||
<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> | ||
Revision as of 14:33, 17 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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