Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and
research findings. The notebook for Pneuumonsensor and Riboregulator are displayed separately.
Pneumosensor
June 2014
Week 1
June 2
Wet lab
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June 3
Wet lab
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June 4
Wet lab
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June 5
Wet lab
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June 6
Wet lab
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Week 2
June 9
Wet lab
· Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600
· Transformation of BBa_B0034 and BBa_E0040 into DH10B
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June 10
Wet lab
· Found reference promoter J23101 in BBa_I20260 from part registry
· Looked for Pcombox gene sequence from the genomic DNA
· Bacterial inoculation of BBa_B0034 and BBa_E0040
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June 11
Wet lab
· Looked for Pcombox gene sequence from the genomic DNA
· Studied the Characteristic, function and mechnism of how promoters work
· Miniprep of BBa_B0034 and BBa_E0040
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June 12
Wet lab
· Looked for Pcombox gene sequence from the genomic DNA
· research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization
· explain of scar
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June 13
Wet lab
· Looked for Pcombox gene sequence from the genomic DNA
· research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization
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Week 3
June 16
Wet lab
· Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for Pcombox gene sequence
· Found a possible Pcombox
· Transformation of BBa_J04450 and BBa_I20260 into DH10B
· Bacterial inoculation of BBa_E0240
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June 17
Wet lab
· Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for Pcombox gene sequence
· Found a possible Pcombox
· Searched for other possible Pcombox sequences
· Miniprep of BBa_E0240
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260
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June 18
Wet lab
· Designed primers for the Pcombox sequences
· Received a Pcombox sequence from Wellcome Trust Sanger Institute
· Miniprep of BBa_J04450(CHL) and BBa_I20260
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260
· Digestion of BBa_J04450(CHL) and BBa_I20260
· Gel electrophoresis of digested products
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June 19
· Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI
· Miniprep of BBa_J04450(CHL) and BBa_I20260
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested product
· Bacterial inoculation of BBa_J04450(CHL)
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June 20
· Amended primers of Pcombox sequences
· Miniprep of BBa_J04450(CHL)
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested product
Week 4
June 23
Wet lab
· Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005
· Primer design for PCR of comX-myc and comW-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain
· Designed plasmid construct for Pcombox
· Bacterial inoculation of BBa_J04450(CHL)
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June 24
Wet lab
· Inoculation of BBa_B0015
· Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp Pcombox
· Designed forward and reverse oligos of 67bp Pcombox
· Transformation of pSB1C3-BBa_I13504
· Miniprep of BBa_J04450(CHL)
· Transformation of BBa_J04450(KAN)
· Bacterial inoculation of BBa_E0240
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June 25
Wet lab
· Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification
· Inoculation of BBa_K880005
· Inoculation of pSB1C3-BBa_E0240
· Miniprep of BBa_E0240
· Digestion of BBa_E0240
· Transformation of BBa_J04450(KAN)
· Bacterial inoculation of BBa_J04450(CHL)
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June 26
Wet lab
· Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI
· Gel electrophoresis of digested BBa_E0240
· Gel extraction and purification of digested BBa_E0240 to obtain the insert
· Miniprep of BBa_J04450(CHL)
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested BBa_J04450(CHL)
· Gel extraction and purification of digested BBa_J04450(CHL) to obtain pSB1C3
· Bacterial inoculation of BBa_K880005
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June 27
Wet lab
· Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of pSB1C3-BBa_E0240
· Miniprep of BBa_K880005
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Week 5
June 30
Wet lab
· Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification
· Gel check of purified BBa_B0015 insert
· Transformation of ligated pSB1C3-K880005.B0015
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July 2014
Week 1
July 2
Wet lab
· Arrival of primers order for comX-myc and comW-FLAG
· Dilution of primers and design of experiment for both comX-myc and comW-FLAG
· Inoculation of ligated pSB1C3-K880005.B0015
· Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240
· Oligo annealing for PcomX
· Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)
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July 3
Wet lab
· PCR of comX-myc using Taq DNA Polymerase with ThermoPol Buffer to check whether primers are functional
· Gel check for comX-myc PCR product (PCR product present)
· PCR of comX-myc using Phusion High-Fidelity PCR Master Mix with HF Buffer
· Gel check for comX-myc PCR product (no PCR product present)
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI
· Received and stored the new common reverse primers for Pcombox and sense oligo and anti-sense oligo of 67bp Pcombox
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested BBa_E0240 and BBa_J04450
· Inoculation of pSB3K3-BBa_J04450
· PcomX Transformation
· PcomCDE Oligo annealing, ligation, transformation
· J04450 Plasmid Extraction
· E0240:Plasmid extraction (6 Falcon tubes); Nanodrop; Digestion by XbaI and SpeI, CutSmart as Buffer (only do 3 tubes); Gel electrophoresis; Gel extraction (INSERT!!! FRONT band); Gel purification
· B0015: Plasmid extraction (3 Falcon tubes); Nanodrop
· comED58E: PCR
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July 4
Wet lab
· PCR of comX-myc using Vent DNA Polymerase, PCR clean up
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI
· comED58E :Phusion PCR
· E0240: Gel purification
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Week 2
July 7
Wet lab
· Digestion of comX-myc with XbaI and PstI
· Gel check of digested comX-myc, digestion clean up
· PCR of comW-FLAG using Vent DNA Polymerase
· Gel check for comW-FLAG PCR product, PCR clean up
· Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI
· Gel purification of digested BBa_E0240 and BBa_J04450
· Submitted primers for Gibson assembly of pSB3K3-100bpPcombox
· Inoculation of pSB3K3-BBa_J04450
· E0240 - Bacterial inoculation
· pSB1C3-PcomX: Bacterial inoculation
· pSB1C3- PcomCDE: Bacterial inoculation, Streaking
· comED58E :PCR cleanup; Digestion with EcoRI and PstI; Ligation with pSB1C3; Transformation
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July 8
Wet lab
· Ligation of comX-myc cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI
· 2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control
· Transformation of ligation products
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested BBa_J04450
· Received pCEPcin from Universite de Toulouse, France
· J04450 (KAN): Digestion by EcoRI and PstI; Gel electrophoresis; Gel extraction
· E0240: Plasmid extraction
· pSB1C3-PcomX: Plasmid extraction
· pSB1C3- PcomCDE: Plasmid extraction
· comED58E: Bacterial inoculation; Ligation (redo); Transformation (redo)
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July 9
Wet lab
· Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc
· Ethanol precipitation of pCEPcin
· Gel purification of digested BBa_E0240
· Gel check for BBa_E0240 insert and pSB3K3 backbone
· PCR of 100bp Pcombox using Phusion polymerase
· Inoculation of pCEPcin
· E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis
· J04450 (KAN): Gel purification; NanoDrop (low conc)
· comED58E:Plasmid extraction; NanoDrop; Gel electrophoresis (not comE); Bacterial inoculation (redo)July
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July 10
Wet lab
· Another PCR of comW-FLAG using Vent DNA Polymerase
· Gel check for comW-FLAG PCR product, PCR clean up
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc
· 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
· Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· PCR of 100bp Pcombox using Phusion polymerase
· Ethanol precipitation of BBa_E0240 insert
· Ligate E0240-PcomX & PcomCDE
· Extract J04450 (KAN)
· Restrict check & run mutagenesis comED58E
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July 11
Wet lab
· Digestion of comW-FLAG with XbaI and PstI, digestion clean up
· Gel check for the PCR product obtained on July 10
· Digestion of pCEPcin at NcoI and XhoI
· Ethanol precipitation of pCEPcin
· Extract J04450 (KAN)
· Ligate pSB1C3 with comED58E
· TransformPcomCDE
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Week 3
July 14
Wet lab
· Ligation of comW-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI
· Transformation of ligation products
· Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc
· Digestion of pCEPcin at NcoI and XhoI
· PCR of 100bp Pcombox using Phusion polymerase
· Gel purification of digested BBa_J04450
· Inoculation of pCEPcin
· PCR of comED58E with only 1 primer → check whether the primers are working or not
· Ligation of pSB1C3-PcomX
· Inoculation of pSB1C3-PcomCDE
· Digest B0015 w/ NotI and SpeI stick ends
· Digest comED58E w/ XbaI and NotI sticky ends:
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July 15
Wet lab
· Digestion check of ligated pSB1C3-K880005.comX-myc
· Digestion of pSB1C3-K880005.comX-myc on SpeI and PstI, but no insert wanted present
· PCR of comX using Vent DNA Polymerase, PCR clean-up
· Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase
· Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)
· Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.comW-FLAG
· Ethanol precipitation of pCEPcin
· Gel check of the PCR product obtained on July 14
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase
· Plasmid extraction of pSB1C3-PcomCDE
· Bacterial inoculation (5 for PcomCDE, 4 for PcomX)
· Bacterial inoculation of B0015 (6 tubes)
· Extract gel B0015 & comED58E
· K880005 dephosphorylation; Digestion with SpeI
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July 16
Wet lab
· Digestion check of ligated pSB1C3-K880005.comW-FLAG
· Colony PCR of pSB1C3-K880005.comW-FLAG
· Gel check of pSB1C3-K880005.comW-FLAG PCR product (no PCR product present)
· PCR of comW using Vent DNA Polymerase (no PCR product present)
· Digestion of comX on XbaI and PstI
· Gel check of the PCR product obtained on July 15
· Gel purification of digested pSB3K3 backbone
· Digestion of pCEPcin2 at XhoI and SalI
· PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase
· Inoculation of pCEPcin2 and pSB3K3-BBa_I20260
· Plasmid extraction of Promoters and B0015
· Colony PCR
· Extract gel B0015 & comED58E by XbaI (comE) and SpeI (B0015)
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July 17
Wet lab
· PCR of comX-myc and comX using Vent DNA Polymerase
· Gel check of comX-myc and comX PCR product, PCR clean-up
· PCR of comW using Vent DNA Polymerase
· Gel extraction and gel purification of comW PCR product
· Dephosporylation of BBa_K880005
· Gel check of the PCR product obtained on July 16, all types of Pcombox shown positive result
· PCR clean-up of Pcombox
· PCR product ligation with pSB1C3
· Bacterial inoculation of B0015, comED58E
· Extract gel B0015 & comED58E - Digestion by XbaI (comED58E) and SpeI (B0015)
· Overnight Ligation
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July 18
Wet lab
· PCR of comX using Vent DNA Polymerase
· Gel check of comX PCR product, PCR clean-up
· Digestion of comX PCR product on XbaI and PstI, digestion clean-up
· Digestion of comX-myc and comW PCR products, digestion clean-up
· Gel extraction and gel purification of digested comX and comX-myc
· Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)
· Digestion of pCEPcin2 (at BamHI and NcoI)
· PCR of 100bp Pcombox using Phusion polymerase
· Gel check of the purified Pcombox obtained July 17
· Gel check of the 100bp Pcombox (PCR product)
· Gel purification of digested pSB3K3 backbone
· Ligation of comED58E
· Gel extract comED58E
· B0015 Gel purification
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Week 4
July 21
Wet lab
· Gel purification of comX and comX-myc
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio
· Ligation of comW into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Colony PCR of pSB1C3-K880005.comW-FLAG using Taq DNA Polymerase
· PCR of 100bp and 150bp Pcombox using Phusion polymerase
· Gel purification of digested pSB3K3 backbone
· Colony PCR of Promoters
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July 22
Wet lab
· 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)
· Inoculation of comX, comW, comX-myc, comW-FLAG
· Gel check of the PCR product obtained on July 21
· Oligo annealing of 67bp Pcombox
· Digestion of pSB3K3-BBa_I20260 at XbaI and PstI
· PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase
· Gel electrophoresis of colony PCR results
· Mutagenesis of comED58E
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July 23
Wet lab
· Digestion check of comX, comW, comX-myc, comW-FLAG colony PCR products on XbaI and SpeI
· Inoculation of comW-FLAG
· Gel check of PCR product obtained on July 22
· Gel purification of digested pSB3K3 backbone
· PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested pSB3K3 backbone
· Gel electrophoresis of colony PCR results
· Mutagenesis of comED58E
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July 24
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and SpeI-HF
· Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone
· PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification
· Digestion of comX-myc and comX on XbaI and PstI-HF
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel Check of PCR product obtained on July 23
· Received pCEPcin2
· Gel electrophoresis of colony PCR and mutagenesis results
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July 25
Wet lab
· Colony PCR of comX-myc using Taq DNA Polymerase
· Gel check of comX-myc PCR product (no PCR product present)
· Gel purification of digested pSB3K3 backbone
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel check on K880005
· Transformation of ligated product (promoters+pSB1C3)
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Week 5
July 28
Wet lab
· Colony PCR of comX-myc using Taq DNA Polymerase
· Colony PCR of comw-FLAG using Taq DNA Polymerase
· Inoculation of comX-myc and comW-FLAG
· Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI
· Colony PCR of promoters-pSB1C3
· Bacterial inoculation of K880005, comED58E
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July 29
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Transformation of comX-myc and comW-FLAG
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Digestion of pCEPcin2 at EcoRI and PstI
· Inoculation of pSB3K3-BBa_J04450
· PCR of 100bp,150bp and 160bp Pcombox using Vent polymerase
· Restrict check K880005
· Promoters colony PCR
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July 30
Wet lab
· Digestion check of comX-myc and comW-FLAG
· Inoculation of comX-myc and comW-FLAG
· Streaking plates of comX-myc and comW-FLAG
· PCR clean-up of 100bp,150bp and 160bp Pcombox
· Digestion of pCEPcin2 at EcoRI and PstI
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Restrict check K880005
July 31
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Redigest comW-FLAG on XmnI and NcoI
· PCR sequencing for comW and comX
· Ethanol precipitation for comW and comX
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· K880005 dephosphorylation & ligation
· Promoters Colony PCR + Sequencing reaction
August 2014
Week 1
Aug 1
Wet lab
· Digestion of pSB1C3-BBa_J04450
· Restrict check Promoters & Sequencing reaction
· Transform K880005
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Week 2
Aug 4
Wet lab
· Digestion of comX-myc on EcoRI-HF and SpeI, gel extraction and gel purification
· Ligation of BBa_B0015 cut on XbaI and PstI into pSB1C3-comX-myc backbone cut on SpeI and PstI
· Transformation of pSB1C3-comX-myc.B0015 ligation product
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Transformation of pSB1C3-B0015.comX-myc ligation product
· Sequencing of comX-myc and comW-FLAG
· Inoculation of comX-myc
· Digestion of 100bp, 150bp, 160bp and 180bp Pcombox at XbaI and PstI
· Ligation of 100bp and 160bp Pcombox with pSB1C3
· Digestion of pCEPcin2 at EcoRI and PstI
· PcomCDE Precipitation
· PcomX Sequencing reaction & precipitation
· Transform comED58E
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Aug 5
Wet lab
· Sequencing result failed
· Transformation of pSB1C3-B0015.comX-myc ligation product failed
· Digestion check of comX-myc on StyI
· Colony PCR of comX-myc
· Inoculation of comX-myc
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Ligation of 150bp, 180bp and 249bp Pcombox with pSB1C3
· Transformation of pSB1C3-150bp Pcombox , pSB1C3-180bp Pcombox and pSB1C3-249bp Pcombox
· Gel purification of digested pSB1C3 backbone
· Inoculation of pCEPcin2
· Send to sequencing: PcomCDE and PcomX
· Bacterial inoculation of comED58E
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Aug 6
Wet lab
· Digestion check of comX-myc on XbaI and PstI
· Gel check of comX-myc PCR product (no PCR product present)
· Sequencing of comW-FLAG using VF2 and VR
· Digestion of comX-myc on StyI
· Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI
· Gel purification of digested pSB1C3 backbone
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Phosphorylation of 67bp Pcombox oligos
· Oligo annealing for 67bp Pcombox
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· Align sequence with expected: PcomCDE and Pcom
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Aug 7
Wet lab
· Digestion of comX-myc on StyI
· Digestion check of comX on SphI and NcoI
· Digestion check of comW on XmnI and NcoI
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp Pcombox
· Gel purification of digested pSB1C3 backbone
· Precipitate after sequencing reaction PcomCDE
· Make glycerol stock for PcomX
· Run colony PCR for comED58E
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Aug 8
Wet lab
· Digestion of comW-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification
· Digestion of comW-FLAG on SpeI and PstI-HF, digestion clean-up
· Sequencing of comX-myc succeeded but mutation at EcoRI site
· Digestion check of comX-myc at EcoRI-HF and PstI-HF
· Ligation of comX and comW into dephosporylated pSB1C3-K880005
· Transformation of comX and comW ligation product
· Digestion of comX-myc at EcoRI-HF and SpeI-HF
· Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI
· Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp Pcombox with pSB1C3 backbone
· Ligate with E0240 PcomX
· Colony PCR for comE mutant
· Check sequence PcomCDE
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Week 3
Aug 11
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Gel purification of comX-myc digested on EcoRI-HF and SpeI
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Restriction check of pSB1C3-Pcombox (100bp, 180bp and 249bp) at XbaI and PstI
· Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp)
· Gel purification of digested pSB1C3 backbone
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Extract pSB1C3 from J04450
· Redo ligation part 1
· Gibson assembly of comED58E
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Aug 12
Wet lab
· Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF
· Dilution of comW-FLAG
· Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up
· Re-ligation of pSB1C3-B0015.comX-myc
· Transformation of pSB1C3-B0015.comX-myc ligation product
· Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc
· Digestion of pCEPcin2 at EcoRI and EcoRV
· Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI
· Ligate K880005 and comE(D58E)
· Inoculate for glycerol stock (PcomX)
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Aug 13
Wet lab
· Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Inoculation of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp)
· Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI
· Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)
· Check new dNTP
· Restrict check of ligation product part 1
· Colony PCR for checking part 2 ligation product and gibson assembly
· Inoculation of PcomX for glycerol stock
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Aug 14
Wet lab
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Transformation of ligated product pSB1C3-K880005.comX-myc.B0015
· Dilution of comW-FLAG
· Digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Ligation of comW-FLAG into pSB1C3-B0015 backbone
· Re-digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Gel check of the digested pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13
· Colony PCR of Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)
· glycerol stock for PcomX
· colony PCR ligation product part 1
· colony PCR gibson assembly
· Run gel for PCR products
· Inoculation of comED58E
·
Aug 15
Wet lab
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up
· Colony PCR of comW-FLAG using Taq DNA Polymerase
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Overnight ligation of BBa_B0015 into pSB1C3-K880005.comX-myc backbone at 16°C
· Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product
· Ligation of comX-myc into pSB1C3-B0015 backbone
· Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015
· Preparation for submittion of samples to be sequenced
· Inoculation of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp)
·
·
·
·
·
·
Week 4
Aug 18
Wet lab
· Transformation of ligated pSB1C3-K880005.comX-myc
· Ligation of comW-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion check of pSB1C3-K880005.comW-FLAG on SpeI-HF and PstI-HF
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015
· Digestion of pSB1C3-K880005.comX-myc on SpeI-HF and PstI-HF, digestion clean-up
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up
· Sent out pSB1C3-Pcombox (100bp, 150bp and 180bp) samples for commercial sequencing
· Digestion of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at SpeI and PstI
· Gel purification of digested pSB1C3-Pcombox (150bp, 160bp, 180bp and 249bp)
· Inoculation of pSB1C3-BBa_E0240
·
·
·
·
Aug 19
Wet lab
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up
· Digestion check of comX-myc on XbaI and PstI-HF
· Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015
· Gel purification of digested pSB1C3-Pcombox (100bp)
· Ligation of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) with BBa_E0240 insert
· Transformation of pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp)
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI
·
·
·
·
Aug 20
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF
· Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI
· Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comW-FLAG
· Gel purification of digested pSB1C3-BBa_E0240
· Digestion of pSB1C3-180bp Pcombox at SpeI and PstI
· Digestion clean-up of pSB1C3-180bp Pcombox
· Ligation of pSB1C3-180bp Pcombox with BBa_E0240 insert
· Transformation of pSB1C3-Pcombox-BBa_E0240 (100bp, 150bp and 180bp)
· Colony PCR for ligated pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp)
· Inoculation of pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp)
·
·
·
Aug 21
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase
· Digestion of comW-FLAG on NotI-HF
· Digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Inoculation of pSB1C3-K880005.comW-FLAG
· Transformation pSB1C3-K880005.comW-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015
· Restriction check of pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp) at NcoI
· Colony PCR for ligated pSB1C3-Pcombox-BBa_E0240 (100bp, 150bp and 180bp)
· Inoculation of pSB1C3-Pcombox-BBa_E0240 (100bp, 150bp and 180bp)
· Received sequencing result of pSB1C3-Pcombox (100bp, 150bp and 180bp)
·
·
·
·
Aug 22
Wet lab
· Digestion check of comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF
· Inoculation pSB1C3-K880005.comX-myc.B0015
· Restriction check of pSB1C3-Pcombox-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI
· PCR of pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp) using Vent polymerase
· Sent out pSB1C3-Pcombox-BBa_E0240 (160bp and 249bp) for commercial sequencing
·
·
·
·
·
·
Week 5
Aug 25
Wet lab
· Ligation of pSB1C3-K880005.comW-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
Transformation of pSB1C3-K880005.comW-FLAG
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
· Received sequencing result of pSB1C3-Pcombox (160bp and 249bp)
· Restriction check of pSB1C3-Pcombox-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI
· Digestion of pSB1C3-Pcombox (100bp, 150bp and 160bp, 180bp and 249bp) at XbaI and PstI
· Gel purification of digested Pcombox-BBa_E0240 (100bp, 150bp and 180bp) insert
·
·
·
·
·
Aug 26
Wet lab
· Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, digestion clean-up
· Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF
· Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Transformation of pSB1C3-K880005.comW-FLAG
· Ligation of comX, 100bp Pcombox-BBa_E0240 and pSB3K3 backbone
·
·
·
·
Aug 27
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification
· Sequencing of pSB1C3-K880005.comW-FLAG.B0015
· Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone
· Transformation of pSB1C3-K880005.comW-FLAG
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG
· Colony PCR of pSB1C3-Pcombox-BBa_E0240 (180bp and 249bp)
· Ligation of comX, 100bp Pcombox-BBa_E0240 and pSB3K3 backbone
· Inoculation of pSB1C3-Pcombox-BBa_E0240 (100bp, 180bp and 249bp), pSB1C3-Pcombox (160bp)
·
Aug 28
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG
· Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone
· Restriction check of pSB1C3-Pcombox-BBa_E0240 (180bp and 249bp)
· Colony PCR of pSB3K3-comX-100bp Pcombox-BBa_E0240
·
·
·
Aug 29
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Sent out pSB1C3-160bp Pcombox for commercial sequencing
· Digestion of pSB1C3-160bp Pcombox at SpeI and PstI
· Colony PCR for pSB3K3-comX-100bp Pcombox-BBa_E0240
· Ligation of pSB1C3-160bp Pcombox with BBa_E0240 insert
·
·
·
·
September 2014
Week 1
Sept 1
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI
· Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP
· Received the sequencing result of pSB1C3-160bp Pcombox
·
·
·
·
·
·
Sept 2
Wet lab
· 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
· Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification
· Restriction check of pSB1C3-160bp Pcombox-BBa_E0240 at XmnI and NcoI
·
·
·
·
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Sept 3
Wet lab
· Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Restriction check of pSB1C3-160bp Pcombox-BBa_E0240 at NcoI and NotI
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·
·
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Sept 4
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification
· Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification
· Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF
· Transformation of pSB3K3-Combox.GFP ligation product
· Digestion of pSB1C3-160bp Pcombox-BBa_E0240 at XbaI and PstI
· Ligation of 160bp Pcombox-BBa_E0240,comX and pSB3K3
·
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Sept 5
Wet lab
· Inoculation of pSB3K3-Combox.GFP
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Week 2
Sept 8
Wet lab
· Digestion check of pSB3K3-Combox.GFP on XmnI
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Sept 9
Wet lab
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Sept 10
Wet lab
· Streak plate for pSB3K3-Combox.GFP
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Sept 11
Wet lab
· Restriction check of pSB3K3-comX-160bp Pcombox-BBa_E0240 at EcoRI and PstI
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Sept 12
Wet lab
· Restriction check of pSB3K3-160bp Pcombox-BBa_E0240 at EcoRI and PstI
· Transformation of pSB3K3-160bp Pcombox-BBa_E0240
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Week 3
Sept 15
Wet lab
· Inoculation of pSB1C3-K880005.comW-FLAG
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Sept 16
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI
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·
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·
·
Sept 17
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG on XbaI and PstI
· Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI
· Digestion check of Combox + GFP on XmnI and PstI
· Restriction check of pSB3K3-160bp Pcombox-BBa_E0240 at NotI and PstI
·
·
·
·
Sept 18
Wet lab
· Ligation of comX-myc into pSB1C3 cut on XbaI and PstI
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
·
·
·
·
·
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Sept 19
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI
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·
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·
·
Week 4
Sept 22
Wet lab
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Ligation of pSB3K3 backbone with 160bp Pcombox-BBa_E0240
·
·
·
·
·
·
Sept 23
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG.B0015 of EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Transformation of pSB3K3-160bp Pcombox-BBa_E0240 ligated on Sept 22
·
·
·
·
·
·
Sept 24
Wet lab
· Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification
· Inoculation of bacterial colonies carrying pSB3K3-160bp Pcombox-BBa_E0240
·
·
·
·
·
·
·
·
Sept 25
Wet lab
· Restrcition digestion of ligated pSB3K3-160Pcombox-BBa_E0240
·
·
·
·
·
·
·
·
·
Sept 26
Wet lab
· Transformation of pSB3K3-160Pcombox-BBa_E0240 sample1
· Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240
·
·
·
·
·
·
·
·
Week 5
Sept 29
Wet lab
·
·
·
·
·
·
·
·
·
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Sept 30
Wet lab
· Digestion of RBS+comX-myc+B0015 on XbaI and PstI
·
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·
Riboregulator
June 2014
Week 1
June 2
Wet lab
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June 3
Wet lab
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June 4
Wet lab
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June 5
Wet lab
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June 6
Wet lab
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Week 2
June 9
Wet lab
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June 10
Wet lab
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June 11
Wet lab
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June 12
Wet lab
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June 13
Wet lab
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Week 3
June 16
Wet lab
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June 17
Wet lab
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June 18
Wet lab
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June 19
Wet lab
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June 20
Wet lab
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Week 4
June 23
Wet lab
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June 24
Wet lab
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June 25
Wet lab
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June 26
Wet lab
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June 27
Wet lab
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Week 5
June 30
Wet lab
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July 2014
Week 1
July 1
Wet lab
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July 2
Wet lab
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·
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July 3
Wet lab
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·
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July 4
Wet lab
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·
Week 2
July 7
Wet lab
. Oligos phosphorilation
. ligation of TAs and CRs
. Colony PCR
. Digest Receptor Backbone pSB1C3
. Agar-LB plates
. Inoculation for transformded BioBricks
. Primer Design for TA construct Gibson Assembly
. Gel check for the colony PCR
. Pbad PCR amplification
July 8
Wet lab
. Transformation of ligated TAs and CRs
. Miniprep previous TAs CRs constructs
. Restrict check previous TAs CRs constructs
. Colony PCR for CRs and TAs
. Re-ligation of TAs and CRs
. Pbad PCR clean up and Digestion
. Ligation of Pbad with pSB1C3
. Sequencing preparation of Pbad
July 9
Wet lab
. Transformation of Re-ligated TAs and CRs
. Colony PCR
. Gibson assembly master mix preparation
July 10
Wet lab
. Sequencing preparation of Pbad
. Miniprep previous TAs CRs constructs
. Restrict check previous TAs CRs constructs
. Colony PCR
. Inoculation for glycerol stocks
. Digest Receptor Backbone , digestion of BBa_ E0240 with EcoR1-HF and Xbal and gel purification
July 11
Wet lab
. Pbad digestion and gel purification, Digestion of PBAD-pSB1C3 with EcoR1-HF and Spe1 and gel purification
. Ligation of pBAD and E0240
. Overlaping PCR design
. Gibson assembly master mix preparation
. Glycerol stock
. Competent cells preparations for DH5a and DH10B
. Run gel for yesterday's restriction check
. Miniprep E0240, mRFP, B0015
. Colony PCR
. One full set re-ligation (short time)
. Miniprep I13401 and K592009
Week 3
July 14
Wet lab
. Gibson assembly master mix preparation
. Trevor's PCR
. Ligation of pBAD an E0240
. Competent cells preparations for DH5a and DH10B
. Prepare receptor backbone for oligo anealing, pSB1C3-J04450 (EcoRI, SpeI)
. One full set of re-ligation (short time)
. One full set of re-ligation (O/N)
. Preparations for Pbad re-Characterization
July 15
Wet lab
. Inoculation for pBAD-GFP-TT
. (2nd time) Colony PCR usning VF2 and VR
. Design primer for pBAD promoter sequencing
. Gibson assembly master mix preparation
. Preparations for Pbad re-characterization
. Competent cells preparations for DH5a and DH10B
. One full set re-ligation (O/N)
. Prepare receptor backbone for oligo anealing
July 16
Wet lab
. Gibson assembly master mix preparation
. Preparations for Pbad re-characterization
. Competent cells preparations for DH5a and DH10B
. Prepare receptor backbone for oligo anealing
. Innoculate succesful oligo annealing colonies
. Oligo annealing products C. PCR?
July 17
Wet lab
. Gibson assembly master mix preparation
. Pbad re-characterization
. Miniprep CRs TAs
. Restric check CRs,TAs
. Oligo annealing products C. PCR
. Prepare receptor backbone for oligo anealing
. Restrict CRs plasmids for GFP.dTT
. Restrict TAs plasmids for dTT
. Restrict GFP.dTT for CRs
. Restrict dTT for TAs
. Ligate CR.GFP.dTT
. Ligate TA.dTT
. Transform Ligated CR.GFP.dTT
. Transform Ligated TA.dTT
. Transform-Ligate Annealed oligos
July 18
Wet lab
. Gibson assembly for amilCP
. Pbad re-characterization
. Colony PCR
. Prepare receptor backbone for oligo anealing
. Restrict GFP.dTT for CRs
. Restrict dTT for TAs
. M9 mininaml medium preparation
. Colony PCR
. plate streaking for pBAD-E0240
. inoculation of TAs and CRs
July 19
Wet lab
. Pbad re-characterization
Week 4
July 21
Wet lab
. Gibson assembly for amilCP
. Miniprep CRs TAs
. Restrict check pSB1C3-TAs/CRs constructs
. Restrict GFP.dTT for CRs
. Restrict Pbad for TAs
. Restrict B0015 EcoR1 and XbaI
. J04450 backbone extraction
. Colony PCR
. inoculation of TAs and CRs
. prepare Pbad L(+) A. concentration gradients for charac
. Restrict CRs
. Restrict TAs-Version1
. Restrict TAs-Version2
. Ligate CR.GFP.dTT
. Ligate TA.dTT
. Transform Pbad.TA
. Transform CR.GFP.dTT
. Transform Gibson assembly
July 22
Wet lab
. Colony PCR
. Miniprep CRs TAs
. Restrict check pSB1C3-TAs/CRs constructs
. Restrict B0015-Version2
July 23
Wet lab
. Inoculation candidate colony or construct
. Miniprep and restrict check innoculations
. Gibson assembly check
. Gibson PCR
. Teach inoue competent cells to main project
. Miniprep of B0015, pBAD-E0240
. glycerol stock (pBAD-E0240)
. Re-Re-ligation of oligo annealing O/N
July 24
Wet lab
. miniprep candidate colony or construct
. Restrict check pSB1C3-TAs/CRs constructs
. Re-Re-ligation trasnformation
. Re-Re-ligation trasnformation V.X
. Gibson assembly check
July 25
Wet lab
. Certification day (safety evaluation)
Week 5
July 28
Wet lab
. oligo phosphorilation
. oligo annealing
. ligation of annealed oligos O/N
. gel for C. PCR
. receptor backbone for annealed oligos
. innoculate last ligation
. Check past locks/keys status
. Pbad sequencing
. transformation of some of the existing riboregulator biobricks
. Benchling Check
July 29
Wet lab
. Miniprep and restrict check innoculations
. Restrict check pSB1C3-TAs/CRs constructs
. Inoculation of CR and TA from distribution kit
. prepare I20260 in PSB1C3
. prepare Side project Buffer
. prepare side project gel red 200:1
. Check digested GFP
. Check digested B0015
. ligate oligos into GFP
. ligate oligos itno B0015
. prepare M9 for characterization
. transform our pbad into DH5alpha
. prepare M9 medium
. transformation of Clon001-010 in pSB1C3 ( with a control)
July 30
Wet lab
. transformation of CLON001-010 in pSB1C3 (B0015, I13401)
. C. PCR for yesterday transfomations (CLON001-010)
. If construct after gel check inoculation, restriction, ligation
. prepare I20260 in PSB1C3
. transform our pbad into DH5alpha
. prepare M9 for characterization
. Teach inoue competent cells to main project
July 31
Wet lab
. Try Gibson assembly mastermix
. Digest Biobrick TA and CR
. PCR clean up? for existing CR and TA biobricks
. run gel for yesterday's colony PCR
. check dNTPs
. Sequencing preparation for posible construct
. colony PCR for 5th
. inoculate candidates
. prepare i20260in psb1c3
August 2014
Week 1
Aug 1
Wet lab
. colony PCR for 5th
. Digest Biobrick TA and CR
. short ligation for TAs CRs
. Transform TAs, CRs
. inoue Buffer
. inoculations if possible
. miniprep CLON005
. Digest 005 Spe1 and Pst1
. ligate CLON 005 with B0015
. prepare backbone : B0015 E, X /// I13401 E,X /// J23102 X, P
. Ligate PBad with Key1 dTT
. digest ptet lock 1 with S and P
Aug 2
Wet lab
. Devise a general blue print for the catalog and feature page
Week 2
Aug 4
Wet lab
. Mini prep 001-010
. Restrict TAs
. restrict CRs
. Restrict pBAD for TAs
. Restrict GFP.dTT for CRs
. Restrict B0015
. Ligate Pbad.TA
. liagte CR.GFP.dTT
. Ligate Pbad.dTT
. Transform TAs, CRs
. inoculate 001-010
. Sequence 001-010
. inoculate B0015
. inoculate constitutive promoter
. Innoculate Pbad with key 1 dTT
. Competent cells preparations DH10B
. Prepare for functional assay of Pbad promoter
. Prepare M9 minimal Medium
. make a second list of oligos to be sent out
. Test out GA mastermix efficiency
. Ligate TAs oligo with B0015
. inoue Buffer
. Sequence 001-010
Aug 5
Wet lab
. Colony PCR for TA.dTTs
. inoculate TA.dTT
. ligation of pBAD and key3
. Restrict TAs
. restrict CRs
. Restrict pBAD for TAs
. Restrict GFP.dTT for CRs
. Ligate Pbad.TA
. liagte CR.GFP.dTT
. Ligate Pbad.dTT
. Transform TAs, CRs
. Prepare M9 medium
. Prepare Innoculations for Pbad characteriation
. Sequecing preapraion
. Digest Ptet lock 1 with S and P
. Glycerol stock for 001-010
. Inoue competent cells
. "Mini Prep pSB1C3-PCRPbad
Aug 6
Wet lab
. Miniprep TA.dTT
. Restriction check for TA.dTT
. Miniprep TAs CRs
. Restrict TAs
. Restrict CRs
. Restrict B0015 for TAs
. Colony PCR for TA.dTTs
. Oligo annealing for TA.dTT
. Inoue competent cells
. Sequencing results
. Ligate PBad with Key1 dTT
. Ligate Ptet Lock 1 (GFP dTT)
. Send PCR Pbad for sequencing
. Assay Pbad promoter
Aug 7
Wet lab
. Gel check for C. PCR
. inculate possible TA.dTT
. Ligation of TA and dTT (4TAs)
. Ligation of pBAD and key dTT
. Gel check for TAs CRs
. competent cells
. miniprep pBAD-dTT
. miniprep pBAD-key3
. Colony PCR for pBAD-dTT and pBAD-key3
. Restriction of pBAD-dTT and pBAD-key3
. prepare backbone and or insert if any // and restrict check
. Preparation for Pbad promoter characterization
. Colony PCR for Pbad with Key 1 dTT
. Colony PCR for Ptet Lock 1 GFP dTT
. Innoculation of ligated DNA
Aug 8
Wet lab
. digestion of pBAD-key3
. Restriction check for pBAD-dTT and pBAD-key3-dtt
. ligation of pBAD-key3-dTT
. Miniprep for CLON007(J), CLON 010(I) and CLON (G)
. Miniprep Pbad Key 1 dTT
. Miniprep Ptet Lock 1 GFP dTT
. Restriction Check for the key 1 and lock 1 mentioned above
. Prepare digestion of key 1 and lock 1 for ligation
. Transform BBa_J13002
. Restriction check CLON007(J), CLON 010(I) and CLON (G)
. digestion of CLON007(J), CLON 010(I) and CLON (G)
. ligation of CLON007(J), CLON 010(I) and CLON (G) with pBAD
. C. PCR for CLON 022-025 TAs.dTT
. ligation of pBAD and TAs
. ligation of GFP and CRs
. Inoue competent cells
. Pbad promoter characterization
Aug 9
Wet lab
. transformation , all in chl, overnight ligation 16C
. Colony PCR gel check
. Check competent cells
Aug 10
Wet lab
. Restriction check for TAs.dTT
. Gel check for C. PCR TAs.dTT
Week 3
Aug 11
Wet lab
. Restriction check for TAs.dTT
. Gel check for C. PCR TAs.dTT
. Colony PCR for CR.GFP.dTTs
. Colony PCR for Pbad.TAs
. Inoculate CR.GFP.dTTs
. Inoculate Pbad.TAs
. Restrict TA.dTTs
. Ligate Pbad.TA.dTTs
. Transform Pbad.TA.dTTs
. Restriction check for Ptet Lock 1 GFP dTT and Pbad Key 1 dTT
. Transform 2014 Kit plate 4 well GF
. Transformation of restricted checked DNA
. Innoculate BBa_J13002
. Innoculate more Pbad dTTs
. sequencing PCR for MCR, MTA
. transformation for backbone with KAN resistant
. inoculation B0034 from glycerol stock B0034
. restriction check
. Ligation of pBAD-key3 into B0015-pSB1AK3 plasmid
. inoculation of pBAD-key3
Aug 12
Wet lab
. transformation pBAD -TA
. Gel for TA.dTTs
. GEl for C. PCR for CR.GFP.dTT
. Digestion of PCR pBAD
Aug 13
Wet lab
. miniprep CR.GFP.dTTs
. miniprep Pbad
. Restrict check CR.GFP.dTTs
. innoculate TA.dTTs
. Gel extraction-purification of constitutive promoter
. Ligate CR.GFP.dTTs into Cosntritutive promoter
. transform, Pbad.Key3
. transform Cosntitutive promoter.CR.GFP.dTTs
. Gibson Assembly mastermix
. Digest Pbad Key dTT and Ptet Lock 1 GFP dTT for 3A assembly
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone
. 3A assembly
. Plan for Pbad characterization
. Prepare stock arabinose solution M9 medium
Aug 14
Wet lab
. Gel check for lock3.GFP.dTT colony PCR I-VI
. check dNTPs
. miniprep TA.dTTs
. Restrict check TA.dTTs
. miniprep Lock3.gfp.dTT
. colony PCR for PBAD-key3-dTT
. Gel extraction-purification of constitutive promoter
. Ligate CR.GFP.dTTs into Cosntritutive promoter
. ligate TA.dTT with Pbad
. transform ligate Pbad.TA.dTTs
. transform Cosntitutive promoter.CR.GFP.dTTs
Aug 15
Wet lab
. Gibson Assembly mastermix
. purify CR.GFP.dTTs
. Ligate CR.GFP.dTTs into Cosntritutive promoter
. Digestion of TA-dTT and PCR clean-up
. Restriction Check for TA-dTT
. transform ligate Pbad.TA.dTTs
. transform Cosntitutive promoter.CR.GFP.dTTs
. make 10x DNA loading Dye
. Order new TA and CR
. Gibson Assembly mastermix
Aug 16
Wet lab
. Miniprep
. Colony PCR for final constructs
Week 4
Aug 18
Wet lab
. Restriction check for Pbad.TA.dTTs
. Restriction check for constitutive promoter.CR.GFP.dTT
. Restriction check for constitutive promoter.RBS.GFP.dTT
. PCR for CR.GFP.dTT
. PCR for RBS.GFP.dTT
. PCR check for constitutive promoter.CR.GFP.dTT
. PCR check for constitutive promoter.RBS.GFP.dTT
. PCR check for Pbad.TA.dTT
. Restrict check TA.dTTs
. Prepare functional assay for RBS, CRs
. Inoculate candidates
. Prepare pSB3K3
. Prepare pSB1C3 for oligo ligation
. Prepare I13401 for oligo ligation
. Prepare B0015 for oligo ligation
. Inoue competent cells
. Prepare for Pbad characterization
. Take over the tank B and C arround 1pm-5;30
Aug 19
Wet lab
. Gel check for O/NPCR products
. Gel check for PCR products (18/08)
. miniprep c.Promoter.Lock 1RBS.GFP.dTT
. miniprep possible candidates
. Restrict TA.dTT
. pSB1C3-I13401 for oligo ligation
. B0015 for oligo ligation
. pSB1C3-J04450 for oligo ligation
. Inoue competent cells
. pBAD-key3-dTT restriction check
Aug 20
Wet lab
. Gel check for Pbad.TA.dTTs
. purification for B0015, I13401
. pSB1C3-J04450 for oligo ligation
. Gel check for PCR products (19/08)
. PCR check for J23102, LOCK 3 GFP dtt
. restrcition check for key1 dtt
Aug 21
Wet lab
. Colony PCR for final constructs
. Ligate UST lock
. restriction check for key1.dTT
. ligate pbad.key1.dTT
. ligate lock 3.GFP.dTT with C. promoter
. Colony PCR for c. promoter.MCR.GFP.dTT
. competent cells
. Glycerol stocks
. transform HKUSTlock.GFP.dTT
. Transform pSB1C3-HKUSTlock
·
Aug 22
Wet lab
. competent cells
. Colony PCR for pSB1C3-HKUSTlock1
. Colony PCR for HKUSTlock1.GFP.dTT
. miniprep PSB3K3-J04550 for pSB3K3 Backbone
. restrict pSB3K3 for 3 pieces assemblage
. ligate TA-CR into pSB3K3
. Transform TA-CR
. colony PCR for PBAD-key3-dTT
. inoculate Pbad.key1.dTT
. gel check for C. PCR on 21/08
. extraction of pSB3K3
. inoculate HKUSTlock1.GFP.dTT
. inoculate pSB1C3-HKUSTlock1
·
Aug 23
Wet lab
. Gel check for TAs
. colony PCR for UST lock in I13401 and pSB1C3
. transform ust key
. Check competent cells
·
Aug 24
Wet lab
. Colony PCR for Pbad.Key1.dTT
. Gel check for Pbad.Key1.dTT
·
Week 5
Aug 25
Wet lab
. Colony PCR for Pbad.Key1.dTT
. PCR check for CR.GFP.dTT
. PCR check for Cons. prom.CR.GFP.dTT
. anneal and ligate ust lock
. anneal and ligate ust key
. transform ust lock1
. transform ust key
. ligation again key 1 into B0015
·
Aug 26
Wet lab
. Colony PCR for "un-named lock"
. inoculate ones which SEEMS like it contains lock
. Colony PCR for "un-named key"
. inoculate ones which SEEMS like it contains key
. colony pcr for Key1.dtt
. Gel check for O/NPCR products
. pcr check for CR.GFP.dTTs
. Inoculate all possible candidates
. Feeling paranoic? Prepare I13401 for CRs
. Streak posible c.p.CR/RBS.GFP.dTT
. make SOC
. single cut check for TAs
. make 5 alpha cell
·
Aug 27
Wet lab
. miniprerp candidates
. pcr check for candidates
. gel check for o/n pcr
. restrict check for ust key 1
. restrick check for key 1
. Pbad for ust key 1
. extract ust lock 1 for j23102
. ligate ust lock and key
. transform ust lock and key
. prepare plates for macroscope
. prepare dna for sequencing
. SOB
. SOC
. oligo annealing
. plasmid PCR for HKUSt lock and key
. overnight digestion of B0034 for control construct
. Digestion of HKUST key-B0015
·
Aug 28
Wet lab
. miniprep locks
. glycerol stocks for locks
. minipreps for B0015 and B0034
. ligation of pBAD-Hkust key-B0015
. digestion of HKUST lock
. ligation of HKUST lock with GFP
. ligation of HKUST key and lock (4th)
. ligation of B0034 with GFP
. colony PCR of HKUST key and lock
. inoculation of key and lock
. miniprep
. Key one restrict check
. key one restriction for inserting pBAD
. Restrict and exytact lock3c rbs and ligate into c.promoter
. ligate lock 3c.GFP.dTT with C. promoter
. transform c. promoterlock 3c rbs.gfp.dtt
. Extract mcr/mta
. ligate MCR and MTA into 3K3
. transform MCR/MTA
. Glycerol stock for locks
. E0240 ligate with C. Promoter
. TB
. parallel ligation and aneling for key 1
. Sequencing preparations
·
Aug 29
Wet lab
. minipreps
. Gel extraction, purification for O/N digestions
. competent cells
. Extract mcr/mta
. ligate MCR and MTA into 3K3
. transform MCR/MTA
. Glycerol stock for locks
. PCR check for biobrick key1
·
Aug 30
Wet lab
. inoculation of 12 falcons
. Colony PCR for pSB3K3 constructs
. Transform key1 O/N
. miniprep
. Competent cells
. AseI check Key 1 Dtt
·
September 2014
Week 1
Sept 1
Wet lab
. restrict check pBAD- Lost your key-dtt
. restrict check pBAD-Lost your key-dtt
. colony PCR for CR.TAs (orthogonality)
. colony PCR for Key1,dtt
. ligate lock3c.GFP.dTT into c.p plasmid
. transform lock3.gfp.dtt
. Colony PCR for CR.TAs (orthogonality)
. Colony PCR for sherLock-GFP-dtt
. restriciton check for Lost Your Key in PSB1C3
. transform pbad.lost your key.dtt
. inoculate orthogonality candidates
. Prepara midi-prep for 3k3
·
Sept 2
Wet lab
. Gel check for O/N colony pcr for key1 and 3k3
. massive prep
. PCR check sense reverse
. colony pcr for orthogonality
. colony pcr for cons. pro. lock3crbs.gfp.dtt
. migrate cpromoter E0240 to psb3k3 with mta key
. inoculation of B0034-GFP-dtt
. inoculation of Sherlock-GFP-dtt
. sequencing for sherlock in pSB1C3
. digestion of B0034-GFP-dtt
. digestion of sherlock-GFP-dtt
·
Sept 3
Wet lab
. digestion of Pbad MTA dTT
. digestion of Pbad dTT
. digestion of J23102 MCR GFP dTT
. digestion of J23102 E0240(B0032 GFP dTT)
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450)
. Ligation of J23102 MCR GFP dTT with Pbad dTT
. Ligation of J23102 E0240 with Pbad MTA dTT
. Ligation of J23102 E0240 with Pbad dTT
. Plasmid PCR for SherLock-GFP-dtt
. ligation of B0034-GFP-dtt
. ligation of SherLock-GFP-dtt
. sequencing
. check plate for arabinose induced mcr mta while singing
. extraction of key and lock
. restriction check for key with lock
. extration of key1 dtt
. ligation of key1 dtt with pBAD
. Gel check for O/N colonyPCR
. Crazy PCR check
. Check 031
. ligate lock3c RBS.GFP.dTT into c.p plasmid
. transform lock3c RBS.GFP.dTT
. Midi prep preparations
·
Sept 4
Wet lab
. Gel purification of digested insert and backbone
. Ligation of J23102 MCR GFP dTT with Pbad dTT
. Ligation of J23102 E0240 with Pbad MTA dTT
. digestion of J23102 E0240(B0032 GFP dTT)
. Transformation of ligated projects mentioned above
. Colony PCR for sherLock-GFP-dtt
. Colony PCR for B0034-GFP-dtt
. Inoculation of successful colonies
. Gel check for O/N Colony PCR
. Gel check for O/N crazy PCR
. Gel check for Erics restriction check
. Inoculation of successful colonies
. Midi-prep preparations
. Gel purification
·
Sept 5
Wet lab
. miniprep/glycerol stocck
. Transform Pbad dTT
. Transform ligated produce of J23102 MCR GFP dTT with Pbad dTT
·
Week 2
Sept 8
Wet lab
·
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·
·
·
Sept 9
Wet lab
·
·
·
·
·
·
Sept 10
Wet lab
. colony pcr
. ligations for final constructs
. inoculations
. Pbad.lost your key.dtt
. sherlock
. gel check for colony pcr
. colony pcr
·
·
·
·
·
Sept 11
Wet lab
·
·
·
·
Sept 12
Wet lab
. colony pcr
. restrict key1.dtt for Pbad insertion
. ligate Pbad with key1.dtt
. transform key1.dTT
. extract pSB3K3
. Prepare TB
. Prepare dextrose for SOC
. check sequencing results
. Digestion of CLON041 for constructs
. ligation
. inoculation
. Digestion of CLON 036
. colony pcr mcr mta
·
Week 3
Sept 15
Wet lab
·
·
·
·
Sept 16
Wet lab
. mini prep Pbad.key1.dtt
. restrict check pbad.key1.dtt
. gel check for O/N c.pcr
. extract pbad.key1
. inoculate every CR.TA construct
. ligate transform key 1 with locks
. miniprep (B1, B2, and B3)
. plasmid PCR B1, B2 and B3 using sense oligo and VR
. Overnight digestion for ones which give positive results
·
·
Sept 17
Wet lab
. purify pbad.key1
. ligate pbad key1 dtt with locks
. transform locks.Pbad.key1.dtt
. 2xYT (agar) AMP
. transform all
·
Sept 18
Wet lab
. Spread a plate
. miniprep for 3 inoculations
. Sense oligo and VR PCR for HKUST Lock-dtt construct
. Overnight digestion of plasmid which gives a band around 1300bp
. EtoHpp for sequencing
. Miniprep construtcs for submission
. innoculation
. transform 031, 034
. Check "Diagonal constructs" and controls
. migrate CR crontruts into 3K3
·
Sept 19
Wet lab
. miniprep lock.key candidate
. ligate problematic cr.key
. restrick check sherlock
. extrations.... madness
·
Sept 20
Wet lab
. miniprep CR.TA
. Colony PCR for CR.TA
. Colony PCR for CR.TA orthogonality
·
Sept 21
Wet lab
. miniprep martching CR.TA
. Restriction check by EcoR1 and Pst1
·
Week 4
Sept 22
Wet lab
. Run Gel check for matching set of CR.TA
. ligate problrmatic sets
. transform rpoblematic sets
. prepare DNA for submission
. Gel purfication for Doyle
. Ligation lock-dtt with cons. promoter
. transformation
. pSB3K3- CRs ligation
·
Sept 23
Wet lab
. pSB3K3- CRs transformation
. Gel extractions
. Gel purifications
. TB
. Agar L (+) plates
. Send DNA to BGI for sequencing
. C. PCR?
. Inoculate possible candidates
. ligate problematic sets
. transform confirmed constructs
. inoculation for HKUST lock Construct
. colony PCR For lock
·
Sept 24
Wet lab
. Competent cells preparations!
. minipreps
. colony PCR
. miniprep for biobrick submission
. send plamsid for sequencing
·
Sept 25
Wet lab
. insane prep
. restriction check
. Gel check for previous PvuII digestions
·
Sept 26
Wet lab
. Gel check PvuII digestions
. Gel check AseI digestions
. 3A assembly for non pSB1C3
. final batch of ligation for all missing component
. prepare DNA for submission
. Extraction for ligation
. Checking constructs-3 constructs using digestion check
. transformation for LOCK-8
. Restriction check for controls for MCR/MTA
. Transformation of confirmed plasmid
·
Sept 27
Wet lab
. C. PCR for H series...
. inoculate possible candidates
. check self ligated 3K3
. Gel check for possible candidates AseI
. Gel check for possible candidates PvuII
. Ligated H6 H7
. transformed H6 H7
. O/N innoculation of MCR/MTA for characterization
·
Sept 28
Wet lab
. Extract pSB1C3
. Etract 036
. miniprep "H" series candidates
. Repeat CHL restrictions
. Repeat CHL ligattions
. Repeat CHL transformations
. Gel check for "7" series using NcoI
. Restrict check AseI
. Restrict check NcoI
. Fluorescence measurement of MCR/MTA for characterization.
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn)
·
Week 5
Sept 29
Wet lab
. Restrict check AseI
. Restrict check NcoI
. mini prep 6 + MCR/MTA (thanks from doyle)
. Restriction check for MCR/MTA in pSB3K3.
. restrict 036
. purify 036
. ligate monkey with sherlock
. Gel check for NcoI and AseI
. Get pSB1C3 bacbone EcoRI and PstI
. Fluorescence measurement of MCR/MTA for characterization (second trial)
. Innoculate Lock and Keys for crude functional assay.
·
Sept 30
Wet lab
. miniprep 11 tubes in shaker c
. Restrict check H" series with NcoI
. Restrict inoculated colonies wiht AseI
. Restrict inoculated colonies wiht NcoI
. Gel purifications
. ligation of H series and some from other series...
. transformtation of 16 confirmed constructs
·
Oct 1
Wet lab
. digestion of key3,HKUSTkey?
. miniprep- final constructs
·
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