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 xx
Wet lab
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June xx
Wet lab
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June xx
Wet lab
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June xx
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
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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
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)
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
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
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
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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
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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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August 2014
Week 1
Aug 1
Wet lab
· Digestion of pSB1C3-BBa_J04450
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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
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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
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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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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
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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
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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
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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
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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)
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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)
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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)
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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
·
·
·
·
·
·
·
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
·
·
·
·
·
·
·
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
·
·
·
·
Sept 5
Wet lab
· Inoculation of pSB3K3-Combox.GFP
·
·
·
·
·
·
·
·
·
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
·
·
·
·
·
·
·
·
·
Sept 11
Wet lab
· Restriction check of pSB3K3-comX-160bp Pcombox-BBa_E0240 at EcoRI and PstI
·
·
·
·
·
·
·
·
·
Sept 12
Wet lab
· Restriction check of pSB3K3-160bp Pcombox-BBa_E0240 at EcoRI and PstI
· Transformation of pSB3K3-160bp Pcombox-BBa_E0240
·
·
·
·
·
·
·
·
Week 3
Sept 15
Wet lab
· Inoculation of pSB1C3-K880005.comW-FLAG
·
·
·
·
·
·
·
·
·
Sept 16
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI
·
·
·
·
·
·
·
·
·
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
·
·
·
·
·
·
·
·
Sept 19
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI
·
·
·
·
·
·
·
·
·
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
·
·
·
·
·
·
·
·
·
·
Sept 30
Wet lab
· Digestion of RBS+comX-myc+B0015 on XbaI and PstI
·
·
·
·
·
·
·
·
·
Riboregulator
June 2014
Week 1
June 2
Wet lab
· Restrcition digestion of ligated pSB3K3-160Pcombox-BBa_E0240
·
·
·
·
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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·
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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
·
·
·
·
July 3
Wet lab
·
·
·
·
July 4
Wet lab
·
·
·
·
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 characterization
. 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
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·
·
·
July 29
Wet lab
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·
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July 30
Wet lab
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July 31
Wet lab
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August 2014
Week 2
Aug 4
Wet lab
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·
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Aug 5
Wet lab
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Aug 6
Wet lab
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·
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Aug 7
Wet lab
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Aug 8
Wet lab
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Week 3
Aug 11
Wet lab
·
·
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Aug 12
Wet lab
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·
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Aug 13
Wet lab
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·
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Aug 14
Wet lab
·
·
·
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Aug 15
Wet lab
·
·
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Week 4
Aug 18
Wet lab
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·
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Aug 19
Wet lab
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·
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Aug 20
Wet lab
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·
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Aug 21
Wet lab
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·
·
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Aug 22
Wet lab
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·
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Week 5
Aug 25
Wet lab
·
·
·
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Aug 26
Wet lab
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·
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Aug 27
Wet lab
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·
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Aug 28
Wet lab
·
·
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Aug 29
Wet lab
·
·
·
·
September 2014
Week 1
Sept 1
Wet lab
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·
·
·
Sept 2
Wet lab
·
·
·
·
Sept 3
Wet lab
·
·
·
·
Sept 4
Wet lab
·
·
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Sept 5
Wet lab
·
·
·
·
Week 2
Sept 8
Wet lab
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·
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Sept 9
Wet lab
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·
·
·
Sept 10
Wet lab
·
·
·
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Sept 11
Wet lab
·
·
·
·
Sept 12
Wet lab
·
·
·
·
Week 3
Sept 15
Wet lab
·
·
·
·
Sept 16
Wet lab
·
·
·
·
Sept 17
Wet lab
·
·
·
·
Sept 18
Wet lab
·
·
·
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Sept 19
Wet lab
·
·
·
·
Week 4
Sept 22
Wet lab
·
·
·
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Sept 23
Wet lab
·
·
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Sept 24
Wet lab
·
·
·
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Sept 25
Wet lab
·
·
·
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Sept 26
Wet lab
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Week 5
Sept 29
Wet lab
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Sept 30
Wet lab
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"
