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 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 3
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 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
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June 24
Wet lab
· Inoculation of BBa_B0015
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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
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June 26
Wet lab
· Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification
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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
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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
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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
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July 4
Wet lab
· PCR of comX-myc using Vent DNA Polymerase, PCR clean up
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July 5
Wet lab
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July 6
Wet lab
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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
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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
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July 9
Wet lab
· Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc
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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
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July 11
Wet lab
· Digestion of comW-FLAG with XbaI and PstI, digestion clean up
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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August 2014
Week 1
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
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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
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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
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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
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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
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Week 2
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
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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
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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
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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
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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
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Week 3
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
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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
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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
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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
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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
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Week 4
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
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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
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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
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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
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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
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Week 5
Aug xx
Wet lab
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Aug xx
Wet lab
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Aug xx
Wet lab
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Aug xx
Wet lab
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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
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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
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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
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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
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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
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Sept 12
Wet lab
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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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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
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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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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
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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
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Sept 24
Wet lab
· Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification
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Week 5
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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
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