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Team:HokkaidoU Japan/Notebook
From 2010.igem.org
since 13 Jul 2010
since 1 Aug 2010
Calendar
| July | ||||||
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| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
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| August | ||||||
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| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
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| September | ||||||
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| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
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Abstract
Monday, July 12
- Our first experiment, transformation of BioBrick devices.
Wednesday, July 21
- Preparation of competent cells
- Single colony isolation of E. coli which had been transformed on Monday, July 12
Monday, July 26
- Cultivation of transformed E. coli for the next day's miniprep
Tuesday, July 27
- Miniprep (Alkaline SDS method)
Monday, August 2
- Restriction enzyme digestion of plasmids which had been purified by miniprep
- Electrophoresis assay
Tuesday, August 3
Wednesday, August 4
Thursday, August 5
Friday, August 6
Monday, August 9
- Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive E. coli and heat-sensitive E. coli
- These are preliminary experiments before starting the main project
Tuesday, August 10
- PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm
Wednesday, August 11
- Preparation for the next day's miniprep
- Estimation of enzyme activity
- Preparation of the glycerol stocks
Thursday, August 12
- Miniprep (1-18F, 2-21H and 2-11P)
- Electrophoresis of the DNA which had been amplified via PCR and miniprep
Friday, August 13
- PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)
Monday, August 16
- Measurement of restriction enzyme activity
- Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)
Tuesday, August 17
- Purification of the DNA solutions via gel extraction
- Preparation of agar medium containing 35 ug/mL chloramphenicol
Wednesday, August 18
- Digestion and gel extraction of 1-2M (retry)
- 3 piece ligation of 1-18F, 1-23L and pSB1C3
- Transformation
Thursday, August 19
- 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)
- Ligation that uses only vector pSB1C3 to estimate its efficiency
Friday, August 20
- PCR amplification of pSB1A3, pSB1C3 and pSB1T3
Monday, August 23
- PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)
- PCR amplification of BioBrick parts using new primers
Tuesday, August 24
- Electrophoresis of PCR products that had been amplified using new primers
- Examination of two ligation kits
- Electrophoresis assay of miscellaneous DNA solutions
- Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium
Wednesday, August 25
- Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium
- Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation
- Digestion of PCR products that had been amplified by using new primers
Thursday, August 26
- Electriphoresis to check whether ligation had been successful
- Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)
Friday, August 27
- Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation
- Retry of 3 piece ligation which was done on August 19th and 20th
- Ligation of vectors to each other
Monday, August 30
- Measurement of restriction enzyme activity using highly purified pUC119
- Digestion of PCR products that had been amplified using new primers
Tuesday, August 31
- Gel extraction, ligation and transformation of pUC119
Wednesday, September 1
- PCR amplification of 1-5A (RFP reporter)
Thursday, September 2
- Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119
Friday, September 3
- Colony PCR of E. coli transformed on previous day
- PCR amplification of 1-3A (RFP reporter)
Saturday, September 4
- PCR amplification of 1-5A (RFP reporter)
- Estimation of the amount of 1-3A PCR product
Monday, September 6
- Ligation of pSB1C3 PCRed from 1-3A and 1-5A（RFP reporter）
Tuesday, September 7
- Concentration check of DNA used for ligation yesterday
- Ethanol precipitation
- Colony PCR of competent cells
Wednesday, September 8
- Colony PCR
- Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3
- PCR of GFP
Thursday, September 9
- 3 piece ligation of HSP, GFP and pSB1C3
Friday, September 10
- 3 piece ligation, continued
Monday, September 13
- AraC promoter purification
- And follow up checks for quality
Tuesday, September 14
- Digestion and ligation of pSB1C3, araC Promoter and GFP
- Ethanol precipitation
Wednesday, September 15
- Observed results of yesterdays transformation
- Transformation using heat shock went well
- Electroporation transformation failed produce colonies
- Did Colony PCR of yesterdays transformed colonies
- Introduced colonies to L(+)Arabinose medium to check if it would show desired function
- Check for results tomorrow
Thursday, September 16
- Observed results of overnight incubation
- Fluorescence was visible when viewed by fluorescence microscope
- Did experiment to see if fluorescence is affected by arabinose concentration
- Scanned GFP intensity of broth containing colonies we isolated yesterday
- Had a free time so amplified some parts for easy training constructs
Friday, September 17
- Construction of GFP marker for a part which will be secreted using T3SS
- Ordered primers for construction for same part
Monday, September 20
Tuesday, September 21
- Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos
Wednesday, September 22
Thursday, September 23
- Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
- Colony PCR of AraC+RBS+pSB1A3
- Electroporetion of BAC plasmid into DH5α MG1655
Friday, September 24
- Miniprep of Arac+RBS+pSB1A3
- Follow quality check
Monday, September 27
- Culture of the BAC clones
Tuesday, September 28
- glycerol-stock of E.coli with salmonella's BAC library vector
- plasmid & GFP-double terminator's Ligation & Transformation
- PCR of E.coli with T3SSsignal and of GFP-double terminator
Wednesday, September 29
- Electrophoresis of T3SS signal and GFP + double terminator
- Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator
- making competent cell of E.coli with SPI2
- PCR of T3SS signal Again
Thursday, September 30
- Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
- Transformation
Friday, October 1
Saturday, October 2
- Colony PCR
- Preparation for Sequencing
Sunday, October 3
- Miniprep
