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Calendar

July
S	M	T	W	T	F	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

August
S	M	T	W	T	F	S
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31

September
S	M	T	W	T	F	S
			1	2	3	4
5	6	7	8	9	10	11
12	13	14	15	16	17	18
19	20	21	22	23	24	25
26	27	28	29	30

October
S	M	T	W	T	F	S
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

Abstract

Monday, July 12

• Our first experiment, transformation of BioBrick devices.

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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)

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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

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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)

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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

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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

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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

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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

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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

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Monday, September 20

Tuesday, September 21

• Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos

Thursday, September 22

• Digestion of pSB1A3, Arabinose promoter and GFP + double terminator
• Ligation & Transformation

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

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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

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After October 3

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