Team:HokkaidoU Japan/NotebookTest

From 2010.igem.org

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==Calendar==
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<div style="clear:both"></div>
<div style="clear:both"></div>
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=Abstract=
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==Abstract==
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===[[Team:HokkaidoU_Japan/Notebook/July12|Monday, July 12]]===
+
===Monday, July 12===
* Our first experiment, transformation of BioBrick devices.
* Our first experiment, transformation of BioBrick devices.
----
----
-
===[[Team:HokkaidoU_Japan/Notebook/July21|Wednesday, July 21]]===
+
===Wednesday, July 21===
* Preparation of competent cells
* Preparation of competent cells
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12
-
===[[Team:HokkaidoU_Japan/Notebook/July26|Monday, July 26]]===
+
===Monday, July 26===
* Cultivation of transformed ''E. coli'' for the next day's miniprep
* Cultivation of transformed ''E. coli'' for the next day's miniprep
-
===[[Team:HokkaidoU_Japan/Notebook/July27|Tuesday, July 27]]===
+
===Tuesday, July 27===
-
* Miniprep
+
* Miniprep (Alkaline SDS method)
----
----
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===Friday, August 6===
===Friday, August 6===
----
----
-
===[[Team:HokkaidoU_Japan/Notebook/August9|Monday, August 9]]===
+
===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
* 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
* These are preliminary experiments before starting the main project
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**Check for results tomorrow
**Check for results tomorrow
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===[[Team:HokkaidoU_Japan/Notebook/September16|Thursday, September 16]]===
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===Thursday, September 16===
-
 
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*Observed results of overnight incubation
*Observed results of overnight incubation
**Fluorescence was visible when viewed by fluorescence microscope
**Fluorescence was visible when viewed by fluorescence microscope
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----
----
-
===[[Team:HokkaidoU_Japan/Notebook/September20|Monday, September 20]]===
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===Monday, September 20===
 +
 
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos
-
===[[Team:HokkaidoU_Japan/Notebook/September22|Wednesday, September 22]]===
+
===Wednesday, September 22===
 +
 
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
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===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===
 +
* Culture of the BAC clones
 +
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===
===[[Team:HokkaidoU_Japan/Notebook/September28|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
 +
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]===
===[[Team:HokkaidoU_Japan/Notebook/September29|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
 +
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===
-
===[[Team:HokkaidoU_Japan/Notebook/October1|Friday, October 1]]===
+
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
 +
*Transformation
 +
 
 +
===Friday, October 1===
 +
 
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===
* Colony PCR
* Colony PCR
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===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===
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* Miniprep
----
----
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===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===
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===[[Team:HokkaidoU_Japan/Notebook/October8|Saturday, October 8]]===
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===[[Team:HokkaidoU_Japan/Notebook/October9|Saturday, October 9]]===
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===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===
----
----

Latest revision as of 17:53, 27 October 2010

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.

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

Monday, October 4

Tuesday, October 5

Wednesday, October 6

Thursday, October 7

Friday, October 8

Saturday, October 9

Sunday, October 10

Monday, October 11

Tuesday, October 12

Wednesday, October 13

Thursday, October 14

Friday, October 15

Saturday, October 16

Sunday, October 17

Monday, October 18

Tuesday, October 19

Wednesday, October 20

Thursday, October 21

Friday, October 22

Saturday, October 23

Sunday, October 24

Monday, October 25

Tuesday, October 26

Wednesday, October 27

Thursday, October 28

Friday, October 29

Saturday, October 30

===Sunday, October 31===
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/NotebookTest"