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# Team:HokkaidoU Japan/Notebook

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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](#Monday.2C_July_12) | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | [21](#Wednesday.2C_July_21) | 22 | 23 | 24 |
| 25 | [26](#Monday.2C_July_26) | [27](#Tuesday.2C_July_27) | 28 | 29 | 30 | 31 |
|  |

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| August | | | | | | |
| S | M | T | W | T | F | S |
| 1 | [2](#Monday.2C_August_2) | 3 | 4 | 5 | 6 | 7 |
| 8 | [9](#Monday.2C_August_9) | [10](#Tuesday.2C_August_10) | [11](#Wednesday.2C_August_11) | [12](#Thursday.2C_August_12) | [13](#Friday.2C_August_13) | 14 |
| 15 | [16](#Monday.2C_August_16) | [17](#Tuesday.2C_August_17) | [18](#Wednesday.2C_August_18) | [19](#Thursday.2C_August_19) | [20](#Friday.2C_August_20) | 21 |
| 22 | [23](#Monday.2C_August_23) | [24](#Tuesday.2C_August_24) | [25](#Wednesday.2C_August_25) | [26](#Thursday.2C_August_26) | [27](#Friday.2C_August_27) | 28 |
| 29 | [30](#Monday.2C_August_30) | [31](#Tuesday.2C_August_31) |  |  |  |  |
|  |

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| September | | | | | | |
| S | M | T | W | T | F | S |
|  |  |  | [1](#Wednesday.2C_September_1) | [2](#Thursday.2C_September_2) | [3](#Friday.2C_September_3) | [4](#Saturday.2C_September_4) |
| 5 | [6](#Monday.2C_September_6) | [7](#Tuesday.2C_September_7) | [8](#Wednesday.2C_September_8) | [9](#Thursday.2C_September_9) | [10](#Friday.2C_September_10) | 11 |
| 12 | [13](#Monday.2C_September_13) | [14](#Tuesday.2C_September_14) | [15](#Wednesday.2C_September_15) | [16](#Thursday.2C_September_16) | [17](#Friday.2C_September_17) | 18 |
| 19 | 20 | [21](#Tuesday.2C_September_21) | [22](#Wednesday.2C_September_22) | [23](#Thursday.2C_September_23) | [24](#Friday.2C_September_24) | 25 |
| 26 | [27](#Monday.2C_September_27) | [28](#Tuesday.2C_September_28) | [29](#Wednesday.2C_September_29) | [30](#Thursday.2C_September_30) |  |  |
|  |

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| October | | | | | | |
| S | M | T | W | T | F | S |
|  |  |  |  |  | 1 | [2](#Saturday.2C_October_2) |
| [3](#Sunday.2C_October_3) | [4](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [5](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [6](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [7](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [8](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [9](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") |
| [10](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [11](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [12](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [13](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [14](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [15](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [16](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") |
| [17](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [18](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [19](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [20](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [21](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [22](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [23](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") |
| [24](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [25](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [26](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [27](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [28](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [29](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") | [30](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") |
| [31](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October") |  |  |  |  |  |  |

## 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](/Team%3AHokkaidoU_Japan/Notebook/August2 "Team:HokkaidoU Japan/Notebook/August2")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August10 "Team:HokkaidoU Japan/Notebook/August10")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August11 "Team:HokkaidoU Japan/Notebook/August11")

* Preparation for the next day's miniprep
* Estimation of enzyme activity
* Preparation of the glycerol stocks

### [Thursday, August 12](/Team%3AHokkaidoU_Japan/Notebook/August12 "Team:HokkaidoU Japan/Notebook/August12")

* Miniprep (1-18F, 2-21H and 2-11P)
* Electrophoresis of the DNA which had been amplified via PCR and miniprep

### [Friday, August 13](/Team%3AHokkaidoU_Japan/Notebook/August13 "Team:HokkaidoU Japan/Notebook/August13")

* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)

---

### [Monday, August 16](/Team%3AHokkaidoU_Japan/Notebook/August16 "Team:HokkaidoU Japan/Notebook/August16")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August17 "Team:HokkaidoU Japan/Notebook/August17")

* Purification of the DNA solutions via gel extraction
* Preparation of agar medium containing 35 ug/mL chloramphenicol

### [Wednesday, August 18](/Team%3AHokkaidoU_Japan/Notebook/August18 "Team:HokkaidoU Japan/Notebook/August18")

* Digestion and gel extraction of 1-2M (retry)
* 3 piece ligation of 1-18F, 1-23L and pSB1C3
* Transformation

### [Thursday, August 19](/Team%3AHokkaidoU_Japan/Notebook/August19 "Team:HokkaidoU Japan/Notebook/August19")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August20 "Team:HokkaidoU Japan/Notebook/August20")

* PCR amplification of pSB1A3, pSB1C3 and pSB1T3

---

### [Monday, August 23](/Team%3AHokkaidoU_Japan/Notebook/August23 "Team:HokkaidoU Japan/Notebook/August23")

* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)
* PCR amplification of BioBrick parts using new primers

### [Tuesday, August 24](/Team%3AHokkaidoU_Japan/Notebook/August24 "Team:HokkaidoU Japan/Notebook/August24")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August25 "Team:HokkaidoU Japan/Notebook/August25")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August26 "Team:HokkaidoU Japan/Notebook/August26")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August27 "Team:HokkaidoU Japan/Notebook/August27")

* 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](/Team%3AHokkaidoU_Japan/Notebook/August30 "Team:HokkaidoU Japan/Notebook/August30")

* Measurement of restriction enzyme activity using highly purified pUC119
* Digestion of PCR products that had been amplified using new primers

### [Tuesday, August 31](/Team%3AHokkaidoU_Japan/Notebook/August31 "Team:HokkaidoU Japan/Notebook/August31")

* Gel extraction, ligation and transformation of pUC119

### [Wednesday, September 1](/Team%3AHokkaidoU_Japan/Notebook/September1 "Team:HokkaidoU Japan/Notebook/September1")

* PCR amplification of 1-5A (RFP reporter)

### [Thursday, September 2](/Team%3AHokkaidoU_Japan/Notebook/September2 "Team:HokkaidoU Japan/Notebook/September2")

* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119

### [Friday, September 3](/Team%3AHokkaidoU_Japan/Notebook/September3 "Team:HokkaidoU Japan/Notebook/September3")

* Colony PCR of *E. coli* transformed on previous day
* PCR amplification of 1-3A (RFP reporter)

### [Saturday, September 4](/Team%3AHokkaidoU_Japan/Notebook/September4 "Team:HokkaidoU Japan/Notebook/September4")

* PCR amplification of 1-5A (RFP reporter)
* Estimation of the amount of 1-3A PCR product

---

### [Monday, September 6](/Team%3AHokkaidoU_Japan/Notebook/September6 "Team:HokkaidoU Japan/Notebook/September6")

* Ligation of pSB1C3 PCRed from 1-3A and 1-5A（RFP reporter）

### [Tuesday, September 7](/Team%3AHokkaidoU_Japan/Notebook/September7 "Team:HokkaidoU Japan/Notebook/September7")

* Concentration check of DNA used for ligation yesterday
* Ethanol precipitation
* Colony PCR of competent cells

### [Wednesday, September 8](/Team%3AHokkaidoU_Japan/Notebook/September8 "Team:HokkaidoU Japan/Notebook/September8")

* Colony PCR
* Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3
* PCR of GFP

### [Thursday, September 9](/Team%3AHokkaidoU_Japan/Notebook/September9 "Team:HokkaidoU Japan/Notebook/September9")

* 3 piece ligation of HSP, GFP and pSB1C3

### [Friday, September 10](/Team%3AHokkaidoU_Japan/Notebook/September10 "Team:HokkaidoU Japan/Notebook/September10")

* 3 piece ligation, continued

---

### [Monday, September 13](/Team%3AHokkaidoU_Japan/Notebook/September13 "Team:HokkaidoU Japan/Notebook/September13")

* AraC promoter purification
* And follow up checks for quality

### [Tuesday, September 14](/Team%3AHokkaidoU_Japan/Notebook/September14 "Team:HokkaidoU Japan/Notebook/September14")

* Digestion and ligation of pSB1C3, araC Promoter and GFP
* Ethanol precipitation

### [Wednesday, September 15](/Team%3AHokkaidoU_Japan/Notebook/September15 "Team:HokkaidoU Japan/Notebook/September15")

* 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](/Team%3AHokkaidoU_Japan/Notebook/September17 "Team:HokkaidoU Japan/Notebook/September17")

* 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](/Team%3AHokkaidoU_Japan/Notebook/September21 "Team:HokkaidoU Japan/Notebook/September21")

* Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa\_B0034 (BBa\_B0034)] made from oligos

### [Thursday, September 22](/Team%3AHokkaidoU_Japan/Notebook/September22 "Team:HokkaidoU Japan/Notebook/September22")

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

### [Thursday, September 23](/Team%3AHokkaidoU_Japan/Notebook/September23 "Team:HokkaidoU Japan/Notebook/September23")

* 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](/Team%3AHokkaidoU_Japan/Notebook/September24 "Team:HokkaidoU Japan/Notebook/September24")

* Miniprep of Arac+RBS+pSB1A3
* Follow quality check

---

### [Monday, September 27](/Team%3AHokkaidoU_Japan/Notebook/September27 "Team:HokkaidoU Japan/Notebook/September27")

* Culture of the BAC clones

### [Tuesday, September 28](/Team%3AHokkaidoU_Japan/Notebook/September28 "Team:HokkaidoU Japan/Notebook/September28")

* 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](/Team%3AHokkaidoU_Japan/Notebook/September29 "Team:HokkaidoU Japan/Notebook/September29")

* 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](/Team%3AHokkaidoU_Japan/Notebook/September30 "Team:HokkaidoU Japan/Notebook/September30")

* Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
* Transformation

### Friday, October 1

### [Saturday, October 2](/Team%3AHokkaidoU_Japan/Notebook/October2 "Team:HokkaidoU Japan/Notebook/October2")

* Colony PCR
* Preparation for Sequencing

### [Sunday, October 3](/Team%3AHokkaidoU_Japan/Notebook/October3 "Team:HokkaidoU Japan/Notebook/October3")

* Miniprep

---

# [After October 3](/Team%3AHokkaidoU_Japan/Notebook/October "Team:HokkaidoU Japan/Notebook/October")

---

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