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Revision as of 11:18, 19 October 2016
Team:HokkaidoU Japan
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We conducted SDS-PAGE with (BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on (pSB1C3) of E.coli (DH5α). Let us explain the 3parts below. BBa_K2015012 part contains lacI expression unit and plac+RBS. The sequences of downstream can be induced strictly by IPTG. BBa_K2015008 consists Self-Assembling Regions(SAR,RADA) and mutated GFP. And BBa_K2015009 This part consists Self-Assembling Region(SAR,P11) and mutated GFP. At first, we made the Gel for SDS-PAGE, with the following the Table. 1. It was necessary to add TEMED finally because the solution was turned into the Gel very fast by TEMED.
The following Table. 2 is about preparing the SDS-PAGE.
We conducted SDS-PAGE with (BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on (pSB1C3) of E.coli (DH5α). Let us explain the 3parts below. BBa_K2015012 part contains lacI expression unit and plac+RBS. The sequences of downstream can be induced strictly by IPTG. BBa_K2015008 consists Self-Assembling Regions(SAR,RADA) and mutated GFP. And BBa_K2015009 This part consists Self-Assembling Region(SAR,P11) and mutated GFP. At first, we made the Gel for SDS-PAGE, with the following the Table. 1. It was necessary to add TEMED finally because the solution was turned into the Gel very fast by TEMED.
Table. 1. Gel assay |
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|
The following Table. 2 is about preparing the SDS-PAGE.
Table. 2. Experimental condition |
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Temperature | IPTG | Concentration (M) | Volume (µL) | Time to culture |
---|---|---|---|---|
37°C | - | - | - | 24 h |
37°C | + | 0.4 | 6 | 24 h |
37°C | + | 2 | 30 | 24 h |
25°C | - | - | - | 16 h |
25°C | + | 0.4 | 6 | 16 h |
25°C | + | 2 | 30 | 16 h |
After we finished cultivating samples, we took 100 µL out of each samples and made the following operation.
- Centrifuge with 13000rpm at 24°C for 2 min
- Remove the supernatant
- Add 50 mL of SDS-Buffer
- Shake for 1 min
- Keep at 100°C for 5 min
- Put on the ice
- Apply 10 µL to SDS
- Run electrophoresis for 1.5 h
- Wash out with MiliQ
- Shake at 24°C with 34 rpm for 1 h
- Dye with QuickCBB
The fig. 1 shows the result of SDS-PAGE.