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− | We made a functonal unit to regulate gene expression. <a href="http://parts.igem.org/Part:BBa_K2015012">BBa_K2015012</a> codes constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>), RBS (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>), LacI (<a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>), dT (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>), PLac (<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a>) and RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig.1). | + | We made a functonal unit to regulate gene expression. <a href="http://parts.igem.org/Part:BBa_K2015012">BBa_K2015012</a> codes constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>), RBS (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>), LacI (<a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>), dT (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>), PLac (<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a>) and RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig. 1). |
Revision as of 02:00, 20 October 2016
Team:HokkaidoU Japan
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We made a functonal unit to regulate gene expression. BBa_K2015012 codes constitutive promoter (BBa_J23101), RBS (BBa_B0032), LacI (BBa_C0012), dT (BBa_B0015), PLac (BBa_R0011) and RBS (BBa_B0034). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig. 1).
We conducted SDS-PAGE with (BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on pSB1C3 of E. coli (DH5α). Let us explain the 3 parts 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, RADA16-I) and mutated GFP. And BBa_K2015009 this part consists Self Assembling Region (SAR, P11-4) 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 made a functonal unit to regulate gene expression. BBa_K2015012 codes constitutive promoter (BBa_J23101), RBS (BBa_B0032), LacI (BBa_C0012), dT (BBa_B0015), PLac (BBa_R0011) and RBS (BBa_B0034). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig. 1).
Fig. 1. Change of expression by IPTG induction left: not induced, right: induced |
We conducted SDS-PAGE with (BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on pSB1C3 of E. coli (DH5α). Let us explain the 3 parts 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, RADA16-I) and mutated GFP. And BBa_K2015009 this part consists Self Assembling Region (SAR, P11-4) 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.
Temperature | IPTG | Concentration (mM) | 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 13000 rpm 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.