Team:HokkaidoU Japan - 2016.igem.org

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).

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.

Table. 1. Gel assay

Materials	Volume
30% Acrylamide	5 mL
Tris-Buffer	2.5 mL
SDS	100 µL
APS	100 µL
TEMED	5 µL
DW	2.295 mL
Total	10 mL

The following Table. 2 is about preparing the SDS-PAGE.

Table. 2. Experimental condition

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.

Team:HokkaidoU Japan/Proof

Team:HokkaidoU Japan