# Team:HokkaidoU Japan/Shuffling Kit/Examples

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fig.5
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fig.5 Each combinations of RBS make different colors

# Demonstrations for Usecase Example

We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!

## Promoter Selector

Let's select the best promoter for Kanamycin resistance by Promoter Selector.

For a demonstration we decided to optimize the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).

If the concentration of Kanamycin was high, the colony with strong promoter will survive. Therefore, only one or two colors of colonies would appear. If the concentration of Kanamycin was low, colonies with weak promoters will be able to survive. This way many colors of colonies would appear (fig.2).

fig.1 Different promoter express each colors
fig.2 Difference of Kanamycin concentration

### Method

Optimum concentration of Kanamycin: in LB is 50 mg/ml We prepared 3 different concentration plates.

• Plate A: Kanamycin 125 mg per plate
• Plate B: Kanamycin 250 mg per plate
• Plate C: Kanamycin 500 mg per plate (optimum concentration)
• Plate D: Kanamycin 1000 mg per plate

Gene Vector: pSB1C3

We cloned Kanamycin resistant gene from pSB1K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).

Culture: 37 °C, for 24h

fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.

### Results

We didn't have time to culture anymore. So we coudn't see the diffference between plates A to D. However,we were able to see different colors on the plate. The colonies were too small to identify the color. The picture below is plate B.

### Conclusion

Our Promoter Selector seemed to work. We got several colors colonies on Kanamycin plates. This means that Kanamycin Resistance gene was successfully ligated. We need more than 24 hrs to identify the colors.

## RBS Selector

Let’s create all combinations by two reporter genes and make various colors on one plate!

The RBS Selector we made, can randomize the strength of RBSs in the operon. For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZα (BBa_I732006) (fig.4). LacZα makes the colony blue. mRFP1 makes the colony red.

fig.4 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006)

When the RBS upstream of mRFP1 was strong and the RBS upstream was weak, the colony should be red. When the RBS upstream of mRFP1 was weak, and the RBS upstream was strong, the colony should be blue. So when if the strength of RBS upstream both genes were the same, colony will be white, purple (fig.5).

fig.5 Each combinations of RBS make different colors

### Method

• Used promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102) and assembled with.
• Spread X-GAL（250 mg）on LBC plate.
• Cultured for 37 °C, 26h.
fig.6 The colonies showed red, blue, white, and purple.

### Results

We got many colored colonies,red, blue, white, and purple.

### Conclusion

We can say that our RBS Selector worked!! The RBSs uptsream 2 genes were randomized and they had many levels of expressions.