# Team:HokkaidoU Japan/Shuffling Kit/Examples

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Promoter Selector

Promoter Selector

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

+

For this demonstration we decided to use 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 indicate the first and second biggest occupancy rate on the plate. +

If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate. - 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). + If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).

# 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 this demonstration we decided to use 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 would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate. If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in 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 pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).

Culture: 37 °C, for 48h

#### Results

fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.
fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.

After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZa expression in Promoter Selector system as negative control to estimate the success of Golden Gate Assembly. We got 0-7 blue colonies which was expressing LacZ alpha.

Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.

In (fig.4), legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=!.

#### Conclusion

Big difference did not appear among each Kanamaicin registance. In these colonies, number of colonies derived from K1084405 (containing K1084010 promoter ) had the most largest rate on each plate. This result suggests that the colonies expressed the lowest amount of Kanamycin resistance gene, and the resource of transcription and translation had been spared to cell growth. Thus, the number of colonies might was the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.

From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.

## RBS Selector

### 4 colors

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.5). LacZα makes the colony blue. mRFP1 makes the colony red.

fig.5 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.6).

fig.6 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.7 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.

fig.8.