Team:HokkaidoU Japan/Shuffling Kit/Examples


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Maestro E.coli

Optimization Kit

Demonstrations for Usecase Example

We will show some interesting demonstrations of our kits, POK and ROK!


What will happen if you optimize a Kanamycin resistance?

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 POK.(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).



Optimum concentration of Kanamycin: in LB is 50 mg/ml We prepared 4 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 POK kit (K1084501, K1084502, K1084503, K1084504, K1084505 ).

Culture: 37 °C, for 24h


We didn't have time to culture anymore. So we coudn't see the diffference beween 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.

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


Our POK 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.


Let’s optimize two reporter genes and make various colors on one plate!

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


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 purple.(fig.5)



  • Used GGA vector (BBa_K1084301) and tandem RBS (BBa_K1084302). and assembled with
  • Spread X-GAL(250 mg)on LBC plate.
  • Cultured for 37 °C, 26h.


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

fig.6 The colonies showed red, blue, deep blue, and purple.


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