To meet the criteria of the Gold Medal, we submitted BBa_K1949060
 and we characterized BBa_R0071, BBa_C0071, BBa_C0171, BBa_K1529300, BBa_K1529310, BBa_K1096001 and BBa_K1096002.

To meet the criteria of the Silver Medal, we submitted BBa_K1949050, BBa_K1949052, BBa_K1949030 and BBa_K1949032.

To meet the criteria of the Bronze Medal, we submitted BBa_K1949000, BBa_K1949001, BBa_K1949100, BBa_K1949101 and BBa_K1949102.

BBa_K1949060 meets the Gold Medal criteria!

We simulated our final genetic circuits and found that the circuits did not work, because compared with Plux, Prhl activity was too weak. (see the Model page and the AHL only assay page). We therefore considered using the improved Prhl (BBa_K1529310, BBa_K1529300) established by iGEM 2014 Tokyo_Tech team, but we noticed it is inappropriate for two reasons (see Rhl_system_assay). Then, we decided to improve Prhl and obtain our original improved Prhl (Noticeable Mutant) (BBa_K1949060). We referred to it as Prhl(NM) hereafter that suited our goal.

Our purpose is to create strong Prhl for our final genetic circuits.

This experiment consists of the three parts below.

1. Improved Prhl by iGEM 2014 Tokyo_Tech team and characterization of rhlR(Fig. 4-1-1).




Fig. 4-1-1 Effect of RhlR by tagging LVA
and The colonies of transformants with a rhlR (left) or a rhlR - LVA (right)

Improvement of the wild type Prhl(Fig. 4-1-2).




Fig. 4-1-2 RFU of GFP / Turbidity of imoroved Prhl mutants

Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech team with our original improved Prhl(Fig. 4-1-3)




Fig. 4-1-3 Comparison of Prhl(NM) by Prhl(LR)

The past improved Prhl did not suit for our final circuits and we could construct the improved Prhl appropriate to our final circuits.

For more information, read this page !

BBa_K1949050 meets the Silver Medal criteria!

amiE codes for protein AmiE. AmiE is an acylase that degrades long chain [N]-acyl homoserine lactone (AHL) molecules with acyl chains longer than six carbons. Prince coli(Fig. 4-1-4) expresses amiE, and Snow White coli resuscitate from its dormancy and wakes up again. We tested the function of AmiE protein that influences the end of the story.

Our objective is to characterize the function of AmiE protein. We prepared three samples shown below. When we tested the AmiE degradation ability with these samples, the results show that C4HSL(refer to it as C4 hereafter) is not degraded by AmiE, but 3OC12HSL(C12) is degraded by AmiE(Fig. 4-1-5).

• PBAD-rbs-amiE(pSB6A1)

• Ptet-rbs-luxR-tt-Plux-rbs-gfp (pSB6A1)

• Ptet-rbs-rhlR-tt-Prhl-rbs-gfp (pSB6A1)




Fig. 4-1-5 AmiE degrades C12 but barely degrades C4

For more information, read this page !

BBa_K1949102 meets the Bronze Medal criteria!

The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, mazE expression was induced by the addition of IPTG (2 mM) after mazF expression was induced by the addition of arabinose(0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.