BBa_K1949060 meets the Gold Medal criteria!
We simulated our final genetic circuits and found that the circuits would 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 was inappropriate for two reasons (see Rhl_system_assay). Then, we decided to further improve this Prhl and obtain our original improved Prhl (Noticeable Mutant) (BBa_K1949060). We referred to as Prhl(NM) from now on that suited our goal.
Our purpose is to create a strong Prhl for our final genetic circuits.
This experiment consists of the three parts below.
Improved Prhl by iGEM 2014 Tokyo_Tech team and characterization of rhlR(Fig. 4-1-1).
Improvement of the wild type Prhl(Fig. 4-1-2).
Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech team with our original improved Prhl(Fig. 4-1-3)
PBAD ‐ rbs ‐ amiE (pSB6A1)
Ptet ‐ rbs ‐ luxR ‐ tt ‐ Plux ‐ rbs ‐ gfp (pSB6A1)
Ptet ‐ rbs ‐ rhlR ‐ tt ‐ Prhl ‐ rbs ‐ gfp (pSB6A1)
Fig. 4-1-1 Effect of RhlR by tagging LVA
and The colonies of transformants with a rhlR (left) or a rhlR - LVA (right)
Fig. 4-1-2 RFU of GFP / Turbidity of imoroved Prhl mutants
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 !
2.Best Basic Part: BBa_K1949050
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.
Fig. 4-1-4 AmiE degrades long chain [N]-acyl homoserine lactone (AHL) molecules
with acyl chains longer than six carbons
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).
Fig. 4-1-5 AmiE degrades C12 but barely degrades C4
For more information, read this page !
Best Composite Part: BBa_K1949102
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.
Fig. 4-1-6 Time vs Turbidity (Stop & Go) and Time vs RFU of GFP (Stop & Go)
It was found from Fig. 4-1-6 that MazF inhibited cell growth. MazE was induced 2 h after mazE expression, and about 8 h later, cell growth which had stopped was resuscitated. From these results, it was suggested that E. coli whose cell growth was inhibited by MazF was able to resuscitate by expression of mazE.
For more information, read this page !
