Team:Tokyo Tech/Proof

Proof of concept

-demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept.

Introduction

The concept of this project concludesare the following 4 points.

These points are expounded sequentially as follows.

1. New cold induible promoter

Here we indicated that the cold inducible promoter is a promoter that depends mainly on temperature.
We performed an experiment using BBa_K1949001.


Fig. 6--1-1. Cold inducible promoter
the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.


In this experiment, each sample was cultivated at 18°C and 37°C, after that their RFU of GFP / Turbidity was measured.
From the experiment results, we found that the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.
From the above result, we were able to show that this promoter is a promoter induced by low temperatures.

2． Rhl system assay

Here shown how we produced the rhl promoter that suited our project best.
We performed an experiment using BBa_K1949060.



Fig. 6--2-1. Our original improved Prhl(NM) is much more sensitive to C4HSL than Prhl(WT).


A single point mutation was inserted in a wild type rhl promoter(BBa_R0071).
In the experiment, an AHL reagent was included in the reporter, and then the RFU was measured.
As a result, the mutant promoters Prhl(NM), which was stronger than a wild type promoter, was newly obtained.


Fig. 6--2-2. The SN ratio of Prhl(NM) is higher than that of past improved Prhl(LR), and Prhl(NM) does not crosstalk to C12 unlike


iGEM 2014 team-Tokyo_Tech has improved the rhl promoter in previous years. However, comparing the Prhl(NM) to the Prhl(LR), Prhl(NM) did not show any crosstalk with C12 and the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the graph shows that Prhl(LR) has crosstalks with C12 at a specific proportion. If we use it in the recreation of Snow White, and if the promoter has crosstalks with C12, it means that the Queen eats the poisoned apple, which she herself produced, and dies. Therefore, this promoter is not used in our project.
The graph also shows that Prhl(NM) almost does not have crosstalk with C12.
Based on the above results, we can say that we found a promoter with strong activity, and it does not have crosstalk with C12, which is the most suitable for our story.

3. TA system

Here shown the inhibition of the E. coli´s growth and translation by MazF and the resuscitation of them by MazE.
We constructed the circuit that includes mazEF.



Fig. 6--3-1. After inhibiting cell growth by MazF, it was resuscitated by expression of MazE.



Fig. 6--3-2. After inhibiting expression of GFP by MazF, it was resuscitated by expression of MazE．


In this experiment, MazF was expressed by arabinose, and after 2 h MazE was expressed by IPTG.
From the results, it was found that the turbidity of the sample without MazE almost did not rise at all. However, it was also found that if IPTG is added, and the MazE is expressed, the E. coli will recover its growth.
Also, if only the MazF is working, the RFU of GFP barely rise. If MazE is induced, the RFU of GFP will rise. (Link: Toxin assay)
As the result above, we concluded that MazF inhibits the E. coli's growth and translation. However, MazE recovers the growth and translation stopped by MazF.
Althrough this experiment, we indicated that the TA system functions properly.

4． AmiE degrades C12

Here it is indicated that AmiE degrades AHL selectively.



Fig. 6--4-1. AmiE degrades C12   Fig. 6--4-2. AmiE barely degrades C４
C12 is greatly degraded. On the other hand, the C4 is barely degradedPrhl(LR).


In this experiment, it was examined whether AHLs would be degraded after the addition of 3OC12HSL(C12) and C4HSL(C4) molecules into a culture of E. coli that expresses AmiE.
From the results, it was found that the C12 added into a culture of AmiE expressing E. coli is greatly degraded. On the other hand, the C4 added into this culture, is barely degraded.
From the above result, we showed that AmiE selectively degraded AHL molecules.