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.

1. New cold induible promoter

Here we indicate that the cold inducible promoter is a promoter that depends mainly on temperature.
a Pcold-gfp was constracted.


Fig. title

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

2． Rhl system assay

Here we indicate how we produced the rhl promoter that suited our project best.



Fig. title

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


Fig. title

iGEM 2014 Tokyo_Tech has already improved the rhl promoter. 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 experiment showed that Prhl(LR) crosswalks with C12 at a specific proportion. If we use it in the recreation of Snow White, and if the promoter crosstalks with C12, it means that the Queen eats the poisoned apple, she herself produced, and dies. So this is not a promoter we can use in our project.
The graph also shows that Prhl(NM) almost does not have crosstalk with C12.
Base 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 we indicate the inhibition of the E. Coli´s multiplication and inhibition of translation by MazF and the restarting of them by MazE.
We constructed a circuit that includes mazEF .



Fig. title

In this experiment, MazF was expressed by arabinose , and after 2 hours MazE was expressed by IPTG.
From the experiment results, it was found that the turbility of the sample without MazE almost didn’t rise at all. However, it was also found that if IPTG is added, and the MazE is expressed, the E. Coli will restart its multiplication.
Also, if only the MazF is working, the RFU of GFP barely rise But if MazE is induced, the RFU of GFP will rise. (Link: Toxin assay)
As the result above, it was concluded that MazF interrupts the multiplication of E. Coli and translation. However, MazE restarted the multiplication and translation stopped by MazF.
Through this experiment, we showed that the TA system functions properly.


Fig. title

4． AmiE degrades C12

Here we indicate that the AmiE decomposes AHL selectively.



Fig. title

In this experiment, it was examined whether AHL would be degraded after the addition of 3OC12HSL and C4HSL molecules into a culture of E. Coli that express AmiE.
From the experiment 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 in this culture, is barely degraded.
From the above result, we were able to show that AmiE selectively degrades AHL molecules.