Proof of Concept

Achievement Gold

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

Fig. 6-3-1-1. 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 results, we found that the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.

The above results indicate that this promoter is induced at low temperatures.

2． Rhl system assay

Here shows how we changed the rhl promoter to the one that suited our project best. We conducted an experiment using BBa_K1949060.

Fig. 6-3-2-1. 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 we measured the RFU.

As a result, the mutant promoter Prhl(NM), which is stronger than a wild type promoter, was newly obtained.

Fig. 6-3-2-2. The Signal-to-Noise (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. However, comparing the Prhl(NM) with the Prhl(LR), the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the graph shows that Prhl(LR) has crosstalk with C12 at a specific proportion. If we use it in the recreation of Snow White, and the crosstalk between the promoter and C12 will mean that the Queen eats the Poisoned Apple, which she produced herself, and dies. Therefore, this promoter cannot be used in our project.

The graph also shows that Prhl(NM) almost have no crosstalk with C12.

Based on the results above, we can say that we found a promoter with strong activity, and it almost have no crosstalk with C12, which is the most suitable one for our story.

3. TA system

Here shows the inhibition of the E. coli´s growth and translation by MazF and the resuscitation of them by MazE. We performed an experiment using BBa_K1949100 and BBa_K1949102.

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

Fig. 6-3-3-2. 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 will be 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. Read mazEF system Assay page.

As the result above, we concluded that MazF inhibits the E. coli's growth and translation. However, MazE resuscitates the growth and translation stopped by MazF.

Althrough this experiment, we indicated that the TA system functions properly.

4． Degredation of C12 by AmiE

Here it is indicated that AmiE degrades AHL selectively. We performed an experiment using BBa_K1949052.

Fig. 6-3-4-1. AmiE degrades C12 Fig. 6-3-4-2. AmiE barely degrades C４
C12 is greatly degraded. On the other hand, the C4 is barely degraded by AmiE.

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.