Team:Shanghai United HS/Proof Of Concept

Shanghai_United_HS

Proof of Concept

Overview

According to the 2017 China Health News[1], the teeth-repairing market in China is large. In China, 98.4% senior citizens, 88.1% adults, 29% teens, 66% children have teeth decay problem. It means that teeth health has become a national problem. How to keep our teeth healthy? Except for Bass Toothbrushing Techniques, the iGEM team Dr. Phage has designed another technique to equip us with strong teeth-cleaning ability due to ClyR, which means people do not have to spend a lot of time on brushing their teeth using the Bass method. And in the meantime, they can make their teeth cleaner.

We plan to construct an engineered bacteria (E. Coli strain: Nissle 1917), which may be functional in secreting protein ClyR under the induction of arabinose released by arabinoxylan. The ClyR is able to wipe out a number of bacteria, especially Streptococcus mutans and S.sobrinus, the major causes of dental caries. This engineered bacteria is designed to add to toothpaste to protect people, especially children and teenagers, from dental caries.

Prototype of our Toothpaste

Supporting Experiment Results

In vitro activity assay of ClyR

Experimental bacteria：E. coli Nissle 1917 transferred with plasmid A and B, Lactobacillus casei subsp. casei (ATCC334)

PROCEDURE

1) The culture of Lactobacillus casei subsp. casei (ATCC334)
2) In vitro assay
The value of OD₆₀₀ was monitored using Multiscan Spectrum (BioTek).

Figure 1. The in vitro assay of ClyR.

The OD₆₀₀ of the Lactobacillus casei subsp. casei cell suspension gradually reduced with the addition of supernatant (Figure 1).

We have successfully obtained the ClyR protein in the culture supernatant of cells carrying plasmids with araB and clyR genes. The concentration of ClyR was low, so more efforts are still needed in future.

The final experiment results proved that ClyR protein is successfully obtained and is able to killing Lactobacillus casei subsp. casei (ATCC334). (Because of the safety rules enforced by iGEM Safety Committee, it is impossible for us to use Streptococcus mutans and S.sobrinus in our experiments.)

In conclusion, the experiment results support our idea to acquire ClyR protein in Nissle 1917 and verify its power in killing bacteria. Thus, we may further proceed our project and try to insert our engineered Nissle 1917 to toothpaste in the future, targeting at making further functional test.