IMPLEMENTATION

The Staphylococcus aureus(S. aureus) contamination is a common problem that we encounter everyday. To address this , our project is designed to utilize protein proximity labeling technology in the concept of real life to inhibit the growth of S. aureus, therefore preserving the food from bacterial contamination. The scientific theory behind our project is that we reduce the growth of S. aureus by blocking down the QS channels in the process of protein biotinylation, which fused protein to bring TurboID to the specific QS site AgrB and AgrC. In order to obtain more biotin and keep the reaction going on, we use engineered Escherichia coli to produce it. Therefore, with enough TurboID-AIP and biotin, the growth of S. aureus will be blocked and our health can be preserved in this way. We identified three potential situations that need our product and the specific treatments are exerted on diverse conditions.

Identification of potential users

Household preservation

We design this type of preservative film with the extracting solution of proteins on it, which can prevent the food from undergoing biofilm effect and the accumulating of bacteria.

Restaurants kitchens’ hygiene

Our special preservative film will prevent food from absorbing S. aureus on it.

Fresh milk transformation

We use silver paper to seal the used glass milk bottles. Therefore, with less bacteria growth inside the bottle, it can be recycled hygienically.

To go one step further

Fig.1 Protein extracting solution and normal preservative film

Fig.2 The use of our product: split protein extracting solution on the film

Fig.3 The protein-treated film can be applied to inhibit the growth of S. aureus

Challenges

In the discussion with Dr. Hailong Gao, we come up with a few questions that need further researches and explanations to certify.

Firstly, we have doubt on whether our target protein has the homologue with other protein, so that we have the possibility to label the wrong protein. However, in the article written by S Balaji et al.(2001)(1), we confirm that our protein don’t have homologue with other ones.

The second question is that how can we preserve the turboID enzyme with the maintainable activity and how can we reduce the rate of protein degradation.

Moreover, the cost in this process is also taken into major consideration. In the process of purifying TurboID from engineering bacteria in the LB broth, we find out that the yield is small. Large amount of engineering bacteria is required to purify only a spot of protein. We apply the protein fusion expression to solve this problem. This technology can help to generate more preferable proteins, slow down protein degradation and keep protein stable.

Protocals of testing S. aureus food contamination

Here, we invent a new way to detect the S. aureus by using the filter bottle. We add the food/liquid to the upper bottle and culture with TurboID-AIP, Biotin and ATP. When we vacuum the liquid in the upper bottle, the bacteria can not get through the 0.2 um filter member. So we incubate the member with streptavidin-HRP for 30 min and followed by washing with PBS. The HRP can react with the SuperSignal™ West Pico PLUS Chemiluminescent Substrate. The membrane can be taken out and imaged to show up the blot on the membrane. Thus, the dark blot on the membrane is the S. aureus.

Fig.4 Detection of S. aureus in food

References:

(1)S Balaji et al.(2001), PALI-a database of Phylogeny and ALIgnment of homologous protein structures, Nucleic acids research, vol.29, pp.61-65