Contribution

Add data to arabinose controlled self lysis system

Arabinose was considered as a safe food ingredient. Therefore, this year we still choose it to be our inducer of self-lysis system. The patient can take a pill of arabinose to start the system and eliminate the engineered bacteria in their intestine. The original design came from team SZ-SHD in 2020 (arabinose induced self- lysis system) and registered as part BBa_K112000. Hence, we add new document to this part.

Change in OD600 indicates the effectiveness of self lysis system.

Firstly, according to the protocol suggested by team SZ-SHD in 2020, we use a constant concentration(2g/L) of arabinose to induce the self-lysis system.

Procedure Comic Strip
Procedure Comic Strip

The OD600 was measured in a 96 well plate using a plate reader, the mean value was the caculated and draw a graph.

We successfully repeated their result and confirmed that this system can worked in our product.

Measure of life cell (CFU) using dilution and plate spraying method

Although OD 600 can indicate the change in cell number, it could not differentiate live cell and dead cells, therefore, we also tried to use dilution and plate spraying method to measure the Colony forming unit (CFU).

The change in CFU also provides a solidary evidence of the effectiveness of lysis system.

Add information to part BBa_K1598002: TPH origin from Human

To better understand the TPH activity in the intestine, we decided to use a hybrid of in silico and in vivo experiments to investigate the TPH activity. The tryptophan hydroxylases (TPH) from human, as well as from mice, zebrafish, and rabbits have been selected and analyzed for their catalytic efficiency in the rate-limiting step of producing serotonin. By predicting the binding affinity of the TPHs to the tryptophan molecule we could predict the catalytic effectiveness of the enzyme selected.

In silico binding affinity prediction

The full-length 3D structures of the four TPHs from human, mouse, zebrafish, and rabbit are acquired as the PDB files from the uniport database under the following entry numbers: P17752; P09810; Q6PBX4; P17290 for each host organism respectively. For entry numbers P09810, Q6PBX4, and P17290, the known x-ray/NMR structures of the proteins are missing. Instead, the Alphafold predicted full-length structure of the proteins was selected for further analysis.

Results for in silico binding affinity prediction

The zebrafish TPH would hypothetically mediate the highest efficiency of tryptophan hydrolysis among the four selected TPHs. Our wet lab experiments and results would, furthermore, help us identify the most potent TPH enzyme to be produced in E. coli expression and used for the final product.

In vitro enzyme activity testing

To measure the HTP activity, we followed the protocol listed in this part page by team UCL in 2015. In the protocol, a continuous fluorometric assay for HTP activity based on the different spectral characteristics of tryptophan and 5hydroxytryptophan is presented.

Results for in vitro enzyme activity testing

Our wet lab experiments and results fit our in silico prediction of HTP activity and show that the zebrafish TPH has the highest efficiency of tryptophan hydrolysis among the four selected TPHs. Therefore, we will choose this strain of HTP in our final product.

Hence, we also submitted a new basic part (BBa_K5009001) for the TPH origin from zebrafish.

More about our design, visit Engineering Success.