Engineering Success

As described above, two surveys were performed. Assessing the validity and relevance of the project was the purpose of the first one. Here, three hypotheses were evaluated. We wanted to check whether mental health has suffered any deterioration after the COVID-19 pandemic. We also wanted to verify the current awareness regarding mental health, compared to the one people had five years ago. Last, we aimed to check whether general habits had changed after lockdowns. Surveys were published and worldwide opinions were gathered and with the input received, we established that our project was of sufficient relevance and was justified (see Results page). After the data interpretation of this first survey, we then launched a second survey with the intention of verifying the awareness of the general population regarding the use of pharmacological solutions to address mental health diseases and whether people trust these solutions. Plus whether people were aware of circular economy solutions, their characteristics and suitability for different applications.

This way, we have completed two full cycles of designing, building, testing and learning from our survey results, that served the purpose of validating our initial hypothesis and giving our project meaning.

Moving on to laboratory work, the team started the first engineering cycle and managed to design the following one.

Designing laboratory strategies usually takes longer. Our team took steps very carefully to guarantee design success. Design of each construction is described below.

The design of the construct for the synthesis of the Tryptophan Decarboxylase enzyme (TDC) was carried out by cloning it into pET28a. pET28a is a non-viral vector of constitutive expression that provides resistance to Kanamycin to the bacteria in which it is introduced. It is 5369 bp long and allows the synthesis of proteins with a His Tag at the N-terminal end. In addition, it has a thrombin cleavage site in case it is desired to eliminate this tail, or to avoid leaving additional residues that could modify the synthesized protein.

The cloning strategy to introduce TDC into the vector is based on conventional restriction enzymes (IIR). In this case, EcoRI and HindIII are used, one at each end. This double digestion ensures that the insert is placed in the correct direction and not in reverse of the reading pattern.

The insert consists of the human TDC sequence optimized for Escherichia coli, flanked by the corresponding restriction sequences, in 5' to EcoRI and in 3' to HindIII, previously added by PCR. In this way, by means of a previous digestion of both the insert and the vector, complementary ends are obtained in both, and by means of the T4 ligase, they are joined, thus obtaining the construction shown here.

The second synthesized enzyme in the project is cloned into a pET15-MHL vector. It is a 7737 base pair long vector containing a cloning region between positions 5214 and 7470. pET15-MHL has ampicillin resistance and the promoter and terminator sequence of phage T7. Moreover, it contains an additional sequence, corresponding to a 6-histidine tail at the N-terminal end, which helps in the purification of the protein of interest.

The cloning strategy performed to obtain the protein of interest is based on the infusion method, whereby by creating complementary regions between the ends of the insert and the vector of interest, and treating the mixture with the corresponding enzymes, a directional recombination is achieved. Insertion of the TPH sequence involves replacement of the SacB gene, which provides negative selection of the original plasmid if the treated bacteria are grown in 5% sucrose-enriched media. Prior to the insertion process, the vector is linearized by the enzyme BseRI whose restriction targets are located at positions 5381 and 7370. The final construction sequence can be checked here.

Comparison of the two cloning methods used:

restriction enzymes (IIR) and ligase-independent cloning, linearizing the plasmid with IIS enzymes.

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Constructions results are available in our Results page in our wiki. Unfortunately, our team was not able to test the enzyme production capabilities of these constructions, and these assays will be performed in future editions by upcoming teams who continue with our project. However, we do know that the constructions did not result toxic to bacterial strains.

For future iterations, we plan to compare the enzyme production capability of our prototype constructions with ones designed based on the iGEM Assembly Standards. To do so, we have chosen a set of constitutive promoters, ribosome binding sites and terminators of different strengths. Following steps involve combining these parts and measuring the protein production via qPCR and western blot assays. The chosen parts can be checked in the table below.

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