The goal of our project is to provide a new solution for semen quality testing using engineered bacteria. Due to the lack of iGEM projects in the field of reproductive health in previous years, we hope to make a brave attempt in this difficult area. We marvel at the great scalability of synthetic biology that allows us to implement whimsical designs in engineered bacteria.

We worked on the project for 20 weeks to design, build and validate our sensing system, information processing system and reporter system, and to assemble these components into the chassis engineered bacteria. In addition, we also designed the corresponding hardware and modeling system to demonstrate the feasibility of our design.

It is worth mentioning that our designed and modified two-component system will be applicable to any protein detection system with corresponding antibodies. Therefore, our project could inspire more diagnosis designs in other application fields with a versatile protein detection system.

We used the two-component system as the detection system for the marker protein.

The first detection system was designed to detect the antibody of marker protein Sp10 representing sperm motility. We modified the PmrB receptor with an affibody that can specifically recognize the Fc fragment of the IgG antibody. Using the antibody as a mediator, we can apply our Affi-PmrB/A system to detecting Sp10 to represent the number of sperms in contact with it. We validated the effectiveness of the constructed Affi-PmrB/A system using IgG antibodies as inducers.

The second detection system was designed to detect EGFR representing sperm fertility. We demonstrated that the fusion protein Affi-nisin could specifically detect and respond to EGFR on the sperm surface.

We constructed a three-state logic gate based on serine integrase Bxb1 and cro/cI system to separate the two signals representing motility and fertility via conditional computation. We verified the inversion efficiency of integrase and the repression/derepression function of cro/cI system.

To meet our demand of household use scenario, we designed our sperm quality test chip to present the results semi-quantitatively in a visual way. For this purpose, we used two color proteins capable of developing color separately under different light sources and designed corresponding colorimetric cards for comparison.

For more details on our design and results, please visit Design and Results pages.

The advantage of our project is that it can perform the entire sperm quality detection process in a single handheld chip. For this purpose, we designed and developed the microfuidic chip-based hardware and verified the possibility of establishing a chemotactic substance concentration gradient in it.

For more details on our design and results, please visit Hardware page.

We have designed and completed two software tools, kmer2vec and Promoter_Transformer.

To examine the application scope in different species of our engineered bacterial system in detecting sperm quality, we designed a rapid DNA/protein sequence comparison software kmer2vec. The kmer2vec software is an novel alignment-free method based on word2vec training, which can be used for rapid multiple sequence comparisons, focusing on phylogenetic analysis and species clustering. With equal accuracy, kmer2vec can speed up the traditional multiple sequence alignment (MSA) method by about 10,000 times.

To perfom computer-based directed evolution of PnisA promoter, we invented a novel deep learning software called Promoter_Transformer to predict the strength of prokaryotes promoters. On the same dataset, the predictive performance of our model outperformed the predictive model in the paper^[1] published on Nucleic Acids Research by 30%.

For more details on our design and results, please visit Software page.

At this point, we have accomplished our goal of validating our overall design in steps, demonstrating the feasibility of our idea. On the Implementation page, you will find our plans and visions for the future development of this project.

We summarize our work this year as a contribution to future iGEMers; we also sort out the iterative process in completing the project, which is a valuable asset in our iGEM process.

[1]Stahl, S., Graslund, T., Eriksson Karlstrom, A., Frejd, F. Y., Nygren, P. A., & Lofblom, J. (2017). Affibody Molecules in Biotechnological and Medical Applications. Trends Biotechnol, 35(8), 691-712.
[2]Kuipers, O. P., Beerthuyzen, M. M., de Ruyter, P. G., Luesink, E. J., & de Vos, W. M. (1995). Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. The Journal of biological chemistry, 270(45), 27299-27304.
[3]Merrick, C. A., Zhao, J., & Rosser, S. J. (2018). Serine Integrases: Advancing Synthetic Biology.ACS synthetic biology, 7(2), 299-310.