Overview

Making useful contributions for future iGEM teams has always been a goal worths striving for. We obtained numerous inspirations and experimental instructions for our project from previous iGEM teams. To continue this tradition, we dedicated to contributing our efforts from wetlab design to drylab simulation in the hope that future iGEM teams can receive as much useful information and guidance as possible.

Today, as the urgency of reproductive health becomes increasingly prominent, efforts to improve human reproductive health through assisted reproductive technology and semen screening become the focus. However, the topic of reproductive health is somewhat remote for iGEMers as high school and college students, and it is even a topic that most of us are unwilling to openly discuss. Projects focusing on human reproductive health can barely be found in previous iGEM teams. One exception is the team of Montpellier 2018, who dipped into the area of reproductive health in their project of antisperm antibody contraception device.

Lack of research on sperm quality test in iGEM and even in the field of synthetic biology as well as the urgency of raising public awareness on sperm quality examination have prompted us to apply synthetic biology to propose a novel diagnostic method for sperm quality examination for household use. Consequently, we believe that one of our primary contributions for future iGEM teams is opening up a new prospect field of applying synthetic biology to tackling sperm quality issues and reproductive health concerns as a pioneer. Perhaps the hardest part of solving reproductive dilemma is to confront it directly. We hope our project will be the first step.

PmrB/A system is a bacterial two-component system originated from Salmonella^[1]. The extracellular iron(III)-binding motif of PmrB receptor can be replaced with an affibody capable of recognizing the Fc fragment of an antibody. By combining the PmrB/A system and the engineered protein affibody recognizing Fc segment of the antibody, we developed a bacterial system capable of detecting any protein signal that can be recognized by its specific antibody. What we achieved is an engineered bacterial system simulating the entire process of ELISA.

The versatility of our Affi-PmrB/A system lies in the fact that affibody is a category of engineered proteins with similar structure and recognition mechanism. Therefore, by simply modifying the affibody fused with PmrB, we could apply our Affi-PmrB/A system to detecting various biomarkers in physiological processes. Currently, the affibodies capable of recognizing antibody and EGFR are already available, which are critical indexes in physiological processes. In summary, our Affi-PmrB/A system is a novel and versatile detection method with the potential of assisting early screening and diagnosis of various diseases through detection of crucial protein biomarkers.

NisK/R system is a bacterial two-component system originated from Lactococcus lactis. In this system, extracellular nisin can bind to histidin kinase receptor NisK and activate the response regulartor NisR to promote transcription through PnisA.^[2] We modified the original NisK/R system by fusing NisA (precursor of nisin) with an affibody capable of recogonizing EGFR. This NisA-affibody Z_EGFR:2377 fusion protein can utilize the convenience of both NisK/R system and affibody, providing an innovative system for genetic modifications aimed for protein signal sensing.

One of our contribution is that we confirmed the feasibility of applying the NisK/R system, which is originated from Lactococcus lactis, in the E.coli. By broadening the application range, we demonstrated huge potentials of the NisK/R system in designing genetic circuits.

Moreover, we developed a versatile method to detect protein signals through our Affi-nisK/R system. By simply combining NisA with other affibodies capable of recognizing target protein or the antibody of target protein to create a fusion protein, our Affi-nisK/R system can respond to any affibody potential targets, possessing unlimited value in diagnostic and therapeutic applications in various fields.

From the starting point of our project, we tried to build a system capable of integrating analysis of multiple signals and inducing corresponding output signals. After investigating around 3000 projects in the iGEM community and existing literatures, we found that although there are many gene circuits that can perform logical operations, they are all originated from bacteria or eukaryotes with rather complicated signaling pathways. Therefore, we tried to think out of the box to find signal processing systems in simpler organisms capable of conducting sophisticated logic operations.

We were very surprised to find that the cro/cI system in bacteriophage can act as a bidirectional switch in determining the fate of phage. The repression and derepression features of cI/Cro system possess the potential of being a “NOT” gate. Moreover, we discovered that site-specific inversion caused by integrase Bxb1 has the potential of inducing 0-1 transformation.

In summary, by combining cro/cI system with integrase Bxb1, we have successfully built a three-state gate circuit. The output structure of the three-state gate circuit is very different from the output structure of the ordinary gate circuit. To be more specific, the three-state gate circuit adds an output control terminal EN (the abbreviation of Enable. Here in our system, EN is the Cro protein) to the circuit. When EN=1, the output depends on the transverter (Bxb1 integrase in our system). When EN=0, all outputs inside the circuit will be kept in the off state.

In contrast to other systems that perform logic operations, our system consists of only two nodes, which is probably the minimum unit that can perform logic operations. We have achieved the most simplified logic computing gene circuit using cro/cI system and integrase Bxb1. Apart from that, our logic gate system can be easily modified to respond to different signals by simply altering the promoters upstream of the Cro protein and integrase Bxb1, demonstrating strong versatility and broad potential applications. It is also worth mentioning that this three-state gate system implements the integration of “AND” “OR” “NOT” gates using extremely simple elements and can be used as a biological calculator. This new integrated system can use specific proteins as input signals, which has not been achieved in previous iGEM projects. It is our unique contribution to the iGEM community.

In our project, we have extended the iGEM project of Northeastern University in 2020, and developed a novel and versatile protein detection method using Affi-PmrB/A system. By altering the upstream receptor and downstream reporter, our Affi-PmrB/A system has the potential of responding to any extracellular protein signal.

Throughout the construction process of affibody, we have developed an efficient workflow of receptor modification and experimental veirification. The workflow begins with designing the sequence of the engineered receptor according to research purpose. Next, structure of the designed sequence will be predicted by Alphafold2 software and compared with the original receptor structure. Moreover, whether the engineered receptor could bind to ligands as expected will be examined using molecular docking simulation software. After structure prediction and molecular docking are confirmed to be correct, we can enter the experimental verification stage.

This workflow of receptor modification greatly reduces the experimental workload by analyzing the feasibility of modification through structural prediction in advance. We believe that this workflow can be adopted by future iGEM teams who are desiring to modify protein receptors in their projects.

In our project, we demonstrated how modeling can be employed to assist in verifying the feasibility of wetlab experiments and hardware design.

In order to assist in verifying the feasibility of our expression system, we designed a response process model to simulate and check its consistency with our experimental expectations. We applied differential equations to build a semi-quantitative model through simplifying the complicated response process into a combination of chemical equations. For instance, we simplified multiple binding equations into the resistance equation because the binding constants are difficult to be determined. Such simplification can reduce the computational burden without sacrificing the accuracy and stability of results. In total, we were able to simulate the whole response process using only three types of equations, namely the combination equation, generation/degeneration equation, and enzymatic equation, transforming complexity into simplicity. The idea and method of constructing a simplified mathematical model to represent a complicated reaction process can be applied to future iGEM projects.

Moreover, in order to examine the feasibility of our microfluidic chip design using a chemokine concentration gradient to induce viable sperms to move ina directional manner, we established a gradient marker model to discuss the formation mechanism of concentration gradient in the microfluidic chip. Our model introduces fluid mechanics, and uses COMSOL, a simulation software commonly used in this field, to show the formation process of concentration gradient. When the model parameters are set, the software can accurately describe almost all the common flow processes. Future iGEM teams intending to employ microfluidic chip in thie projects can then use the software to predict experiment results in advance. In addition, our model can be applied to observing the distribution of pollutants in the water under experimental conditions, contributing our efforts to protecting the environment.

For more details on our modeling, please visit modeling page.

In the hope of designing a sperm quality detection chip capable of integrating two different protein signals and presenting the detection results in a visual color representation through an engineered bacterial system, we applied microfluidic chip consisting of microchannels with chemokine gradients to induce viable sperms to enter the detection area. We adopted the method of finger driving liquid flow to overcome the defect that traditional microfluidic technology requires external power pump to drive liquid flow, so that our chip-based sperm quality test product can be used at home.

In addition, we realized the automatic screening of high-motility sperms by establishing a chemokine concentration gradient in the detection device. Such a screening method can be applied to other detection scenarios where the target cell has certain chemotaxis and motility.

For more details on our microfluidic chip, please visit hardware page.

kmer2vec

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 based on k-mer theory and word2vec embedding. A biological sequence can be viewed as an article, and each k-mer is equivalent to a word in the article. Therefore, using the word2vec method in natural language processing (NLP) for training, we can get vector representations of different k-mers, and then we can study the relationship between biological sequences. This software tool can be applied to performing multiple sequence comparison, which is much faster than the traditional alignment method.

Moreover, there are many other applications of this algorithm. For instance, DNA sequence comparison is the basis of bioinformatics and can be used for phylogenetic analysis, species clustering, and homologous gene searching. The kmer2vec method can speed up multiple sequence comparison, making it possible to analyze large genomes.

Promoter_Transformer

To perfom computer-based directed evolution of PnisA promoter, we invented a software called Promoter_Transformer to predict promoter strength. Promoter_Transformer regards the promoter sequence as text, performs word segmentation through the k-mer method, and then extracts the base information of the promoter sequence for training. We selected the top10 promoter sequences with the highest predictive strength as alternative optimized sequences, and verified this prediction results by experimental work.

The promoter is one of the core components in synthetic biology, which can control the strength of transcription and thus the strength of gene expression. Therefore, finding a promoter with suitable strength is crucial for constructing an expression vector. Our software can be very useful for other projects since it can get promoters with suitable strength based on computational methods.

We added more information to existing parts and documented new parts througout our project. We hope that our contribution to the iGEM Parts Registry can be beneficial to future iGEM teams. Here we list our parts involved in our project.

[1]Hua, J., Jia, X., Zhang, L., & Li, Y. (2020). The Characterization of Two-Component System PmrA/PmrB in Cronobacter sakazakii. Frontiers in microbiology, 11, 903. https://doi.org/10.3389/fmicb.2020.00903
[2]Ge, X., Teng, K., Wang, J., Zhao, F., Zhang, J., & Zhong, J. (2017). Identification of Key Residues in the NisK Sensor Region for Nisin Biosynthesis Regulation. Frontiers in microbiology, 8, 106. https://doi.org/10.3389/fmicb.2017.00106