Engineering

Design:

     The bacteria-microalgae ratio refers to the proportion of biomass or cell number between bacteria and microalgae in the interaction system. It is a crucial factor that affects the stability and efficiency of the symbiotic relationship. Different ratios can lead to varying material exchanges and information transmissions, thereby influencing growth, metabolism, and function of this interaction system.

     Generally speaking, a lower bacteria-microalgae ratio benefits the growth of microalgae, as bacteria can provide regenerative nutrients and growth stimulants, while also suppressing the natural enemies and pathogens of microalgae. Conversely, a higher ratio favors the growth of bacteria as microalgae provide light energy sources, as well as increase the pH and dissolved oxygen content of the water.

     However, it should be noted that this rule is not universally applicable in all cases. Several studies have demonstrated that different types or states of bacteria or microalgae exhibit varying adaptability and sensitivity to the bacteria-microalgae ratio, sometimes yielding contradictory outcomes. For instance, certain bacteria possessing nitrogen fixation ability or antibiotic production capabilities can stimulate microalgae growth at higher bacteria-algae ratios. Similarly, heat or salt-tant microalgae producing protective substances may enhance bacterial proliferation at lower bacteria-microalgae ratios.

Build:

     Consequently, determining the optimal bacteria-microalgae ratio necessitates considering multiple factors, such as the type, characteristics, state, and environmental conditions of both bacteria and microalgae. In our project, we aimed to conduct experiments and modeling by employing density ratios between microalgae and bacteria.

Figure 2 Experimental schematic diagram of the interaction ratio between different bacteria and microalgae

Test:

     We designed a gradient density ratio experiment based the characteristics of experimental materials. P. tricornutum and Q. aquimaris were cultured in ratios of 1:1, 1:3, 1:5, 1:7, and 1:9 respectively, while conventional culture conditions were employed for microalgae as a control group. In this study, we determined an optimal symbiotic ratio by considering factors such as biomass, lipid accumulation, and photosynthetic efficiency.

Figure 3 Effect of different microalgae-bacteria interaction ratios on lipid accumulation in microalgae

Learn:

     During Design Cycle 1, we observed that diverse bacteria-microalgae interactions can potentially impede nitrogen-phosphorus removal in the water while concurrently facilitating biomass accumulation in P. tricornutum. However, these interactions exhibit a favorable impact on lipid accumulation and overall biomass growth in microalgae.

Design:

     In the bacteria-microalgae interaction system, mutual interactions between P. tricornutum and Q. aquimaris can occur through material exchange, information transmission, and structure formation. To further investigate this system, we employed a commonly approach involves co-culture and separation culture for comparative analysis.

Build:

     We implemented microbial immobilization embedding schemes based on previous research findings to investigate the forms of interaction between P. tricornutum and Q. aquimaris. Furthermore, in order to minimize potential confounding factors associated with microbial immobilization embedding schemes, we designed relevant hardware equipment and auxiliary devices.

Test:

     In terms of designing the experimental group, we established a total of five treatment groups to investigate interactive effects under different cultivation conditions. Microalgae and bacteria were co-cultivated in an optimal ratio (AB). Microalgae were individually immobilized and co-cultivated with bacteria(AO）, while bacteria were individually immobilized and co-cultivated with microalgae(BO). Additionally, bacteria and microalgae were immobilized separately(ABO) or together(ABS) , and co-cultivated subsequently.

Figure 4 Experimental schematic diagram comparing different immobilization strategies for the interaction between bacteria and microalgae

     The consumption rate of total nitrogen in the culture medium along with the accumulation of lipids and pigments in microalgae were utilized as evaluation indicators. Regrettably, during the initial design phase, we overlooked the challenges associated with sampling and detection in the immobilization system, which hindered some anticipated evaluation indicators from proceeding normally, potentially leading to an incomplete assessment process.

Figure 5 Experimental Phenotype Diagrams

     Fortunately, our hardware device version 1.0 designed for microbial embedment proved instrumental in this evaluation experiment. The utilization of this device for producing microbial immobilized beads resulted in more uniform spheres prepared at a faster pace compared to manual embedding, significantly reducing time costs.

Figure 6 Effects of different immobilization interaction modes on lipid accumulation in microalgae

Figure 7 Microbial Embedding Device v1.0

Learn:

     Detecting lipids and pigments of immobilized interaction system poses a challenging task. We acknowledge that a small part of cells rupture caused by sodium citrate during dissociation of immobilized balls is temporarily inevitable.

     Surprisingly, our findings reveal that separate immobilization culture of bacteria（BO） exhibit a significant reduction in microalgae lipid accumulation but a notable increase in the chloroplast volume and pigment accumulation compared to control group.

     Furthermore, we also realize that there are some issues with the current microbial embedding device: potential operational risks associated with high voltage (110v), long delivery pipelines (resulting in certain wastage of embedding materials), and relatively complex operational logic requirements. Based on these feedback, our experimental team intends to implement iterative updates addressing these concerns.

Design:

     The Design Cycle 1 revealed that different species exhibit varying efficiencies in nutrient absorption, such as nitrogen and phosphorus. Consequently, the utilization of bacteria-microalgae interaction for water remediation may not yield the desired outcomes.

     In order to fully leverage the beneficial characteristics of bacteria-microalgae interaction, while also addressing the rapid absorption and utilization requirements for elements like nitrogen and phosphorus in aquatic environments. To achieve this, we intend to employ synthetic biology techniques starting from microalgae for modification.

Build:

     Base on the previous research on enhancing the capacity and lipid accumulation of microalgae, we have cloned and constructed and overexpression systems for three isozymes of fucosidase (BBa_K4688002 to BBa_K4688004) in the genome of P. tricornutum，which were subsequently labeled with fluorescence for convenient localization.

Figure 8 Schematic diagram of engineering strain construction and transformation process

Test:

     By designing specific primers, we successfully amplified and cloned BBa_K4688002 to BB_K4688004. Subsequently, utilizing the infusion method, we ligated the obtained fragments into the expression vector provided by the 2022 CHINA-FAFU team. Finally, microalgae strains were validated at the DNA, RNA, and Protein levels.

Figure 9 Cloning of the target gene

Figure 10 Colony PCR validation of the overexpression system constructed in microalgae

（PtFUCO1 (A),PtFUCO2 (B), PtFUCO3 (C)overexpression vectors）

Learn:

     In the construction of engineered microalgae strains, the electroporation introduces some instability in the transforming efficiency, which will be further optimized to enhance transformation. Fortunately, we have successfully obtained an overexpressed engineered microalgae strain for one of the fucosidases.

Design:

     Based on the engineered microalgae strains obtained in Design Cycle 3 and relevant conclusions combined with Design Cycles 1-2, we evaluated the physiological and biochemical indicators of FUCO3 microalgae strain.

Build:

     We conducted experiments to evaluate the environmental remediation performance of individual engineered microalgae strains and bacteria-microalgae interaction system comprising these engineered microalgae strains in conventional culture media or artificially simulated wastewater. Furthermore, we employed microbial embedding device v2.0 to facilitate the execution of these experiments.

Figure 11 Experimental schematic diagram for testing themediation performance of engineered microalgae

Figure 12 Microbial Embedding Device v2.0

Test:

     After testing, we observed significant enhancements in the nitrogen removal efficiency, lipid accumulation and photosynthetic efficiency of the engineered microalgae strains when cultivated individually or in the bacteria-microalgae interaction system. Furthermore, the improved microbial embedding device 2.0 exhibited enhanced safety (12v), more streamlined pipeline design, and more user-friendly operational logic during experimentation, thereby providing favorable hardware support for microbial immobilization.

Learn:

     Due to the exceptional characteristics of the engineered strain of microalgae, we aim to further elucidate the molecular mechanisms underlying algal lipid accumulation, nutrient element utilization (specifically nitrogen and phosphorus), as well as fucoxanthin accumulation mediated by fucosidase. Moreover, we intend to explore its potential commercial value.

Design:

     Given the outstanding performance of BBa_k4112013 and BBa_k4112014 in microalgae bioremediation utilizing which designed by our former CHINA-FAFU team, we aim to further explore the potential bioremediation characteristics achievable by integrating our previous research with the ongoing development of engineered microalgae and bacteria-microalgae interaction system.

Build:

     We intend to conduct comprehensive validation of the transfer of BBa_k4112013 and BBa_k4112014 vectors into appropriate engineered microalgae strains during Design Cycle 3. Furthermore, we enhance the bioremediation strategies by integrating these engineered microalgae strains into a bacteria-microalgae interaction system.

Test:

     We have completed the transformation of engineering microalgae strains at present, but due to the time-consuming growth cycle and identification process of microalgae, we have not yet achieved positive outcomes. Nevertheless, our research in this field will persist.

     In this prestigious competition, our team meticulously selected a topic aligned with our interests and skillfully components along with standard molecular to construct and rigorously test a sophisticated biological system.

     First and foremost, we would like to express my heartfelt gratitude to the organizing committee and the competition platform for providing us with this exceptional opportunity to showcase our innovative achievements and research capabilities.Throughout the entire process, we diligently adhere to the iterative “Design-Build-Learn cycle” by continuously testing, and learning. This approach guided us inrefiningour project through a systematic and iterative refinement process.

     Secondly, we extend our sincere appreciation to our esteemed professors and scholars who have been instrumental in guiding us on this journey. Their invaluable advice and feedback have enabled us to achieve breakthroughs both technically and theoretically. Their expertise and extensive experience have profoundly influenced our learning and growth.

     Lastly, we want to convey our deep appreciation for our remarkable team members who have proven themselves as outstanding collaborators. We worked together cohesively during the competition, supporting each other's progress every step of the way. This competition not only enhanced our research abilities but also improved our teamwork skills and communication prowess. We gained a wealth of knowledge and experience while forging many new friendships along this incredible journey.

     Overall, we attribute our accomplishments in the iGEM competition to both its inherent excellence as well as the exceptional support rendered by our guiding professors and scholars who enabled us to effectively navigate through every stage of the Design Cycle involved in this ambitious endeavor.

     If you want to learn more about other aspects of our engineering journey have a look at our Hardware and Results page!