Contribution
We are dedicated to the green bioproduction of medium-chain fatty acids and their derivatives. In this year’s iGEM competition, we have made significant efforts and accomplished a lot. We have gained valuable knowledge and experiences, and we hope that all of our work can be helpful and inspiring to future iGEM teams. We have collected all the components of our contributed project. We have successfully completed the preliminary construction of the green synthesis framework for medium-chain fatty acids and their derivatives, laying a foundation for future teams dedicated to this research field. To achieve better industrial production, we have utilized model fitting to optimize the fermentation process and designed hardware solutions to address challenges such as difficulties in oil-water mixing and fermentation product separation, which were not easily obtained in the past. Additionally, we have achieved remarkable achievements in inclusivity. We have collaborated with some teams to initiate the production of synthetic biology picture books, aiming to help autistic children easily access and understand synthetic biology. We hope that more teams will join us in constantly expanding its content in the future.
In our biobricks, we have contributed three enzymes, alkB, alkG, alkT, and the structural protein scaffold SpyTag/SpyCatcher. The alkB, alkG, and alkT enzymes are used for the synthesis of 10-hydroxydecanoic acid. alkB converts decanoic acid into 10-hydroxydecanoic acid, alkT consumes NADH and transfers electrons to alkG, and alkG provides electrons to alkB. GDH is a cofactor involved in the synthesis of 10-hydroxydecanoic acid, and its role is to provide more NADH to alkT. SpyTag/SpyCatcher is a pair of protein scaffolds that can specifically recognize each other, and they can be used to form multi-enzyme complexes. We used SpyTag/SpyCatcher to link alkT with GDH to enhance catalytic efficiency. It is worth mentioning that part BBa_M45424 documents the catalytic reaction using the alkB in the alkane hydroxylase system of Pseudomonas putida GPo1. Based on our experimental results, we have supplemented the optimal conditions for inducing the expression of this protein.
We have re-designed a new red light switch in yeast based on references and previous iGEM teams' designs. This new switch exhibits a higher red light response efficiency. But due to time constraints, we have not been able to verify it yet. However, we hope that it can provide design ideas for future teams.Additionally, considering that the yeast fermentation process results in a turbid broth, which affects the transmission of red light, we conducted modeling to calculate the optimal red light intensity, taking into account the partial weakening of red light. This calculation was done to assist in the experiment, and the results were added to part BBa_K801043.
Additionally, we have designed and validated a new suicide switch called the NeuAc riboswitch. The sequence of the NeuAc riboswitch consists of an RNA aptamer and a hammerhead ribozyme. The principle of action is that NeuAc binding to the RNA aptamer causes a conformational change in the entire switch, leading to self-cleavage of the hammerhead ribozyme, creating a gap. Then, under the action of the ReJ endonuclease in E. coli cells, the downstream linked gene is cleaved, thereby reducing the abundance of mRNA for the downstream expression gene. We chose φX174 as the lysis protein for E. coli suicide.While verifying the NeuAc ribose switch, we measured that approximately 2-3 hours after induction with IPTG, φX174 was able to completely kill Escherichia coli. This provides additional experimental data to validate the functionality of part BBa_K4156083.
Overall, we have made significant contributions to the iGEM project and hope that our work can inspire and benefit future teams.
We have had very long struggles in the wetlab with a few fragments, and for future teams handling with the same parts might find these tips useful:
BBa_K4711026：During the induction of expression for these three proteins, we observed significant differences in their expression levels. However, the RBS we used is already strong for these three proteins. Despite this, it still caused significant variations in protein expression, which can have an impact on the final fermentation catalysis. Due to time constraints, we were unable to redesign the RBS for further experimentation. Therefore, we suggest that future teams intending to use this part should redesign the RBS to ensure minimal differences in expression levels for these three proteins.
BBa_K4711041：During the validation process of this suicide switch, measuring OD values is often required. However, due to the excessive concentration gradients set, the procedure for sampling and measuring OD values becomes cumbersome. Therefore, we recommend using a 96-well plate or a 24-well plate for cultivation and measurement. However, when we actually measured using a 96-well plate, we found that the limited growth space and nutrient conditions in the small wells caused the Escherichia coli growth curve to shorten, leading to cell death within 3-4 hours. Therefore, we adjusted our sampling frequency from once every hour in the first experiment to once every half an hour in the second experiment. As a result, we recommend that future teams use a 24-well plate to ensure sufficient growth environment for Escherichia coli and achieve optimal data measurement.
Through modeling, we have been able to help resolve many issues in the wet lab experiments, such as the intensity of red light and the selection of the linker, among others. This approach has allowed us to avoid setting up additional variables and has resulted in a reduction in experimental time while achieving optimal results. It is worth mentioning that, due to the semi-continuous fermentation approach adopted in this project, we attempted to utilize a mathematical model to simulate the actual fermentation process. This has enabled us to adjust various input values and separation times to achieve maximum yield. This model can serve as a reference for future teams working on fermentation-related projects. Below is the complete flowchart of our model.
Figure 1 Model summary diagram
In industrial production, oil substances often need to be emulsified with other culture media to improve factors such as oxygen solubility and bacterial contact area, ultimately leading to increased productivity. However, current emulsification equipment is often expensive and bulky, and the cost of linking multiple large-scale fermentation tanks is also a concern. To address this, we have designed a unique multiphase flow bioreactor impeller to achieve optimal mixing efficiency.
The conventional separation method for MEL involves using a 1L graduated cylinder for settling and separation, which is inefficient. As a result, we took this opportunity to develop a unique device for the separation of similar organic macromolecules required for our project.
These devices will provide inspiration for future teams dedicated to industrial-scale production.
Figure 2 3D modeling of hardware
During our project, we took various actions, covering different groups, promoting synthetic biology knowledge to a wider public, and promoting our project.
The conventional separation method for MEL involves using a 1L graduated cylinder for settling and separation, which is inefficient. As a result, we took this opportunity to develop a unique device for the separation of similar organic macromolecules required for our project.
Including:
1.Synthetic Biology Picture Book Activity
2.Official account activities
3.Video Science Popularization
4.Stall promotion
5.High school summer camp
We focus on the education of children with autism.Education is a key means to help children with autism integrate into society, but currently the education foundation for children with autism is relatively weak, with a large shortage of teachers and a low level of talent specialization. We continuously improve the format of our picture books through communication with experts in relevant fields to provide better access to synthetic biology for children with autism. We also invite teams from around the world to join us in constantly refining our picture books.
By collaborating with educators, psychologists, language experts, and other professionals, we ensure that our picture books are effective and beneficial in terms of education and cognition. Their expertise and experience are invaluable for improving and developing our picture books.
We actively invite teams and individuals from around the world to participate in our project and share their insights and suggestions. This collaborative approach allows us to gather more creativity and professional knowledge, continuously enhancing the quality and educational effectiveness of our picture books.
Our goal is to provide an engaging, understandable, and interactive learning tool for children with autism, helping them better understand the concepts of synthetic biology and sparking their interest in science. Through continuous improvement and collaboration with global teams, we believe we can make a greater contribution to the education of children with autism.
Figure 3 Images from the scene