In UCAS-China's project this year, we use the tools of synthetic biology, we constructed a cell-free enzymatic reaction system for the degradation of caffeine to theacrine, and demonstrated our engineering success. At the same time, we have contributed some brand new parts to the iGEM community and also improved existing ones.
In March 2021, Team UCAS-iGEM conducted a brainstorming session on project options for iGEM 2021. Team members brainstormed and combined their life experiences and synthetic biology knowledge to come up with several novel projects.
Some team members focused on the current big social trend of weight loss and fitness, where low-energy foods are becoming a new demand for people's diets. However, the current meal replacements face the problem of expensive but single taste. Referring to the project of "Food production using microorganisms in interstellar travel" designed by the iGEM team in previous years, we proposed a microbial meal replacement solution called " Gut Treasure ". Gut Treasure micro meal replacement is a new type of meal replacement powder. Bifidobacterium strains that are resistant to oxygen, acid and bile are screened out through directed evolution, whey protein-secreting particles and vitamin-secreting plasmids are introduced into the strains for synthesis. Other nutrients necessary for the human body are introduced into the optogenetic system at the same time, as well as the production of different flavor substances which is controlled by adjusting of the wavelength of light. However, due to the complicated experimental process and the high workload of this project, it was difficult for us to finish it completely in one year. Therefore, with the advice of our PI, we made it an alternative project for the future.
Some team members were inspired by the currently popular foldable phone screens and wanted to develop a microbial-based flexible screen for cell phones. Using the luciferin-luciferase system from bacteria, using oxygen in the air as an oxidant to release chemiluminescence, and using pyocyanin and ferrocyanide as redox intermediates to regulate the metabolism of engineered bacteria The luminous brightness of engineering bacteria can be adjusted horizontally. However, with our subsequent information research, we found that such an idea was really difficult to realize, so we put this project idea on hold.
Based on their life experiences, other students proposed ideas such as “Biosynthetic Spider Silk Protein”, “Formaldehyde Removal”, “Super Toothpaste” and so on. However, most of the projects were shelved or rejected due to poor feasibility or the fact that more mature products are currently available in the market.
Then, at a group meeting, one of our members had too much coffee the night before, which caused insomnia, and he slept through the group meeting the next day. Inspired by this, we decided to explore the effects of caffeine on the human body. Through questionnaires and crowd interviews, we found that caffeine sensitivity is plaguing many people. After literature research and discussions with our instructor, we found the enzyme system that degrades caffeine, and from there, we built a comprehensive system to help users manage their caffeine intake. Our project DeCaffi was born.
From the outset of the project's design, we were committed to changing the world by our work in synthetic biology. There are many people who have trouble sleeping and even have palpitations due to excessive coffee or milk tea consumption. We are keen to facilitate the removal of caffeine from drinks by means of synthetic biology, so that people can drink them more freely without fear of mental hyperactivity.
In the enzymatic reaction we first chose, the caffeine is demethylated and gradually degraded, but unfortunately the reaction produces formaldehyde. This clearly does not meet the requirement of being edible. We then continued our literature research and finally identified the enzymatic pathway that degrades caffeine to theacrine in two steps.
Subsequently, at the Beijing District Meeting in April, there were constructive suggestions on the originality of our project from other iGEM partners. During this process, we gradually revised and clarified our initial ideas, drawing up the blueprint for our project.
During the Design part, we transformed our ideas from theoretical concepts to a feasible project.
First, we list all the molecular biology elements, i.e. enzymes and coenzymes, required to move from caffeine to bitter theophylline. Second, we look for suitable synthetic biology production methods to obtain these elements. Third, we wanted to construct complete reaction pathways and cell-free systems. Finally, we wanted to improve the heat resistance of the cell-free system so that it could function effectively in the coffee environment.
Construction and Verification
With a practical plan and a concrete idea in mind, we actually build our system in the laboratory. We produced the enzymes and coenzymes using synthetic biology methods and used both directed evolution and PROSS prediction to improve heat resistance. We then needed to test the effectiveness of our efforts to improve heat resistance. Due to the effects of the epidemic, our labs were not open the entire time, which resulted in only partial completion of our work in this section.
The following sections describe the design of our project and the progress we have made until now.
Since the plasmids were already designed and ordered from the gene company, we did not need to do the gene extraction and enzymatic cleavage, but simply transformed the plasmids into the prepared receptor E. coli. This saved us time and allowed us to complete part of the work despite the limited lab opening time.
In the experiment, we used E. coli BL21 strain which is suitable for expressing the protein and verified the transformation success using colony PCR and extraction of plasmid for PCR after transferring the plasmid.
Bacteria cultivation & Induction of protein expression
We used a lactose manipulator in the gene line to control the expression of the target protein, so the addition of the commonly used inducer Isopropyl-beta-D-thiogalactopyranoside (IPTG) to the bacterial broth allows the engineered bacteria to start producing our protein of interest. However, the production of such a large molecular weight protein is obviously a very burdensome task for the engineered bacteria and can interfere with their own normal growth and reproduction. We do not want to limit the protein yield by having low numbers of engineered bacteria, so we cannot add IPTG at the initial start of the culture; however, if we add it too late, too much metabolic waste will accumulate in the bacterial broth, which is also detrimental to the production of the target protein. Therefore, we need to find the right time to add the inducer to maximize the yield of the target protein.
Currently, our engineered bacteria have been cultured and confirmed that our target proteins have been produced by protein electrophoresis after cell crushing. These strains will be further expanded and cultured for subsequent protein extraction, directed evolution, etc.
Optimization on traditional methods：SUMO tag
In previous igem projects, the most common method used to purify proteins was the 6-his tag. in this process, a small tag consisting of six histidines is added to the end of the protein so that the protein gains affinity for the Ni column. Subsequently, the target protein can be extracted from the cell fragmentation solution by affinity chromatography.
This method is simple and fast, but has some disadvantages. For one thing, there is a possibility of forming inclusion bodies during the production of target proteins by engineered bacteria, which makes the process of purification tedious; for the other, despite the small size of the 6-his tag, it may still change the spatial structure of the enzyme, thus affecting its activity. Therefore, when designing the plasmid, we tried to find a better purification method.
Finally, we found this ideal method - SUMO tag.
The small ubiquitin-like modifier (SUMO), as a post-translational modification in eukaryotic cells, functions in many cellular processes. But more importantly, it has been shown that SUMO can also be added to the N-terminal end of recombinant proteins as a tag, significantly improving the stability and solubility of recombinant proteins and reducing inclusion body production, thus improving the production efficiency of engineered bacteria. In addition, after the target protein is extracted from the cell fragmentation fluid, the SUMO tag can be cut off completely with the specific protease ULP1, getting a product that is identical to the original protein sequence.
In our project, due to the large molecular weight of the target protein, the yield and activity may be lower when produced using engineered bacteria. Thus, the role of SUMO tag in improving the stability and solubility of recombinant proteins becomes even more significant to us. Therefore, we chose to use SUMO tags and attach them to the N-terminal end of the target protein in the plasmid, despite the fact that it would prolong the protein purification process.
Methods：Well-established process from article
We have found detailed protocols for the purification of proteins using SUMO tags, although it is not commonly used due to the long process. Overall, the purification step is divided into three steps: Ni column affinity chromatography, ULP1 digestion, and again Ni column affinity chromatography.
In the first step, the fusion protein of SUMO and the target protein is bound to the Ni column with the help of the 6-his tag on SUMO and thus separated from the cell fragmentation solution; in the second step, after one day of enzymatic cleavage, the SUMO tag is cleaved off the target protein by the ULP1 enzyme; in the third step, the enzymatically cleaved mixture containing the three components (SUMO, ULP1 and the target protein) is again flowed through the Ni column again. Since both SUMO and ULP1 enzymes contain 6-his tags, they will both be bound to the column, so we only need to look for the target protein in the solution before eluting the protein from the column.
Unfortunately, we have not yet completed the purification of the protein because of the limited opening hours of the laboratory due to anti-epidemic measures. However, with the availability of detailed experimental steps and successful precedents in the reference literature, we are confident that we will complete this work in the near future.
For S-adenosylmethionine (SAM) production, we hope to produce engineering bacteria that can efficiently produce SAM by using the tools of synthetic biology. We chose Pichia pastoris strain GS115 as the engineering strain because it can efficiently produce SAM. According to the literature, the synthesis of Sam by strain GS115 mainly depends on sam1 and sam2 genes. Since the expression of sam2 is not inhibited by the product, it is selected to increase the synthesis of SAM by enhancing the expression of sam2 gene. The enhancement was achieved by introducing exogenous sam2 gene. In addition, SAM can be converted to cysteine under the catalysis of cystathionine-β-synthase (CBS), which will consume SAM and reduce its accumulation. Therefore, we inactivated the CBS gene by inserting a DNA sequence into the CBS gene, so as to inhibit the decomposition of SAM. Finally, we hope to adjust the nutrient addition mode of engineering bacteria in order to explore the best conditions for engineering bacteria to produce SAM and further improve the yield of SAM.
Error-prone PCR is used to increase the frequency of mutations during amplification by adjusting the reaction conditions, such as increasing the concentration of magnesium ions, adding manganese ions, changing the concentration of four dNTPs in the system, or applying low-fidelity DNA polymerase, when DNA polymerase is used for target gene amplification. After that, the DNA sequences containing different mutations will be separately transformed into E. coli to build a mutation library, from which the proteins with enhanced heat resistance can be screened using appropriate high-throughput screening methods.
In our project, we plan to mutate the Cdh gene with library building using error-prone PCR and make it react with caffeine, and measure the reaction rate and enzyme activity by the change in absorbance of coenzyme Q0 before and after the reaction. However, again due to laboratory opening time constraints, this part of the experiment has not yet been started, but the references we found could prove its feasibility.
In our project, we used PROSS algorithm to enhance the thermal stability of CkTcS.
As an algorithm dedicated to calculating and improving protein stability, PROSS is based on phylogeny and energy estimation. Firstly, by analyzing and comparing a large number of homologous sequences, i.e., MSA analysis, we can ﬁnd the conserved sites and co-evolution sites in the protein sequence. According to evolutional theory, the most conservative amino acid tends to stabilize the protein or play an important role in the function of the protein and amino acids that rarely or never appear may damage protein stability or biological functions. There may be disulﬁde bonds, hydrogen bonds and other interactions between co-evolved amino acid sites. These interactions may play a role in maintaining protein conformation. Therefore, by this step, we can screen out harmful mutations and delineate legal mutations.
We plan to enhance our protein Tm to close to 343 K after sequence modification by PROSS. Currently, we have completed the synthesis and transformation of the new plasmid containing the modified CkTcS and await future protein purification and activity validation.
To use the enzyme CkTcs and Cdh efficiently and prevent them from leaking into the coffee, we need to immobilize these enzymes on the de-caffeine filter. In the end we succeeded in immobilising the enzyme on acetate paper and here are the results of our experiments.
Fig1.Acetate paper under activation
Limited to the experiment condition and lab opening policy, we did not obtain the ideal statistics and results. Thus, we have a future plan to make up our project, and get further results. Future plan includes the expression and the purification of protein, the rational design in some specific protein and other considerations.
How to express protein efficiently in E.coli system is an essential step that needs to be broken through. We expressed them in E.coli system this year but we did not obtain the ideal results. By searching the literatures, we found that it is inefficient in prokaryotes for the expression of protein so it is better to achieve this goal in yeast system. Limited to the time and other factors, we did not realize it. Therefore, it is a promising design belonging to our future plan.
The rational design can calculate and simulate the structures of protein so that we can design some specific proteins according to our requests. Pross and AlphaFold2 are useful instruments to finish it. However, we did not know clearly how to use AF2 so we finally choose Pross to predict the sequences and the structures. Our next step is to learn to use AF2 to predict them because AF2 has higher accuracy and better algorithm. Besides, directed revolution is also an effective way to process those unclear proteins which can not be found in any database. In order to achieve this goal, we need to refer to more references and make our future plan.
What’s more, online education platform and offline education should be taken into consideration, which play an indispensable role in the development of our future team members. We try to complete it in order to guarantee our inheritance. All we plan in the future is making iGEM UCAS-China better.
Peroutka Iii, R. J., Orcutt, S. J., Strickler, J. E., & Butt, T. R. (2011). SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes. Methods in molecular biology (Clifton, N.J.), 705, 15–30. https://doi.org/10.1007/978-1-61737-967-3_2
Ulrich H. D. (2009). SUMO Protocols. Preface. Methods in molecular biology (Clifton, N.J.), 497, v–vi. https://doi.org/10.1007/978-1-59745-566-4