Team:XHD-Wuhan-China/Poster

Poster: XHD-Wuhan-China

Mars-PhD: decrease Mars soil pH with Deinococcus radiodurans

Team: XHD-Wuhan-China

Student Team Member

Tianda Fan, Lishuo Jin,Ze Jiang ,Ling Chen,Zimeng Yang,Mutian Yang,JIALU YU,Cui Yuchen Zixin Zhou,Minfei Xiong,xiyiao Qin,Jingyi Hu,Mengzhen Xiao, Hu keer, Dingchen Ding, Zhuoyang Chen

PIs: WenZhang Chi Xu

Instructors: Shun Hong CaiYun Zhang

Advisors: ShiDi Xiao. Ting Geng

Introduction

The goal of our project is to dissolve insoluble phosphates on Mars. Mars-PhD was produced to complete it. It can solubilize phosphate on Mars effectively and improve the content of phosphorus.

The content of phosphorus in plants is second only to nitrogen and potassium, which is very important for the growth and development of plants. Mars contains a large amount of phosphate, the total amount is about 5-10 times the total amount of phosphate on Earth. But the phosphorus on Mars mainly exists in the form of dolomite and chloroapatite, which are insoluble phosphates. To meet the needs of crops growth, it is necessary to release the phosphorus element from insoluble phosphates. Phosphate-dissolving microorganisms are a type of microorganisms that can convert poorly soluble phosphate in the soil into soluble phosphate. it mainly secretes small molecular organic acids (involving Pyrroloquinoline quinone(PQQ) and Glucose dehydrogenase(GDH)) to decrease the pH of the soil, thereby promoting the dissolution of phosphate. However, neither phosphorus-dissolving microorganisms nor Escherichia coli can cope with the harsh environment on Mars.

Deinococcus radiodurans (DR) has special bacterial structure, super DNA repair ability, efficient antioxidant defense system and cell purification system, can survive on Mars. In response to strong radiation on Mars, we consider using DR as biological chassis to construct genetic circuit related to phosphate dissolution, so that it can dissolve insoluble phosphate on Mars. Besides DR can synthesis PQQ by its own. Therefore, we just need to transform the glucose dehydrogenase (gcd) gene into DR. It then can synthesize GDH enzyme which along with PQQ can catalyze the production of gluconic acid, decrease the pH and solubilize phosphate. In addition, in order to improve the efficiency of dissolution, we also transformed the synthesized gabY gene into DR to enhance the binding efficiency of GDH and PQQ and increase the activity of GDH. Our team hopes to provide new solutions to transform the Martian soil and explore the possibility of growing crops on Mars.

Inspiration

Exploration of Mars has never been stopped because Mars seems the only possible plan B for human habitation within the solar system. And 2020 is the unusual year of Mars exploration, many countries have launched Mars rovers to conduct exploration missions to Mars. One of the important tasks is to explore the possibility of humans migrating to Mars, and whether humans can be self-sufficient on Mars with only a small amount of supplies. On Mars, agricultural crops are the main source of food, so the normal growth of plants is crucial. But the phosphorus on Mars mainly exists in the form of dolomite and chloroapatite, which are insoluble phosphates. We hope that the soils on Mars could be used for crop production. Our project is a trial to promote the dissolution of mineral phosphate in Martian soil and make it suitable for farming.

We used Deinococcus radiodurans (DR) as biological chassis to construct genetic circuit related to phosphate dissolution, so that it can dissolve insoluble phosphate on Mars. Besides DR can synthesis PQQ by its own. Therefore, we just need to transform the glucose dehydrogenase (gcd) gene into DR. It then can synthesize GDH enzyme which along with PQQ can catalyze the production of gluconic acid, decrease the pH and solubilize phosphate.

Design

The Martian environment is extremely harsh, so we use Deinococcus radiodurans (DR) as the biological chassis. In view of the insoluble phosphate of Martian soil, we designed phosphate dissolution systems to dissolve phosphate to meet the needs of plants growth. The DR R1 used in the experiment has amazing stress resistance and a relatively mature gene expression system. Using the shuttle plasmid pRADK, the exogenous gcd and gabY genes can be transferred into DR and successfully expressed to complete the phosphate dissolution systems.

Deinococcus radiodurans

DR has superb radiation resistance and resistance to various extreme environments. It is the most likely known bacteria to survive on Mars. DR has special cell wall structure (6-layer cell walls structure, thick peptidoglycan layer); unique nucleoid structure (very compact nucleoid, multiple copies); efficient DNA damage repair mechanism; super strong antioxidant system; cell purification system. These unusual characteristics make DR the best biological chassis for this project, and it was transformed into a bioreactor.

Figure 1. The phenotype of Deinococcus radiodurans.

Phosphate dissolving systems

We design three phosphate dissolving systems (Phosphate dissolving system, Phosphate dissolving Plus system, GDH-Pro system) in DR. These three systems have different degrees of phosphate dissolving capabilities, and can deal with different levels of soil of insoluble phosphate. After transforming these systems separately to DR, glucose is catalyzed to gluconic acid, pH is decreased, and the phosphorus is released.

Implementation

In this project, we successfully constructed three phosphate dissolving systems with different efficiencies: Phosphate dissolving system, Phosphate dissolving Plus system, and GDH-Pro system, and verified the function of three phosphate dissolving systems to decrease pH and dissolve phosphate. Deinococcus radiodurans (DR) with function of dissolving phosphate, can be applied to the following aspects:

Transformation of Martian soil

Phosphates on Mars belong to insoluble phosphates, which is not easy to be directly absorbed and utilized by plants. In order to meet the growth needs of crops, phosphorus must be released from insoluble phosphorus minerals. However, ordinary microorganisms can’t adapt to the harsh environment of Mars. Our modified DR can survive in the outdoor soil of Mars, directly transform the surface soil of Mars, so that the absorbable phosphorus content can meet the needs of normal plant growth, and can be applied to Mars exploration and migration.

New chassis organism and expression vector for iGEM

In the past years, many teams had plans to transform the Martian soil. However, the chassis organisms they used were all Escherichia coli, and the actual survival chance on Mars was very low. The DR we used is the most promising species to survive on Mars, and has the strongest reliability in the actual application scenarios of soil modification on Mars. At the same time, we introduced DR into the iGEM species library for the first time, and provided mature gene expression systems, providing an important solution for environmental modification in extreme environment.

As a supplement to phosphate fertilizer

Phosphorus is an essential mineral element for plant growth, and the content of phosphorus in soil that can be directly absorbed and utilized by plants is very low. Most of phosphorus exists in the form of insoluble inorganic phosphorus. However, the large use of phosphate fertilizer is easy to cause serious pollution to the environment. Therefore, bioavailability of soil insoluble phosphorus has been a hot topic for researchers for a long time. It is a preferred method to release insoluble phosphorus from soil by microorganisms with phosphate solubilizing effect, so as to reduce the input of phosphate fertilizer and enrich phosphorus in soil.

Engineering

We use Deinococcus radiodurans (DR) as the biological chassis. In view of the insoluble phosphate of Martian soil, we engineered phosphate dissolution systems to dissolve phosphate to meet the needs of plants growth. The DR R1 used in the experiment has amazing stress resistance and a relatively mature gene expression system. Using the shuttle plasmid pRADK, the exogenous gcd and gabY genes can be transferred into DR and successfully expressed to complete the phosphate dissolution systems.

Phosphate dissolving system

The gcd gene of Escherichia coli expresses GDH, GDH is a membrane-bound quinone protein that catalyzes the conversion of glucose to gluconic acid, generating hydrogen protons, and decreasing the pH value. but it needs to be combined with Pyrroloquinoline quinone (PQQ) coenzyme, then the holoenzyme can function normally. DR constitutively synthesize PQQ, which can form holoenzymes with GDH from E. coli to catalyze the production of gluconic acid. Therefore, we recombined the gcd gene from E. coli and the DR shuttle plasmid pRADK to form a gcd-pRADK recombinant plasmid. Then, we transform it into DR, so that the successfully transformed DR can realize the combination of GDH and PQQ and catalyze the conversion of glucose to gluconic acid.

Figure 1. Constitution of PGroES-RBS-gcd gene circuit

Phosphate dissolving system

gabY promotes the combination of PQQ coenzyme and GDH, increases the effective concentration of the holoenzymes, and enhances its catalytic efficiency. We know the sequence of the gabY gene, and synthesized the gabY gene fragment through a biological company. Then the gabY fragment was recombined with pRADK to form gabY-pRADK. Then we transform it into DR containing gcd-pRADK plasmids, so that DR containing gabY-pRADK and gcd-pRADK plasmids can decrease the pH to a greater extent and promote the dissolution of phosphate.

Figure 2. Constitution of PGroES-RBS-gabY gene circuits.

GDH-Pro system

RiboJ is a self-cleaving ribozyme that can remove the UTR sequence at the upstream 5'end. RiboJ has 75 nt, which comprises the sTRSV-ribozyme which is used to cut the 5'-UTR sequence in the promoter with an additional 23-nt hairpin immediately downstream to help expose the RBS. We constructed the PGroES-Riboj-RBS-gcd gene circuit: connect RiboJ to the downstream sequence of the promoter PGroES to enhance the expression of downstream gcd genes, increase the content of GDH, and promote the dissolution of phosphate.

Figure 3. Constitution of PGroES-Riboj-RBS-gcd gene circuit.

Model

Glucose dehydrogenation and hydrogen ion production

In order to dissolve the insoluble phosphate in the soil, we plan to decrease the pH of the soil, promote the dissolution of phosphate, and release the metal ions in the phosphate to meet the needs of normal plant growth. The purpose of this modeling is to screen out a suitable glucose dehydrogenase (GDH) for experiment which can efficiently complete the conversion of glucose to gluconic acid in low pH environment. It is expected that under the conditions of low pH and substrate concentration, the reaction rate catalyzed by GDH remains high. GDH enzyme with optimal kinetic characteristics is selected from E. coli, Sulfolobus solfataricus, Bacillus megaterium, Lysinibacillus sphaericus, Bacillus amyloliquefaciens.

Figure 1. Reaction of glucose to gluconic acid.

Glucose dehydrogenation reaction is shown in Figure 1, to search the optimal concentration of substrate, the Michaelis-Menten equation was used:

Figure 2. Assumes enzyme(Et) concentration of 0.05 Mm, comparing rate of reaction of glucose dehydrogenation from 5 different species.

GDH from E. coli has the highest reaction rate when the substrate concentration is low. At the same time, the overall reaction rate is high, and its optimal pH is low, so it can have high enzyme activity when hydrogen ion is released in the reaction. In general, it is more appropriate to choose GDH enzyme from E. coli.

In addition, we constructed the Phosphate dissolving system (PGroES-gcd) during the experiment, and measured the pH change of the solution in the TGY liquid medium, as shown in the Figure 4. The experimental results show that the pH value decreased from 7.0 to 4.8 within 12 hours. The reason may be that the effective concentration of GDH holoenzyme in the experiment is low, the consumption of glucose is insufficient. We fit the experimental result data with the theoretical model, and get the theoretical prediction result as shown in the Figure 5.

The result show that after 12 hours, the amount of hydrogen ions generated is relatively stable, which is the same as the experimental result, except that the final content of hydrogen ions is higher than the experimental result. This may be due to glucose also participates in other metabolic processes and will not all be converted into gluconic acid. The experimental results have a guiding role in the revision of the modeling and can improve the theoretical model.

Figure 4. Changes in pH value of TGY liquid medium culturing DR R1 and DR containing gcd-pRADK respectively from experiment. (Left)

Figure 5. Time course kinetic analysis of Glucose dehydrogenation according to experiment result by modeling. (Right)

Discussion

In this model, the GDH enzyme concentration is regarded as a constant amount. However, in practice, GDH can only function with the participation of the pyrroloquinoline quinone (PQQ) Coenzyme, so the level of binding between GDH and PQQ will also affect the reaction rate. The combination of GDH and PQQ may be regulated by gabY, which affects the effective concentration of the enzyme, thereby changing the reaction rate. Moreover, after introducing the above genes into Deinococcus radiodurans, it is not certain that the catalytic effect and reaction rate will be the same as in E. coli.

Collaboration

We team XHD-Wuhan-China have been active in the in-depth collaboration with other teams in the 2020 iGEM competition. We have close contact with XHD-ShanDong-China, HZAU-China, WHU-China and have received a lot of their help in return. These collaborations have played an indispensable role in our path of better completing our other teams’ projects.

HZAU-China Helped us to contact Professor Tian Bing of the school of life sciences of Zhejiang University and asked him to provide us with pRADK, pRADG shuttle plasmids and plasmid profiles for free. This successfully promoted our project implementation.

WHU-China provided contact information of the organization purchasing Deinococcus radiodurans

Then we successfully contacted this institution and purchased Deinococcus radiodurans. They Gave us some suggestions on how to conduct human practices .They Offered a great help in experimental design and safety

In addition, we held a meet up with three teams in Wuhan to share our project plans, experiences and difficulties. These cooperation and exchanges greatly promote the smooth progress of our project.

XHD-ShanDong-China and we are brother teams, and we have a lot of in-depth cooperation throughout the whole season of iGEM competition in 2020. We learn the basic knowledge of synthetic biology together, and we hold weekly project meetings to learn from each other.During the summer experiment, we helped each other in the common basic experimental technology They help us deal with Wiki editing technical problems, provide video editing help and support, etc.

In the process of human practices work, they helped us solve the application problem of wechat.

human Practices

Introduction

What's the meaning of our project? From the beginning of the iGEM preparation, we struggled to find the answer. In order to let society, know about our project, we used many different ways to show our progress and our achievements. Thus, we conducted our human practices, including the survey and expert interviews, field research, to seek the social impact we could create and explore how society affects our projects.

During the expert interview, we gradually adjusted and revised the project design. We first visited Professor Ma, an expert in synthetic biology.

He was very supportive of our project and told us that synthetic biology plays a very important role in the Mars transformation plan, allowing us to clarify the goal of using microorganisms to transform the Martian soil.The subsequent interview with Professor Chen allowed us to clarify the details of the project. She introduced us to the phosphate-solubilizing microbes and the GDH protein expressed by the gcd gene.We then focused on converting insoluble phosphates into soluble phosphates.

Professor Tian Bing and Dr. Dai helped us complete the selection of chassis organisms. When we considered changing the chassis organisms, it was Professor Tian Bin who recommended DR. After our teacher's guidance, we found that DR can synthesize PQQ, this unexpected gain greatly promoted our project.According to our original plan, we started to construct our gene circuit using plasmids commonly used in E. coli (pSB1C3 or pUC19) and transform them into DR, but the result failed. Then they sent us pRADK, pRADG shuttle plasmid and plasmid profiles, which enabled us to successfully construct gene expression systems and promoted our project implementation.

In order to let the public know more about our project and synthetic biology, we have carried out some educational activities. First of all, we conducted a public prestation of synthetic biology for the college students of the Huazhong Agricultural University and then went to Wuhan Britain-China School to deliver prestation for high school students.

For the general public, we also went to Touma International Speech Club to carry out synthetic biology science education and project promotion.

Results

Phosphate dissolving system

In order to realize the function of PGroES-RBS-gcd gene circuit, we construct the plasmid gcd-pRADK. Theoretically, after gcd-pRADK is transformed to DR, DR can secrete gluconic acid, gradually decrease the pH of the solution, and promote the dissolution of phosphate. pH changes are shown in the Figure 1. In addition, phosphate rings are shown in the Figure 2. The results show that DR containing Phosphate dissolving system can decrease pH and dissolve phosphate to a great extent.

Figure 1. Changes in pH value of TGY liquid medium culturing DR R1 and DR containing gcd-pRADK respectively. (Left)

Figure 2. Phosphate ring in PKO solid medium culturing DR R1 and DR containing gcd-pRADK respectively. Left: DR R1; Right: DR containing gcd-pRADK. (Right)

Phosphate dissolving Plus system

We design an enhanced Phosphate dissolving system (Phosphate dissolving Plus system) which has two plasmids: gcd-pRADK, gabY-pRADK. Theoretically, after transforming gabY-pRADK to DR containing gcd-pRADK, the effective concentration of the holoenzyme formed by GDH and PQQ coenzymes will increase, and the catalytic efficiency will be higher. pH changes are shown in the Figure 3. In addition, phosphate rings are shown in the Figure 4. The results show that the Phosphate dissolving Plus system has stronger abilities to decrease pH and dissolve phosphate than the Phosphate dissolving system.

Figure 3. Changes in pH value of TGY liquid medium culturing DR R1, DR containing gcd-pRADK and DR containing pRADK2 respectively. (Left)

Figure 4. Phosphate ring in PKO solid medium culturing DR R1, DR containing gcd-pRADK and DR containing pRADK2 respectively. Left: DR R1; Middle: DR containing gcd-pRADK; Right: DR containing pRADK2. (Right)

GDH-Pro system

We designed a gcd gene expression enhancement system, inserting the RiboJ sequence downstream of the PGroES promoter, and exposing RBS during the translation process to enhance the expression of the gcd gene. We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK, and then transformed it into DR. Theoretically, the DR containing the gcd-pRADK-RiboJ plasmid will express more GDH, will have higher catalytic efficiency, greater decrease in pH, and greater range of phosphate ring. pH changes are shown in the Figure 5. In addition, phosphate rings are shown in the Figure 6. These results show that the GDH-Pro system has stronger abilities to decrease pH and dissolve phosphate than the Phosphate dissolving system.

Figure 5. Changes in pH value of TGY liquid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively. (Left)

Figure 6. Phosphate ring in PKO solid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively. Left: DR containing gcd-pRADK; Right: DR containing gcd-pRADK-RiboJ. (Right)

Conclusion

We have successfully constructed three phosphate dissolving systems (Phosphate dissolving system, Phosphate dissolving Plus system, GDH-Pro system) in DR. These three systems have different degrees of phosphate dissolving capabilities, and can deal with different levels of soil of insoluble phosphate, and the comparison of the effects of the three phosphate dissolution systems is shown in the Figure 7, Phosphate dissolving power is sequentially enhanced. The functions of the three systems are in line with expectations, except that the effects of the Phosphate dissolving Plus system and the GDH-Pro system are similar, indicating that although the method of enhancing the phosphate dissolving ability is different, the phosphate dissolving ability has been enhanced.

DR is most likely to survive on Mars, and our modified DR has the ability to improve soil phosphorus content. Therefore, we can modify the phosphorus content of soil on Mars to meet the needs of plants growth and provide basic conditions for human survival on Mars.

Figure 7. Phosphate dissolving results of Phosphate dissolving system, Phosphate dissolving Plus system, GDH-Pro system. Left 1: Control; Left 2: Phosphate dissolving system; Right 2: Phosphate dissolving Plus system; Right 1: GDH-Pro system.

Acknowledgments

Future work

In this project, we completed most of the experiments. We successfully constructed three phosphate dissolving systems with different efficiencies: Phosphate dissolving system, Phosphate dissolving Plus system, and GDH-Pro system, and verified the function of three phosphate dissolving systems to decrease pH and dissolve phosphate, we also completed evaluation and comparison of various systems. However, we have not yet verified whether the DR with a phosphate dissolving system still has amazing resistance to radiation and various extreme environments, which determines whether our modified DR will survive on Mars.

In the future, we will use DR R1 and modified DR to test the resistance in harsh environments such as radiation exposure, high temperature environment and low temperature environment switching. Ensure that the transformed DR has the same resistance to radiation and various extreme environments, and can adapt to the harsh environment of Mars. Cultivating the modified DR in the Martian soil is the prerequisite for transforming the Martian soil, so being familiar with the composition of the Martian soil and simulating the Martian soil is an essential preliminary preparation. Later, we will also test whether the modified DR can grow normally in the simulated Martian soil.

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Acknowledgments

Thanks and acknowledgement to all the people and organizations who have supported and helped us during this iGEM season.