Team:XHD-Wuhan-China/Proof Of Concept
Proof of Concept
Project and design
In view of the insoluble phosphate of Martian soil, we designed phosphate dissolution systems to dissolve phosphate to meet the needs of plants growth. Considering the harsh environment of Mars, we use Deinococcus radiodurans (DR) as biological chassis, besides DR can synthesis Pyrroloquinoline quinone (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.
Results
Deinococcus radiodurans culture
We cultivated DR in the laboratory, then placed it on a plate and observed the growth process of DR. The results are shown in the Figure 1. In order to further understand the growth characteristics of DR, we need to determine the growth curve of DR. In order to determine the growth curve of DR, DR was cultured in TGY liquid medium at 28°C and 200rpm in a shaker, and the OD600 value of the bacterial solution was measured by spectrophotometer. The growth curve of DR is shown in the Figure 2. Compared with E. coli, DR has a longer growth cycle and takes a long time to culture.
Figure 1. DR streak separation and culture.
Figure 2. The growth curve of DR.
Phosphate dissolving system
As mentioned in our DESIGN page, Phosphate dissolving system has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015). In order to realize the function of gene circuit, we construct the following plasmid gcd-pRADK (Figure 3). 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. The DR containing gcd-pRADK and wild-type DR were cultured in TGY liquid medium, and the pH was measured every 1 hour, the results are shown in the Figure 4. In addition, DR containing gcd-pRADK and wild-type DR were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 5. The results show that DR containing Phosphate dissolving system can decrease pH and dissolve phosphate to a great extent.
Figure 3. Design of phosphate dissolving plasmid (gcd-pRADK).
Figure 4. Changes in pH value of TGY liquid medium culturing DR R1 and DR containing gcd-pRADK respectively.
Figure 5. Phosphate ring in PKO solid medium culturing DR R1 and DR containing gcd-pRADK respectively. Left: DR R1; Right: DR containing gcd-pRADK.
Phosphate dissolving Plus system
We design an enhanced Phosphate dissolving system (Phosphate dissolving Plus system) which has two plasmids：gcd-pRADK, gabY-pRADK. And gabY gene circuit has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gabY(BBa_K3560001), TT(BBa_B0015). In order to realize the function of gene circuit, we construct the following plasmid gabY-pRADK (Figure 6). 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. We cultured DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR in TGY liquid medium, and measured pH every 1 hour. The results are shown in the Figure 7. In addition, DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 8. The results show that the Phosphate dissolving Plus system has stronger abilities to decrease pH and dissolve phosphate than the Phosphate dissolving system.
Figure 6. Design of Phosphate dissolving Plus plasmid (gabY-pRADK).
Figure 7. Changes in pH value of TGY liquid medium culturing DR R1, DR containing gcd-pRADK and DR containing pRADK2 respectively.
Figure 8. 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.
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. The circuit has parts: PGroES(BBa_K3560002), RiboJ(BBa_K2066535), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015). We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK (Figure 9), 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. We cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in TGY liquid medium respectively, and measured the pH every 1 hour. The results are shown in the Figure 10. In addition, the DR containing gcd-pRADK-RiboJ and the DR containing gcd-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 11. These results show that the GDH-Pro system has stronger abilities to decrease pH and dissolve phosphate than the Phosphate dissolving system.
Figure 9. Design of GDH-Pro plasmid (gcd-pRADK-RiboJ).
Figure 10. Changes in pH value of TGY liquid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively.
Figure 11. 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.
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 12, 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 in the Martian environment, 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 12. 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.
References
Ahemad, Munees. Phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: a review[J]. Biotech, 2015, 5(2):111-121.
Adcock C T , Hausrath E M , Forster P M . Readily available phosphate from minerals in early aqueous environments on Mars[J]. Nature Geoence, 2013, 6(10):824-827.
Clifton K P , Jones E M , Paudel S , et al. The genetic insulator RiboJ increases expression of insulated genes[J]. Journal of Biological Engineering, 2018, 12(1).
