Team:XHD-Wuhan-China/Design
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 system
Through mathematical modeling and reference data, we simulated the activity of GDH enzyme from 5 species, and finally screened out the glucose dehydrogenase (GDH) most suitable for this project, which come from E. coli. Details in Model
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.
Figure 2. Reaction of glucose to gluconic acid.
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 3. Design of phosphate dissolving plasmid.
Phosphate dissolving Plus 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 4. Design of phosphate dissolving Plus plasmid.
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 5. Secondary structure of RiboJ.
Figure 6. Constitution of PGroES-Riboj-RBS-gcd gene circuit.
Figure 7. Design of GDH-Pro plasmid.
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.
Babu-Khan S , Yeo T C , Martin W L , et al. Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia.[J]. Applied & Environmental Microbiology, 1995, 61(3):972-978.
Xiao-Fei C , Meng N , Fei F , et al. Research Progress on the Radiation-resistant Mechanisms of Deinococcus radiodurans[J]. Biotechnology Bulletin, 2017.
Khairnar N P , Misra H S , Apte S K . Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization.[J]. Biochemical & Biophysical Research Communications, 2003, 312(2):303-308.
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).
