Team:XHD-Wuhan-China/Improvement
Improvement
Improved part design
We improved part BBa_K2717007, which consist of RBS and gcd gene. In order to further enhance the express of GDH, based on the existing part BBa_K2717007, we construct a new composition part BBa_K3560006. Insert 75-nt RiboJ into downstream of PGroES and expose downstream RBS to improve the translation efficiency of gcd.
Construction of gcd-pRADK-RiboJ
We cloned gcd gene sequence from E. coli MG1655, synthesized the RiboJ sequence by a biological company, and then homologously recombined with the linearized pRADK plasmid (Figure 1). We successfully recombined the gcd-pRADK-RiboJ plasmid and then transformed it into Deinococcus radiodurans (DR). The correctness of the obtained recombinant plasmid was identified by restriction enzyme digestion(Figure 2).
Figure 1. Design of GDH-Pro plasmid (gcd-pRADK-RiboJ).
Figure 2. ElectropHoresis of plasmid gcd-pRADK-RiboJ with enzyme digestions.
Growth curve influenced by GDH
We cultured R.r R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively in TGY liquid medium, and then determined the growth curve. As shown in Figure 3, DR with high GDH expression has faster growth rate.
Figure 3. The growth curve of R.r R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ.
Application to decrease pH
Glucose dehydrogenase (GDH) can catalyze the conversion of glucose to glucic acid, decrease the pH of the solution, and promote the dissolution of phosphate. In addition, our improved part can increase the effective concentration of GDH. Compared with PGroES-gcd, DR containing gcd-pRADK-RiboJ decrease the pH at a greater speed and range (Figure 4).
Figure 4. Changes in pH value of TGY liquid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively.
Application to dissolve phosphate
Because PGroES-RiboJ-gcd can decrease the pH of the solution, we cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in PKO solid to observe the size of the phosphate-dissolving ring, evaluating the ability of DR to dissolve phosphate. As shown in the figure, the result show that DR containing gcd-pRADK-RiboJ has a stronger ability to dissolve phosphate (Figure 5).
Figure 5. 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 improved BBa_K2717007 with PGroES promoter and RiboJ sequence, and determined the growth curve in DR, the results showed that BBa_K3560006 expressed higher GDH content, and DR containing this part grew faster. And the above results show that we have successfully constructed the GDH-Pro system in DR. Compared with Phosphate dissolution systems, the GDH-Pro system has faster pH decrease speed and greater range, and has stronger ability to dissolve phosphate.
Our results demonstrated that the function of PGroES-RiboJ-gcd part has been improved with higher activity than original part, which can express more GDH, improve the efficiency of dissolving phosphate, and it is more suitable for the transformation of soil.
