Improvement
Improved part design
We improved part PGroES-DrRBS-gabY (BBa_K3560005)
We improved part BBa_K3560005, which consist of RBS and gabY gene. In order to further enhance the solubility of phosphate,
based on the existing part BBa_K3560005, we construct a new composition part(BBa_K4413993). The composition part consist of PGroES(BBa_K3560002),
RiboJ(BBa_K2615996),RBS(BBa_K3560003), gabY(BBa_ K3560001), TT(BBa_B0015).
Insert 75-nt RiboJ into downstream of PGroES and expose downstream RBS to improve the translation efficiency of gabY.
Construction of gabY-pRADK-RiboJ expression system
The RiboJ sequence and gabY were synthesized by a biological company, and then double enzyme digestion recombine plasmid with the linearized pRADK plasmid. We successfully recombined the gabY-pRADK-RiboJ plasmid(Figure 1) and then transformed it into Deinococcus radiodurans(DR). The correctness of the obtained recombinant plasmid was identified by restriction enzyme digestion (Figure 2) and PCR (Figure 3).
Figure 1. Design of gabY-pRADK-RiboJ plasmid.
Figure 2. ElectropHoresis of plasmid gabY-pRADK-RiboJ with enzyme digestions.
Figure 3. ElectropHoresis of plasmid gabY-pRADK-RiboJ with PCR.
Growth curve of DRs
The growth curves of three strains of DR : D.r R1, DR (pRADK-gabY), and DR (gabY-pRADK-RiboJ) were determined within 48 hours. The above strains were cultured using TGY liquid medium and the OD600 of the bacterial suspension was determined by spectrophotometer; The results showed that the plasmids did not significantly affect the growth of DR(Figure 4).
Figure 4. The growth curve of D.r R1, DR (pRADK-gabY) and DR (gabY-pRADK-RiboJ).
Verification of the pH decrease function
gabY promotes the combination of PQQ coenzyme and Glucose dehydrogenase (GDH), increases the effective concentration of the holoenzymes, and enhances its catalytic efficiency. 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 concentration of GabY. Compared with PGroES-gabY, DR containing gabY-pRADK-RiboJ decrease the pH at a greater speed and range (Figure 5).
Figure 5. Changes in pH value of TGY liquid medium culturing D.r R1, D.r containing gabY-pRADK and D.r containing gabY-pRADK-RiboJ respectively.
Application to dissolve phosphate
Because PGroES-RiboJ-gabY can decrease the pH of the solution, we cultured DR containing gabY-pRADK-RiboJ or DR containing gabY-pRADK in PKO solid to observe the size of the phosphate-dissolving ring to evaluate the ability of DR to dissolve phosphate. As shown in the figure, the results show that DR containing gabY-pRADK-RiboJ has a stronger ability to dissolve phosphate (Figure 6).
Figure 6. Phosphate ring in PKO solid medium culturing D.r R1(left), D.r containing gabY-pRADK(middle) and D.r containing gabY-pRADK-RiboJ(right) respectively.
Conclusion
We improved BBa_K3560005 with RiboJ sequence, and determined the growth curve of DR. Our results demonstrated that the function of PGroES-RiboJ-gabY part has been improved with higher activity than original part, can express more GabY, improve the efficiency of dissolving phosphate.