Difference between revisions of "Part:BBa K3560004"

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PGroES-DrRBS-gcd

The gcd gene of Escherichia coli expresses glucose dehydrogenase (GDH), GDH is a membrane-bound quinone protein that catalyzes the conversion of glucose to gluconic acid, generates hydrogen protons, and decrease the pH value. but it needs to be combined with Pyrroloquinoline quinone (PQQ) coenzyme, then the holoenzyme can function normally. Deinococcus radiodurans constitutively expresses PQQ, which can form holoenzymes with GDH from E. coli to catalyze the production of gluconic acid.

Sequence and Features

Introduction

We successfully verified three important phosphate dissolution systems in the project. Phosphate dissolving system shows considerable ability to dissolve phosphate. As time goes by, the pH value of the solution gradually decreases, and the dissolving range of phosphate becomes larger. In addition, the Phosphate dissolving Plus system also shows a more efficient ability to dissolve phosphate. As for the GDH-Pro system, we strengthen the expression of GDH, increase the effective concentration of enzymes, and promote the dissolution of phosphate. The functions of the above-mentioned systems are all realized in Deinococcus radiodurans(DR), which meets the needs of soil modification on Mars.

Experiment and Results

Phosphate dissolving system in Deinococcus radiodurans As mentioned in our DESIGN page, Phosphate dissolving system has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (figure 1). In order to realize the function of gene circuit, we construct the following plasmid gcd-pRADK (figure 2). And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (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.

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

Figure 2. Design of phosphate dissolving plasmid (gcd-pRADK).

Figure 3. ElectropHoresis of plasmid gcd-pRADK with enzyme digestions.

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.

Figure 4. Changes in pH value of TGY liquid medium culturing D.r R1 and D.r containing gcd-pRADK respectively.

Figure 5. Phosphate ring in PKO solid medium culturing D.r R1 and D.r containing gcd-pRADK respectively. Left: D.r R1; Right: D.r containing gcd-pRADK.

Conclusion

These results indicate that we successfully constructed a Phosphate dissolving system in DR. Compared with DR R1, DR containing gcd-Pradk can significantly decrease the pH value of the solution and dissolve the phosphate in the medium, which proves that the modified DR has the potential to transform the Martian soil.

Reference

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.