Composite

Part:BBa_K4413993

Designed by: Grace(Ziyu) Xu   Group: iGEM22_Bioplus-China   (2022-10-05)
Revision as of 23:20, 8 October 2022 by Lavenderxc (Talk | contribs)

PGroES-RiboJ-DrRBS-gabY

gabY is the gene used for promotes the combination of PQQ coenzyme and GDH, increases the effective concentration of the holoenzymes, enhances GDH’s catalytic efficiency, and we insert RiboJ between promoter and RBS to increase Translation efficiency.With the over-expression of gabY, it can decrease the pH to a greater extent and promote the dissolution of phosphate.

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. plasmid contenting PGroES-Riboj-RBS-gabY gene circuit was transformed into Deinococcus radiodurans. Enhance the expression of downstream gabY genes, increase the content of GabY, and promote the dissolution of phosphate.

Introduction

BBa_K3560005 contains RBS and gabY genes. We plan to improve this part, and constructed the improved part (BBa_K4413993). We inserted a RiboJ sequence in the upstream of gabY and downstream of the PGroES promoter. In addition, we described the functions of this part in Deinococcus radiodurans (DR), including the growth curve and the ability to decrease pH and dissolve phosphate.


Experiment and Results

We designed a gabY 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 gabY gene. The composition part consist of PGroES(BBa_K3560002), RiboJ(BBa_K2615996),RBS(BBa_K3560003), gabY(BBa_ K3560001), TT(BBa_B0015)(Figure 1). The target fragment was synthesized by a biological company, and then we double enzyme digestion recombine plasmid with the linearized pRADK plasmid.We successfully recombined the gabY-pRADK-RiboJ plasmid (Figure 2), and then transformed it into DR. And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (Figure 3) and PCR(Figure 4). Theoretically, the DR containing the gabY-pRADK-RiboJ plasmid will express more GabY, will have a higher catalytic efficiency, a greater decrease in pH, and a greater range of phosphate ring.

Figure 1. Constitution of PGroES-RiboJ-RBS-gabY gene circuits.
Figure 2. Design of gabY-pRADK-RiboJ plasmid.
Figure 3. ElectropHoresis of plasmid gabY-pRADK-RiboJ with enzyme digestions.
Figure 4. ElectropHoresis of plasmid gabY-pRADK-RiboJ with PCR.


We cultured DR R1, DR containing gabY-pRADK and DR containing gabY-pRADK-RiboJ respectively in TGY liquid medium, and then determined the growth curve. As shown in Figure 5, the plasmids did not significantly affect the growth of DR. Then, we cultured DR containing gabY-pRADK-RiboJ and DR containing gabY-pRADK in TGY liquid medium respectively, and measured the pH. The results are shown in the Figure 6. In addition, the DR containing gabY-pRADK-RiboJ and the DR containing gabY-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 7.

Figure 5. The growth curve of D.r R1, DR (pRADK-gabY) and DR (gabY-pRADK-RiboJ).
Figure 6. Changes in pH value of TGY liquid medium culturing D.rR1, D.r containing gabY-pRADK and D.r containing gabY-pRADK-RiboJ respectively.
Figure 7.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 in DR, the results showed that BBa_( BBa_K4413993) expressed higher GabY content. The above results show that we have successfully constructed the PGroES-Riboj-RBS-gabY gene circuit in DR. Compared with original part , the improved part has faster pH decrease speed and greater range, and has stronger ability to dissolve phosphate.

References

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).

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):824-82


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 476
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 190
    Illegal NgoMIV site found at 390
    Illegal NgoMIV site found at 482
    Illegal NgoMIV site found at 648
  • 1000
    COMPATIBLE WITH RFC[1000]


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