Part:BBa_K3560006
PGroES-RiboJ-DrRBS-gcd
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-gcd gene circuit was transformed into Deinococcus radiodurans. Enhance the expression of downstream gcd genes, increase the content of GDH, and promote the dissolution of phosphate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 190
Illegal AgeI site found at 1880 - 1000COMPATIBLE WITH RFC[1000]
Introduction
BBa_K2717007 contains RBS and gcd genes. We plan to improve this part, and constructed the improved part BBa_K3560006. We inserted a RiboJ sequence in the upstream of gcd 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
GDH-Pro system
We designed a gcd 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 gcd gene. The circuit has four parts: PGroES(BBa_K3560002), RiboJ(BBa_K2066535), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (Figure 1). We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK (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). Theoretically, the DR containing the gcd-pRADK-RiboJ plasmid will express more GDH, 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-gcd gene circuits.
Figure 2. Design of GDH-Pro plasmid (gcd-pRADK-RiboJ).
Figure 3. ElectropHoresis of plasmid gcd-pRADK-RiboJ with enzyme digestions.
We cultured DR 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 4, DR with high GDH expression has a faster growth rate. Then, we cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in TGY liquid medium respectively, and measured the pH every 1 hour. The results are shown in the Figure 5. In addition, the DR containing gcd-pRADK-RiboJ and the DR containing gcd-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 6.
Figure 4. The growth curve of DR R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ.
Figure 5. Changes in pH value of TGY liquid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively.
Figure 6. 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, and compared with Phosphate dissolution systems, the GDH-Pro system has a stronger pH decrease speed and range, and has a stronger ability to dissolve phosphate.
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.
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).
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