Part:BBa_K5115082

ribozyme connected nik operon

This composite part combines BBa_K5115077(ribozyme+RBS+nikA+stem-loop), BBa_K5115078(ribozyme+RBS+nikB+stem-loop),BBa_K5115079(ribozyme+RBS+nikC+stem-loop), BBa_K5115080(ribozyme+RBS+nikD+stem-loop)and BBa_K5115081(ribozyme+RBS+nikE+stem-loop). We introduced this ribozyme-assisted polycistronic co-expression system from 2022. By inserting ribozyme sequences between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.

With this design, we achieve co-expression of BBa_K5115077(ribozyme+RBS+nikA+stem-loop), BBa_K5115078(ribozyme+RBS+nikB+stem-loop),BBa_K5115079(ribozyme+RBS+nikC+stem-loop), BBa_K5115080(ribozyme+RBS+nikD+stem-loop)and BBa_K5115081(ribozyme+RBS+nikE+stem-loop) at similar level.

The ribozyme-connected nikABCDE operon we use facilitates the import of nickel ions, essential for sustaining Ni/Fe hydrogenases in E. coli under anaerobic conditions, where nickel is required for hydrogenase activity. The NikABCDE transporter consists of five proteins that work together to import nickel from the environment, despite its low natural availability ^[1]. In our design, the ribozyme linkage aids in fine-tuning the expression of the nik operon, ensuring efficient nickel uptake for cellular processes.

Usage and Biology

The ribozyme-connected nikABCDE operon encodes a high-affinity nickel transport system in Escherichia coli that is essential for the activation of nickel-dependent enzymes like hydrogenases. The operon consists of five genes: nikA, nikB, nikC, nikD, and nikE. NikA functions as the periplasmic nickel-binding protein, while nikB and nikC form the membrane channel that transports nickel ions across the inner membrane. NikD and nikE provide energy through ATP hydrolysis to drive nickel transport.

This transport system ensures efficient nickel uptake, which is crucial for hydrogenase activity, especially under low-nickel conditions. Mutations in the nik genes disrupt hydrogenase activity due to impaired nickel transport ^[2].

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from one E. coli (DH5α) colony.

M: DNA Marker; Lanes 1-5: Corresponding bands for nikB, nikE, nikD, nikC, and nikA (pointed by red arrowheads), demonstrating successful assembly and integrity of the ribozyme-connected nikABCDE as designed. Primers for these PCR are listed on https://2024.igem.org/fudan/parts."

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 2256
Illegal AgeI site found at 5432
1000
COMPATIBLE WITH RFC[1000]

References

↑ Dosanjh, N. S., & Michel, S. L. (2006). Microbial nickel metalloregulation: NikRs for nickel ions. Current opinion in chemical biology, 10(2), 123–130. https://doi.org/10.1016/j.cbpa.2006.02.011
↑ Navarro, C., Wu, L. F., & Mandrand-Berthelot, M. A. (1993). The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Molecular microbiology, 9(6), 1181–1191. https://doi.org/10.1111/j.1365-2958.1993.tb01247.x

[1] Dosanjh, N. S., & Michel, S. L. (2006). Microbial nickel metalloregulation: NikRs for nickel ions. Current opinion in chemical biology, 10(2), 123–130. https://doi.org/10.1016/j.cbpa.2006.02.011

[2] Navarro, C., Wu, L. F., & Mandrand-Berthelot, M. A. (1993). The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Molecular microbiology, 9(6), 1181–1191. https://doi.org/10.1111/j.1365-2958.1993.tb01247.x

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