Difference between revisions of "Part:BBa K5115082"

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ribozyme connected nik operon

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]. The ribozyme-assisted polycistronic co-expression system can ensure that each cistron can initiate translation with comparable efficiency. For more information, please check part wiki of 2022 Fudan iGEM.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.

M: DNA Marker; Lanes 1-5: Corresponding bands for nikB, nikE, nikD, nikC, and nikA, demonstrating successful assembly and integrity of the ribozyme-connected nikABCDE operon as designed.

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

[1]

[2]

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 | '''Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.
-M: DNA Marker; Lanes 1-5: Corresponding bands for nikB, nikE, nikD, nikC, and nikA, demonstrating successful assembly and integrity of the ribozyme-connected nikABCDE operon as designed.
+M: DNA Marker; Lanes 1-5: Corresponding bands for ''nikB'', ''nikE'', ''nikD'', ''nikC'', and ''nikA'', demonstrating successful assembly and integrity of the ribozyme-connected nikABCDE operon as designed.
 '''