Part:BBa_K5115082

ribozyme connected nik operon

Introduction

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 . 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 .

Characterization

Agarose gel electrophoresis

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

Figure 2. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 20 mg/L Ni²⁺. The graph shows the percentage of Ni²⁺ absorbed by E. coli expressing different constructs after 5 hours of growth in a medium containing 20 mg/L Ni²⁺ (E. coli strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 20 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0306, Dunnett’s post-test).

Figure 3. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 30 mg/L Ni²⁺. The graph shows the percentage of Ni²⁺ absorbed by E. coli expressing different constructs after 5 hours of growth in a medium containing 30 mg/L Ni²⁺ (E. coli strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 30 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0052, Dunnett’s post-test).

Figure 4. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 50 mg/L Ni²⁺. The graph shows the percentage of Ni²⁺ absorbed by E. coli expressing different constructs after 5 hours of growth in a medium containing 50 mg/L Ni²⁺ (E. coli strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 50 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0020, Dunnett’s post-test).

Sequence and Features

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]