Part:BBa_K5115038

ribozyme connected: MTA, Hpn, RcnR_C35L

Introduction

This composite part combines BBa_K5115035(ribozyme+RBS+MTA+stem-loop), BBa_K5115036(ribozyme+RBS+hpn+stem-loop)and BBa_K5115033(ribozyme+RBS+RcnR_C35L+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 MTA, hpn, RcnR_C35L at similar level. MTA is a protein that can bind with nickel ions to reduce its toxicity to the E.coli. The hpn is a protein that can sequester metals that accumulate internally to reduce nickel's toxicity to the E.coli. RcnR_C35L can regulate the nickel ion channel proteins in the cell membrane to tune the nickel ion transport rate.

Usage and Biology

This part is eventually chosen as a component of mineral nickel module, tuning the nickel ion transport rate and reducing nickel's toxicity to the E.coli.

Characterization

Figure 1. 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, leaky expression, no IPTG induction). 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 at 37°C with a rotating speed at 220 rpm. With out parts for Ni²⁺ uptake, there was no significant difference in the efficiency of nickel absorption between the modified E. coli and control.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 935
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 198
  
• 1000
  COMPATIBLE WITH RFC[1000]