Part:BBa_K5115040

ribozyme connected: RcnR_C35L, MTA, Hpn

Introduction

This composite part combines BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop), BBa_K5115035(ribozyme+RBS+MTA+stem-loop) and BBa_K5115036(ribozyme+RBS+Hpn+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

To ascertain which order the three proteins should be assembled for the best results, we create six different parts containing all the sequential possibilities. We finally confirm that the order of the three parts don't affect much on their effect. We choose to use ribozyme connected: MTA, Hpn, RcnR_C35L at last.

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 303
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 582
  
• 1000
  COMPATIBLE WITH RFC[1000]