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 toxity to the E.coli. The Hpn is a protein that can sequester metals that accumulate internally to reduce nickel's toxity 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 toxity to the E.coli.

Characterization

Growth curve of E.coli

Figure 1: Comparison of E. coli Growth curve with and without hpn in 20 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 20 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

Figure 2: Comparison of E. coli Growth curve with and without hpn in 50 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 50 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

Figure 3: Comparison of E. coli Growth curve with and without hpn in 100 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 100 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

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]