Part:BBa_K5115068

mineral, nickle module

Introduction

This part is made up of BBa_K5115038(ribozyme connected: MTA, Hpn, RcnR_C35L) and BBa_K5115086(NixA-F1v) wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. In BBa_K5115038, the three proteins each are wrapped with the same structure.

By inserting BBa_K4765020 before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA . To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS . In 2023, we extensively tested various stem-loops using BBa_K4765129. For parts we made this year, this strong protective stem-loop sequence was used.

As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used T7 RBS, from bacteriophage T7 gene 10 . It is an intermediate strength RBS according to our 2022 results, which allows us to change it to a weaker J6 RBS or a stronger B0 RBS if needed, enabling flexible protein expression levels between various ribozyme connected parts.

With this design, we achieve co-expression of MTA, Hpn, RcnR_C35L and NixA-F1vat similar level.

Hpn is a His-rich putative nickel storage protein plays a crucial role in nickel detoxification. Hpn may sequester metals that accumulate internally via a passive equilibrium mechanism (from a high external metals environment).

MTA, sourced from Pisum sativum, is a cysteine-rich protein known for its high binding affinity for various heavy metals, including nickel. By sequestering excess nickel ions, MTA further reduces the potential cytotoxic effects associated with elevated nickel levels . Hpn BBa_K1151001, derived from Helicobacter pylori, is characterized by its high histidine content. Its structure allows it to exist in various multimeric forms in solution. The primary function of Hpn is to bind nickel ions, with the ability to sequester up to five Ni²⁺ ions per monomer in a pH-dependent manner (optimal at pH 7.4).By binding and storing excess nickel, Hpn prevents harmful interactions between nickel ions and cellular machinery, thereby promoting the survival and functionality of E. coli in nickel-rich conditions . This dual strategy, combining Hpn and MTA, enhances the overall nickel absorptivity of our engineered bacteria while simultaneously minimizing the harmful effects of nickel accumulation.

Figure 1. Hpn works with MTA to alleviate the toxity of nickel against E. coli.

RcnR_C35L BBa_K5115000,a nickel-responsive transcriptional regulator, can optimize nickel uptake by modulating gene expression. RcnR is a tetrameric transcriptional repressor that responds to the binding of Ni(II) ions by releasing DNA, resulting in the expression of RcnA, which is responsible for nickel export from the cell . By inhibiting RcnA, RcnR ensures that intracellular nickel ion concentrations remain elevated, thereby enhancing the effectiveness of our nickel absorption system. As to the nickel transporting protein, we choose to use NixA BBa_K5115071, a monomeric protein which is specifically adapted for nickel transport . In our design, we don't simply introduce NixA into the E. coli. Instead, we linked it with F1v BBa_K5115085, optimizing NixA's dimerization to enhance its transporting ability.

Figure 2. The working principle of RcnR_C35L.

NixA-F1v is F1v linking with NixA's C end. NixA is a high-affinity nickel transporter in helicobacter pylori. It belongs to the NiCoT family of transporters and facilitates the import of Ni²⁺ ions across the bacterial cytoplasmic membrane. F1v is a signal transducer, binding NixA at its C end. The working procedure of F1v need the use of AP20187, which is a small molecule inducers. The addition of AP20187 to live cells expressing a F1v-tagged fusion protein induces self-association of the fusion protein by promoting the interaction of the dimerization domains.

Figure 3. The working procedure of AP20187 and F1v according to the iDimerize™ Inducible Homodimer System User Manual. The F1v is combined with the target protein, which is NixA in our experiment, and the AP20187 can induce self-association of the NixA.

Usage and Biology

This part is designed to raise the absorption of nickel ions and decrease nickel's toxicity to the E. coli, which is the basic working module of our project. This part ensures that the nickel ions in the wastewater can be locked inside the E. coli, laying the foundation for the following processing steps.

Our part demonstrates nickel enrichment capabilities by maximizing nickel ion influx and minimizing nickel efflux. The concept of increasing tolerance and retaining metal ions within the cytosol can be applied to a wide range of other heavy metals. This strategy can be utilized by teams working on heavy metal bioaccumulation projects in the future.

Characterization

Viability test

Before we test the nickel absorption ability of the E. coli, we must make sure that the E. coli can survive in the waste water. Hpn being the major detoxifying part, we launched experiments on E. coli expressing Hpn compared to E. coli without Hpn expression in mediums containing different concentration of nickel ions.

Figure 4: 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. 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 5: 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. 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 6: 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. 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.

Ni absorption experiment

In order to accurately measure the concentration of nickel ions, we adopted the spectrophotometry and used dimethylglyoxime as a color developer, which can specifically bind with Ni. For more details about its protocol, please visit our experiments wiki.

Figure 7. Close-up view of the spectrophotometer we used.

Figure 8. Every centrifuge tube contained a medium waiting to be made spectrophotometric.

Ni absorption with a single part introduced

Multiple proteins are used in our design. We launched independent experiments to test their nickel absorbing ability.

Figure 9. 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 10. 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 11. 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).

Ni absorption with two parts combined

Firstly, we compared whether there was an effect of Hpn on the function of NixA-F1v.

Figure 12. Comparison of Ni²⁺ Uptake Efficiency, with and without Hpn. 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, 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. The results indicate that E. coli expressing Hpn demonstrated higher Ni²⁺ uptake efficiency compared to E. coli without Hpn expression.

Next, we compared whether there was an effect of RcnR_C35L on the function of NixA and nik-ribozyme.

Figure 13. Comparison of Ni²⁺ Uptake Efficiency, with and without RcnR_C35L. 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. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. RcnR_C35L refers to a mutation in which cysteine (C) at position 35 in the RcnR protein was substituted with leucine (L). The results indicate that E. coli expressing RcnR_C35L consistently has higher Ni²⁺ uptake efficiency compared to E. coli without RcnR_C35L expression.

Ni absorption with three parts combined

We combined MTA, Hpn, RcnR_C35L using our ribozyme-assisted polycistronic co-expression system (pRAP). To ascertain which order the three proteins should be assembled for the best results, we created six different parts containing all the sequential possibilities.

Figure 14. 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.

Ni absorption with four parts combined

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Figure 15. 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 for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. All constructs increase Ni²⁺ uptake significantly compared to the control. Among them, NixA-F1v and Hpn are the most important parts for the nickel uptake efficiency.

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]