Throughout our project, we encountered problems in Parts, Measurement, Software, Hardware, Human Practices, Inclusivity and Entrepreneurship, we solved them following iterations of the DBTL cycle (Design, Build, Test, Learn). Below are the steps we went through to develop our nickel module - part number BBa_K5115068.

Cycle 1

Cycle 1 Design

To make our Escherichia coli efficiently absorb nickel ions, we need active nickel uptake, which includes a nickel transport protein called nikABCDE^[1]. Because nikA is the rate-limiting enzyme, according to our previous observations, we put it as the last one when we connect these CDS with the Ribozyme-Assisted Polycistronic expression system (pRAP)^[2].

As for the other nickel transport protein NixA (BBa_K5115071)^[3], we also made dimerizable versions (BBa_K5115086, BBa_K5115087), using a well-studied rapamycin-induced protein dimerization system ^[4].

Cycle 1 Build

We built on the work done previously, BBa_K2652006 and created four new parts to test our nickel uptake module. These include ribozyme-connected nik operon BBa_K5115082, NixA BBa_K5115071 (additional stop codon was added when testing its own), NixA-F1v BBa_K5115086, and F1v-NixA BBa_K5115087 (F1v is short for FKBP F36V mutant).

Cycle 1 Test

To evaluate how well our E. coli absorbs nickel, we measured the nickel concentration in the bacterial supernatant after making the standard curve. A lower nickel concentration in the supernatant indicates that more nickel has been absorbed by E. coli. This method allows us to calculate the percentage of nickel that E. coli uptakes during culture.

Figure 1: Comparison of Ni²⁺ Uptake Efficiency by Different E. coli
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).

As illustrated in Figure 1, NixA-F1v demonstrates the highest efficiencies for nickel uptake among these parts. We also conducted nickel absorption tests with various starting Ni²⁺ concentrations (20 mg/L and 50 mg/L), leading to the same results.

Cycle 1 Learn

NixA-F1v performs better than F1v-NixA, likely due to its fused F1v could reside better in the cytosol. We learn this by using AlphaFold3 to analyze its structure and identify factors contributing to its effective nickel uptake. However, NixA-F1v achieved 25% nickel absorption during the 5-hour culture, and we are not fully satisfied. We speculate this may be due to E. coli facing survival pressure in nickel-rich environments.

Cycle 2

Cycle 2 Design

We aim to reduce the survival pressure of E. coli by enhancing its tolerance, thereby improving its nickel absorption efficiency. We discovered the Hpn protein derived from H. pylori, which contains a 60-residue histidine that can bind to nickel ions and lower intracellular nickel ion levels^[5]. This protein forms multimers and exhibits changes in its α-helix and β-fold secondary structure upon binding to nickel.

Cycle 2 Build

We created the part ribozyme connected Hpn (BBa_K5115036) and connected this part with NixA-F1v (BBa_K5115086) in pRAP.

Cycle 2 Test

We evaluated the nickel uptake efficiency of these E. coli in a medium with an initial concentration of 50 mg/L of nickel. As shown in Figure 2, the nickel absorption ability of the Hpn + NixA-F1v bacteria was significantly higher than NixA-F1v alone.

Figure 2: 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. 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.

Cycle 2 Learn

We confirmed that expressing Hpn does enhance E. coli's tolerance to nickel and improves overall nickel uptake efficiency, resulting in over 45% absorption with an initial nickel concentration of 50 mg/L.

Cycle 3

Cycle 3 Design

Could we improve further? We discovered that wild-type E. coli expresses RcnA^[6], especially at high Ni²⁺ concentration, will efflux nickel as a protective mechanism. While it was not successful for us to knockout rcnA in E. coli, fortunately, over-expression of a mutant RcnR (C35L) could limit the induction of rcnA^[7] and avoid E. coli's ability to efflux nickel. RcnR is a transcription factor, responds to nickel ions, and increases rcnA expression. Its C35L mutant avoids its nickel sensing, thus no RcnA.

Cycle 3 Build

We built the part RcnR C35L (BBa_K5115000), with the Cys residue at position 35 changed to Leu. This alteration prevents RcnR from releasing from DNA upon high nickel, thereby limiting RcnA's induction under high nickel concentrations. Ultimately, this ensures the absorbed nickel remains within our engineered E. coli.

Cycle 3 Test

After making the constructs, we measured the efficiency of nickel uptake by bacteria. As shown in Figure 3, RcnR^C35L improved nickel absorption in E. coli, both with nik-ribozyme and NixA. This result supports our design by inhibiting nickel efflux, effectively trapping Ni²⁺ within the cells.

Figure 3: 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.

Cycle 3 Learn

Through our design, build, and tests, we found that:

1. Among all the nickel transport proteins, our engineered dimerized NixA-F1v is the most effective.
2. Expressing Hpn reduces the burden on E. coli by enhancing its tolerance to nickel, leading to improved nickel absorption.
3. RcnR^C35L helps to retain absorbed nickel in E. coli by inhibiting its efflux.

Next, we integrate these parts and create our nickel module (BBa_K5115068), which effectively increases nickel concentration in the cytosol and provides a suitable condition for our hydrogenases. Please refer to our Part Collection page to see how it is used.

Summary

These DBTL cycles are just one example of our project engineering processes. Other examples are available on our Measurement, Software, Hardware, Human Practices, Inclusivity and Entrepreneurship pages.

References

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1. Dosanjh, N. S., & Michel, S. L. (2006). Microbial nickel metalloregulation: NikRs for nickel ions. Curr Opin Chem Biol, 10(2),123-130. https://doi.org/10.1016/j.cbpa.2006.02.011 ↩︎

2. Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2023). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143.https://doi.org/10.1021/acssynbio.2c00416 ↩︎

3. Hernandez, J. A., Micus, P. S., Sunga, S. A. L., Mazzei, L., Ciurli, S., Meloni, G. (2024). Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori. Chem Sci, 15(2), 651-665. https://doi.org/10.1039/d3sc05135h ↩︎

4. Clackson, T., et al. (1998). Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci U S A, 95(18):10437-42. https://doi.org/10.1073/pnas.95.18.10437 ↩︎

5. Ge, R., Watt, R. M., Sun, X., Tanner, J. A., He, Q.-Y., Huang, J.-D., & Sun, H. (2006). Expression and characterization of a histidine-rich protein, Hpn: Potential for Ni2+ storage in Helicobacter pylori. Biochemical Journal, 393(Pt 1), 285–293. https://doi.org/10.1042/BJ20051160 ↩︎

6. Koch, D., Nies, D. H., & Grass, G.(2007). The RcnRA (YohLM) system of Escherichia coli: a connection between nickel, cobalt and iron homeostasis. Biometals, 20(5), 759-771. https://doi.org/10.1007/s10534-006-9039-6 ↩︎

7. Higgins, K. A., Chivers, P. T., & Maroney, M. J. (2012). Role of the N-terminus in Determining Metal-Specific Responses in the E. coli Ni- and Co-Responsive Metalloregulator, RcnR. Journal of the American Chemical Society, 134(16), 7081–7093. https://doi.org/10.1021/ja300834b ↩︎