Difference between revisions of "Part:BBa K5115067"

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mineral, F module

Introduction

This part is made up of BBa_K5115063(hox and hyp, with EP targeted hoxF) and BBa_K5115060(ribozyme+RBS+cso without csoS3)+stem-loop). The hoxs and hyps make up the Ni-Fe hydrogenase and the csos make up the carboxysome. In our design, the hydrogenase is encapsulated into the carboxysome by EP. All the subunits of the proteins constructed into our ribozyme-assisted polycistronic co-expression system. 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 hydrogenase, carboxysome and EP at similar level. These parts make up the Ni-Fe hydrogenase with the hoxF targeted by EP and carboxysome. The former will be directed into the latter, creating a stable environment for the Ni-Fe hydrogenase to work in.

The Ni-Fe hydrogenase we use is an enzyme that functions in vivo bidirectionally for NAD⁺ reduction and NADH oxidation coupled to H₂ uptake and H₂ production, respectively. ^[1] In our design, the Ni-Fe hydrogenase works mainly to restore the nickel to a zero valence, which can help reduce nickel toxicity and collect nickel particles. The Ni-Fe hydrogenase is made up of six major and three auxiliary subunits. The former includes hoxF, hoxU, hoxY, hoxH, hoxW and hoxI, while the latter includes hypA, hypB and hypF.

The hoxF and the hoxU form the module of NADH dehydrogenase. The hoxF is a hydrogenase subunit responsible for electron transport. The most important group in hoxF is FMN-b, which has the ability of switching electron. Under anaerobic conditions, NADH is oxidized to NAD⁺ on the surface of hoxF subunit. In the meanwhile, the electrons generated in this reaction travel through a series of processes to the hoxH, completing the reduction of the hydrogen ion. Under aerobic conditions, NAD⁺ is reduced to NADH on the surface of the hoxF subunit. The electron transferring is contrary to former. ^[2] The hoxU houses a 2Fe-2S cluster and is responsible for the role of conducting electrons between hoxH and hoxF. ^[3]

The hoxY and the hoxH form the module of catalytic center.The hoxY houses a [4Fe-4S] cluster of the site, and an FMN group (FMN-a) near the Ni-Fe site in the hoxH. It is also responsible for the role of conducting electrons between hoxH and hoxF.^[4] The most important site in hoxH is the [NiFe] -hydrogenase active site, which is composed of Ni and Fe particles coordinated with cysteine residues, cyanide and carbon monoxide. ^[5] It is the most central component of our intracellular conversion of nickel ions. On its surface, oxidation and reduction of hydrogen gas happens alternately according to different oxygen status.

The rest of the subunits may work together to ensure that the hydrogenase can assemble and function well. It's worth noting that hypA and hypB can cooperate to precisely guide and insert the nickel ions into the hydrogenase catalytic center.^[6]

Through the synergistic integration of the hox and hyp subunits, our system effectively enhances hydrogen production and enables the reduction of nickel ions into nanoparticles, thereby maximizing the efficiency of nickel recovery from industrial wastewater.

The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes. EP supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes. This targeting mechanism is essential for protein encapsulation within these structures, aiding in the assembly of a functional, proteinaceous shell that sequesters enzymes or other proteins, ensuring efficient catalysis or protection from environmental stress^[7].

The csoS operon, originating from the Halothiobacillus neapolitanus, encodes a series of proteins essential for the assembly of α-carboxysomes, a type of microcompartment that facilitates the sequestration and concentration of enzymes involved in carbon fixation, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)^[8]. In literature, α-carboxysomes have been extensively studied and successfully utilized in Escherichia coli for enhancing carbon fixation efficiency and optimizing metabolic pathways. The csoS operon includes key structural proteins including csoS4B, csoS1C, csoS1A, csoS1B, csoS1D, csoS4A, and CsoS2, which play crucial roles in forming the shell and encapsulating cargo enzymes, including those required for hydrogen production. The operon serves as a model for synthetic biology applications, particularly in constructing nanoreactors capable of enhancing catalytic functions through encapsulation of heterologous enzymes. The successful expression of this operon in E. coli demonstrates its potential for industrial and biotechnological applications, enabling the creation of efficient microbial systems for sustainable bioprocessing.^[9]

Usage and Biology

The F module harnesses the collaborative power of hydrogenase enzymes, carboxysome compartments, and encapsulation peptides to drive an innovative approach for nickel reduction in E. coli. This integrated module not only advances the biotechnological potential of engineered microorganisms but also addresses environmental concerns related to nickel contamination by converting harmful ions into less toxic nanoparticles.

Characterization

Agarose gel electrophoresis

We firstly make sure that our genes are successfully introduced into the E.coli

In our design, we choose to remove the subunit csoS3 from the cso operon. Previous studies have shown that it is not essential for carboxysome assembly or function. Deleting this subunit can release some burden of the engineered E.coli^[10]. The result of PCR shows that the removal of csoS3 was rather successful.

Figure 1. Agarose gel electrophoresis of PCR products amplified from E. coli (DH5α) colonies. M: DNA Marker. (A) Lanes 1-8: Amplification of specific regions corresponding to csoS2, csoS3, csoS4A, csoS4B, csoS1C, csoS1A, csoS1B, and csoS1D, demonstrating the presence of the expected subunits derived from the α-carboxysome plasmid. (B) Lanes 1-8: Primers as in (A) were used for amplification. Please note no specific band in lane 2, which is due to the removal of csoS3 from the operon. Also, bands in (B) 3-8 are all smaller than (A) 3-8. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

To have our Ni-Fe hydrogenase directed into the carboxysomes, we deleted hoxF from the original hydrogenase operon and create another part to accommodate the hoxF-GS-EP which wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. We run this PCR test to examine the result of the hoxF removal.

Figure 2. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony. M: DNA Marker. Lanes 1-8: Corresponding bands for hoxF, hoxU, hoxI, hoxH, hoxW, hoxY, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp operon as designed. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Figure 3. Agarose gel electrophoresis of PCR products amplified from one E. coli (DH5α) colony. M: DNA Marker. Lanes 2-8: Amplification of specific regions corresponding to hoxU, hoxY, hoxH, hoxW, hoxI, hypA, and hypB, demonstrating successful assembly and integrity of the ribozyme-connected hox and hyp, without hoxF (no band in lane 1) as designed. Lane 4, the band for hoxH appears faint, pointed by the red arrowhead. Lane 7, there are non-specific bands, besides the red arrowhead pointed hypA. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.

Fluorescence microscopy results

Our experiments validated the role of EP by first fusing it with the StayGold fluorescent protein to assess its effectiveness in directing protein localization to the α-carboxysome. The fluorescence microscopy results confirmed successful incorporation of StayGold into the carboxysome shell, indicating that EP effectively directs proteins into the compartment. Subsequently, we incorporated EP with core assembly proteins of hydrogenase to examine nickel nanoparticle formation. Analysis of nickel particle distribution within E. coli revealed that EP facilitated the assembly of the α-carboxysome shell around the hydrogenase, leading to the formation of well-defined nickel nanoparticles. These results demonstrate that EP not only directs protein localization but also supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes.

Figure 4. Fluorescence images of E. coli expressing stayGold fused with EP, without or with cso-S3. Images were captured using spinning disk confocal with a 150x objective lens, as described on our Experiments page. Bacteria in A-C only express stayGold fused with EP BBa_K5115057, while bacteria in D simultaneously express BBa_K5115057 and BBa_K5115065. 1 mM IPTG was added to A,B only. Images without scale bar are 5x5 µm square, unless specifically indicated below. (A) The entire image field is shown (41.27x41.27 µm square), with brightfield image on the left, and green fluorescence image on the right. (B) Four regions in (A) are enlarged, showing uniform distribution of green fluorescence. (C) Although no IPTG was added, leaky expression from the promoter is sufficient to fill bacteria with green. (D) With all carboxysome components expressed, stayGold fused with EP concentrated to the carboxysome. Leaky expression from the promoter is sufficient to drive 1 or 2 carboxysome formed within each bacteria.

Hydrogenase-carboxysome function test

During our experiment, both F and U modules in BL21DE3 failed to be induced by arabinose. We switched to BL21AI and tried to induce it with arabinose again. This time, U module still failed to induce, but F module succeeded. We tried to run a protein electrophoresis to show the protein translation status. However, we can't find distinct bandings on the protein gel. We suspect that this failure results from too much burden the plasmid has brought to our E.coli. To learn more about the burden, please visit our software wiki. Our CDSs are not designed with any tags, making it difficult to run any western-blot experiments. As a result, we eventually choose to use electron microscope to show the results of our design.

Figure 5. Transmission electron microscopy images of negative stained E. coli expressing MINERAL F module. Osmium tetroxide and uranyl acetate were used for the staining A-E. Scale bar shown on the image. (A) Overview of E. coli cells. (B) Sections of bacteria, filled with carboxysome-sized regions (CSR) surrounded by electron dense dots. In one cell, all visual CSR are circled by yellow dash lines. (C) The size of CSR are various, with two examples circled. (D,E) For cells with less electron dense dots, CSR are clear, with the cell in C fully packed with CSR and the cell in D sparsely packed. (F) No uranyl acetate staining. The image confirms that the electron dense dots throughout A-E are not salt crystals but actual metallic particles, which we believe are Ni particles. Three 80-nm square regions are enlarged, showing polyhedral outline of CSR, with Ni particles surrounded.

Hydrogenase-cabo safety test

To activate hydrogenase to reduce nickel

Sequence and Features

References

1. ↑ Teramoto, H., Shimizu, T., Suda, M., & Inui, M. (2022). Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains. International Journal of Hydrogen Energy, 47(52), 22010–22021.
2. ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
3. ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
4. ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
5. ↑ Chan, K.-H., Lee, K.-M., & Wong, K.-B. (2012). Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation. PLOS ONE, 7(2), e32592.
6. ↑ Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.
7. ↑ Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448.
8. ↑ Oltrogge, L. M., Chaijarasphong, T., Chen, A. W., Bolin, E. R., Marqusee, S., & Savage, D. F. (2020). Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation. Nature structural & molecular biology, 27(3), 281–287.
9. ↑ Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448.
10. ↑ Baker, S. H., Williams, D. S., Aldrich, H. C., Gambrell, A. C., & Shively, J. M. (2000). Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus. Archives of Microbiology, 173(4), 278–283.