Composite

Part:BBa_K5115052

Designed by: Yi Shi   Group: iGEM24_Fudan   (2024-09-19)


ribozyme connected hox and hyp, without hoxF

contributed by Fudan iGEM 2024

Introduction

This composite part combines BBa_K5115012(ribozyme+RBS+hoxU+stem-loop),BBa_K5115013(ribozyme+RBS+hoxY+stem-loop),BBa_K5115014(ribozyme+RBS+hoxH+stem-loop),BBa_K5115015(ribozyme+RBS+hoxW+stem-loop),BBa_K5115016(ribozyme+RBS+hoxI+stem-loop),BBa_K5115017(ribozyme+RBS+hypA+stem-loop),BBa_K5115018(ribozyme+RBS+hypB+stem-loop)and BBa_K5115019(ribozyme+RBS+hypF+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 BBa_K5115012(ribozyme+RBS+hoxU+stem-loop),BBa_K5115013(ribozyme+RBS+hoxY+stem-loop),BBa_K5115014(ribozyme+RBS+hoxH+stem-loop),BBa_K5115015(ribozyme+RBS+hoxW+stem-loop),BBa_K5115016(ribozyme+RBS+hoxI+stem-loop),BBa_K5115017(ribozyme+RBS+hypA+stem-loop),BBa_K5115018(ribozyme+RBS+hypB+stem-loop)and BBa_K5115019(ribozyme+RBS+hypF+stem-loop) at similar level. These subunits, if added with hoxF, make up the Ni-Fe hydrogenase.

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.

In this part, we choose to delete hoxF, but it doesn't means that hpxF isn't important in our design. Instead, the hoxF is rather crucial for that we utilize it to EP in BBa_K5115061. The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes[7]. Linking EP with hoxF, we hope that EP can import the hydrogenase into the carboxysome, creating a stable environment for the enzyme to work.

Characterization

Agarose gel electrophoresis

contributed by Fudan iGEM 2024
Figure 1. 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.


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 485
    Illegal BglII site found at 563
    Illegal BamHI site found at 956
    Illegal XhoI site found at 493
    Illegal XhoI site found at 685
    Illegal XhoI site found at 1163
    Illegal XhoI site found at 3402
    Illegal XhoI site found at 5231
    Illegal XhoI site found at 6393
    Illegal XhoI site found at 8232
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1256
    Illegal NgoMIV site found at 1936
    Illegal NgoMIV site found at 4543
    Illegal NgoMIV site found at 4650
    Illegal NgoMIV site found at 4920
    Illegal NgoMIV site found at 5766
    Illegal NgoMIV site found at 5998
    Illegal AgeI site found at 446
    Illegal AgeI site found at 5718
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 267
    Illegal BsaI site found at 429
    Illegal BsaI site found at 3056
    Illegal SapI.rc site found at 378


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.
[edit]
Categories
Parameters
None