Part:BBa_K5115060
ribozyme+RBS+cso(without csoS3)+stem-loop
Contents
Introduction
This composite part is composed of cso without csoS3 coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting BBa_K4765020 before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.[1] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.[2] 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.[3] 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.
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)[4]. 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.[5]
Usage and Biology
The csoS operon composite part is designed for use in E. coli to facilitate the expression and assembly of α-carboxysomes. These microcompartments are advantageous for engineering metabolic pathways, especially in enhancing the efficiency of carbon fixation and enzyme activity. The operon includes genes that encode shell proteins, such as CsoS1A and CsoS1B, which form the structural framework of the carboxysome. Additionally, the CsoS2 protein is vital for organizing Rubisco within the carboxysome, playing a role analogous to that of CcmM in β-carboxysomes, where it links cargo and shell assemblies.
Research indicates that the CsoS2 protein exists in two isoforms—CsoS2A and CsoS2B—differing in size and functionality, with the longer form being particularly important for the proper assembly of the empty α-carboxysome shells. Experimental results have shown that deletion of CsoS2 results in the failure to form shell structures in E. coli, underscoring its necessity in carboxysome assembly. The C-terminus of CsoS2 has been identified as an encapsulation peptide (EP) that facilitates the incorporation of cargo enzymes into the shell, allowing for the construction of functional nanoreactors.
By employing this composite part in E. coli, researchers can harness the natural assembly mechanisms of carboxysomes to create efficient systems for producing biofuels and other valuable bioproducts. The incorporation of enzymes such as [FeFe]-hydrogenase into the α-carboxysome shell offers a promising strategy for enhancing hydrogen production while protecting these sensitive enzymes from oxygen, thereby optimizing catalytic activities within a controlled microenvironment.
In our part, we choose to remove the subunit csoS3 from the BBa_K5115034(csoS operon). CsoS3 encodes the β-carbonic anhydrase enzyme, which is not necessary in our design. Plus that previous studies have shown that it is not essential for carboxysome assembly or function.[6] Deleting this subunit can release some burden of the engineered E.coli.
Characterization
Agarose gel electrophoresis
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| 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.org/fudan/parts. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 208
Illegal NotI site found at 5126 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 366
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 874
Illegal AgeI site found at 1825
Illegal AgeI site found at 2506
Illegal AgeI site found at 3460 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 266
References
- ↑ Eiler, D., Wang, J., & Steitz, T. A. (2014). Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proceedings of the National Academy of Sciences, 111(36), 13028–13033.
- ↑ Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143.
- ↑ The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Olins PO, Devine CS, Rangwala SH, Kavka KS. Gene, 1988 Dec 15;73(1):227-35.
- ↑ 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. https://doi.org/10.1038/s41594-020-0387-7.
- ↑ 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. https://doi.org/10.1038/s41467-020-19280-0.
- ↑ 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.
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