During our project we both designed new parts and adapted parts already in wide use in Rhodococcus opacus PD630. Our main contributions in parts include a small library of constitutive promoters, a reporter gene, a variety of parts composing Rhodococcus opacus toolkit and Loxport construct: a composite part device to mediate site-specific gene integration in Rhodococcus chassis.
Best Basic Part
The pB2 Promoter (BBa_259G7NV9) is not only a newly developed DNA part that expands the genetic toolkit available for Rhodococcus opacus PD630, but also a gateway for discovering and optimizing additional promoters derived from other strains. As a constitutive promoter, it enables both the study and application of novel genes within pollutant degradation pathways.
One of the main limitations in this field has been the lack of constitutive promoters beyond pNit—the only one currently available—which restricts continuous pollutant degradation under different growth conditions. In contrast, pB2 provides an alternative to inducible systems such as the pTip promoter, which requires activation by thiostreptone.
We designed pB2 as an optimization of p2 constitutive promoter in Rhodococcus jostii RHA1. The p2 promoter was identified by Round, Roccor and Eltis in 2019 [4], and we optimized it following the same approach used for pM2 in the same paper. We developed a reporter system based on sfGFP fluorescence, which we used to characterize a small library of constitutive promoters.
We believe that pB2, together with the other promoters we tested, represents a fundamental tool for introducing and expressing new genes in R. opacus. Additionally, we developed a system that takes advantage of Cre recombinase, which is pre-inserted into the genome, ensuring precise integration and control. Combining these two new tools, the use of R. opacus in bioremediation will be much easier.
Moreover, pB2 is one of the selectable options when building plasmids with our metabolic pathway prediction tool, CAPE, making it a versatile and valuable addition to synthetic biology applications in pollutant degradation.
Our Part Library
Constitutive Promoters
We tested a small library of constitutive promoters for Rhodococcus opacus PD630. Their expression levels can be found in this graph.
| Name | Registry Code | Part Type | Origin |
|---|---|---|---|
| pNit | BBa_25AYM82O | Promoter | pNit plasmid [1] |
| pB2 | BBa_259G7NV9 | Promoter | Rhodococcus jostii RHA1 [4] |
| pB3 | BBa_25RYU8SQ | Promoter | New |
| p2 | BBa_25V63Y8N | Promoter | New |
| pLac | BBa_K4156079 | Promoter | New characterization of promoter, from [5] |
Reporter Gene
We also implemented an optimized version of sfGFP as a reporter for promoter studies.
| Name | Registry Code | Part Type | Origin |
|---|---|---|---|
| Domesticated sfGFP | BBa_25BE2EBO | Reporter | Domestication of part BBa_J428326 |
Primers
Many successful PCR reactions were obtained by using these primers, which allowed us to amplify both reporters and promoters.
| Name | Registry Code | Part Type | Origin |
|---|---|---|---|
| sfGFP_f | BBa_25TME224 | Primer | New |
| sfGFP_r | BBa_25RAR337 | Primer | New |
| p2_f | BBa_25Q98NV7 | Primer | Adapted from [4] |
| p2_r | BBa_25CLTZOI | Primer | Adapted from [4] |
| pB2_r | BBa_25P5QYBQ | Primer | New |
| pB3_r | BBa_25SVD1UA | Primer | New |
| pLac_f | BBa_25GDIXQU | Primer | New |
| pLac_r | BBa_25P1T7OX | Primer | New |
Rhodococcus Toolkit from pTip and pNit Plasmids
We also used already established toolkits for Rhodococcus. We added to the registry many parts that we implemented in our constructs.
| Name | Registry Code | Part Type | Origin |
|---|---|---|---|
| pTipA | BBa_25J70TBC | Promoter | pTip plasmid [1] |
| Lg-10 RBS | BBa_25JK5LID | RBS | pTip plasmid [1] |
| Lg-10 RBS+lox71 | BBa_257JXMK4 | RBS | New fusion between pTip plasmid [1] and loxLE [3] |
| Cre recombinase | BBa_250HXRZ2 | Coding | Codon optimized from [3] |
| ThcA terminator | BBa_25CPY605 | Terminator | pTip plasmid [1] |
| ThioR Translational Unit | BBa_25R4ZFOV | Translational Unit | pTip plasmid [1] |
| Inverted TipAL Translational unit | BBa_25DOSDGD | Translational Unit | pTip plasmid [1] |
| Homology region UP | BBa_25I699K3 | Homologous region | ROCI 1 region [2] |
| Homology region DOWN | BBa_25MX9A49 | Homologous region | ROCI 1 region [2] |
| spacer | BBa_2563RGJO | Miscellaneous | pTip plasmid [1] |
Composite Part
We developed a construct for production of a gene-integrating chassis of Rhodococcus opacus PD630.
| Name | Registry Code | Part Type | Origin |
|---|---|---|---|
| Loxport | BBa_2566C7GE | Device | New |
References
▼- [1]Nakashima N, Tamura T. Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression. Applied and Environmental Microbiology. 2004;70(9):5557-5568. doi: https://doi.org/10.1128/aem.70.9.5557-5568.2004
- [2]Anthony WE, Carr RR, DeLorenzo DM, et al. Development of Rhodococcus opacus as a chassis for lignin valorization and bioproduction of high-value compounds. Biotechnology for Biofuels. 2019;12(1). doi: https://doi.org/10.1186/s13068-019-1535-3
- [3]Kitagawa W, Hata M. Development of Efficient Genome-Reduction Tool Based on Cre/loxP System in Rhodococcus erythropolis. Microorganisms. 2023;11(2):268-268. doi: https://doi.org/10.3390/microorganisms11020268
- [4]Round JW, Roccor R, Eltis LD. A biocatalyst for sustainable wax ester production: rewiring lipid accumulation in Rhodococcus to yield high-value oleochemicals. Green Chemistry. 2019;21(23):6468-6482. doi: https://doi.org/10.1039/c9gc03228b
- [5]Hetzler S, Bröker D, Steinbüchel A. Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains. Applied and Environmental Microbiology. 2013;79(17):5159-5166. doi: https://doi.org/10.1128/aem.01214-13