DNA Parts
In Synthetic Biology, genetic toolkits – composed of promoters, RBS, terminators, and reporter genes – are essential for the expression of new synthetic genes. For this reason, in addition to our main project, we decided to provide the community with fundamental tools for engineering Rhodococcus opacus PD630, a strain that we strongly believe has significant potential, particularly in the field of bioremediation.
Figure 1 shows four new constitutive iGEM promoters and one previously existing (pLac), which had never been characterized in R. opacus PD630 and will be explained below. Our experimental setup measured 3 technical replicates of 3 different biological replicates of R. opacus PD630, each harboring a plasmid carrying the same RBS – sfGFP – terminator (BBa_25JK5LID - BBa_25BE2EBO - BBa_25CPY605) structure, to establish a comparative benchmark.
To evaluate this phenomenon, we conducted a fluorescence emission analysis employing our sfGFP fluorescence analysis protocol in Rhodococcus opacus. This assessment focused on the signal of a reporter gene BBa_25BE2EBO (adapted from BBa_J428326 iGEM part), described down below.
pLac Promoter
Regarding the strong promoter pLac BBa_K4156079, our experiments confirm for the first time its ability to drive protein expression in Rhodococcus opacus PD630. This is a relevant contribution, since no data were previously available on the use of this well-known promoter in this strain. Given its wide diffusion in molecular biology labs and its inclusion in the iGEM Distribution Kit, pLac now represents an easily accessible tool for teams interested in engineering Rhodococcus.
p2, pB2 and pB3 Promoters (BBa_25V63Y8N, BBa_259G7NV9, BBa_25RYU8SQ)
Regarding our newly designed promoters p2, pB2, and pB3, we demonstrated their ability to drive protein expression in Rhodococcus opacus PD630, with p2 showing even higher activity than the widely used constitutive promoter pNit. The pB2 and pB3 promoters were derived from p2, the promoter of a division cluster transcriptional repressor in Rhodococcus jostii (Round et al., 2019), by generating shorter variants through PCR-based truncation. This strategy, inspired by the minimization of the p10 promoter described in the same study, was intended to yield more compact and efficient regulatory parts. While pB2 proved successful and represents a valuable contribution to the community, pB3—likely shortened excessively—did not perform as expected. Overall, p2 and pB2 emerge as strong, reliable promoters that expand the genetic toolbox available for engineering Rhodococcus.
pNit Promoter (BBa_25AYM82O)
Regarding the constitutive promoter pNit, originally developed by N. Nakashima and T. Tamura (2004), we confirmed its reliability as a robust transcriptional element in Rhodococcus opacus PD630. Over the years, this promoter has established itself as a benchmark in the field: indeed, it has been routinely used as a positive control in comparative studies of constitutive promoters in Rhodococcus strains, such as the comprehensive investigation conducted by Round, Roccor, and Eltis in 2019.
In our experiments, we characterized the activity of pNit using a sfGFP (BBa_25BE2EBO) based fluorescence reporter assay. Specifically, sfGFP was expressed under the control of pNit in the pNit-QT1 vector, enabling a reliable evaluation of promoter strength within the R. opacus background. This assessment confirmed pNit as a strong and constitutive promoter, providing the necessary reference framework for comparing our newly designed promoters, including pB2, thereby consolidating its role as the historical standard for gene expression in Rhodococcus.
sfGFP (BBa_25BE2EBO)
The super folded green fluorescent protein (sfGFP) has excitation at 488 nm and emission at 510 nm. We obtained it from BBa_J428326. In the iGEM Registry, this sequence is typically found as part of a composite part that includes a promoter and RBS, but for our purposes we isolated only the protein-coding sequence, in order to directly evaluate promoter activity. To comply with iGEM standards, we removed restriction sites incompatible with Golden Gate Assembly by introducing silent mutations through site-directed PCR mutagenesis. Specifically, primers were designed with single mismatches at the BsrGI and SapI sites, allowing us to eliminate these sites without altering the amino acid sequence of the protein.
After constructing this modified version, we validated its functionality in Rhodococcus opacus PD630. The reporter was expressed successfully, producing a strong fluorescent signal, which confirmed its suitability as a standalone part for promoter characterization. Importantly, this version of sfGFP can now be directly reused in new constructs as an individual iGEM part, rather than being confined to a plasmid backbone where it previously served only as a negative control (losing fluorescence upon insertion). With this contribution, sfGFP is now available as a standardized and functional reporter for expression studies in Rhodococcus.
References
▼- Nakashima, N., & Tamura, T. (2004). 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, 70(9), 5557-5568.
- Round, J. W., Roccor, R., & Eltis, L. D. (2019). A biocatalyst for sustainable wax ester production: re-wiring lipid accumulation in Rhodococcus to yield high-value oleochemicals. Green Chemistry, 21(23), 6468-6482.