Bologna - iGEM 2025

Insertion of LoxPort into Rhodococcus opacus PD630

Our goal was to build and test a system based on our construct, called LoxPort. This cre recombinase expressing construct was designed to perform site-specific recombination on lox sequences, to facilitate gene insertion. The construct was designed to be integrated in Rhodococcus opacus PD630 genome via double homologous recombination. Several changes were made to our original plan to improve amplification, assembly, and plasmid stability.

Using Q5 high-fidelity polymerase, we successfully amplified the DNA fragments containing homology regions. The PCR bands matched the expected sizes, confirming correct amplification. However, PCR experiments for linear construct that we first ordered for synthesis never yielded a good amplification.

To make the process easier, the construct was then divided into smaller fragments. Each fragment was amplified correctly, but fusion PCR on them never yielded a full construct. This has been mainly attributed to the complexity of the region to be amplified.

Linear construct amplification was also attempted by cloning into the pGEM vector, but no correct recombinant plasmids were obtained. Although verification PCR reactions worked well, but isolation of plasmid DNA gave no results. A similar result was obtained using ExTaq polymerase: amplification succeeded, but assembly in both pGEM and Topo vector failed.

To solve these issues, the construct was redesigned with reduced GC content and synthesized directly by Twist Bioscience in a medium-copy plasmid. This optimized version was stable, easy to amplify, and gave reliable DNA yields. This confirmed that the main problems before might have been caused by high GC content and by the choice of plasmid backbone.

Next, we tried integrating the LoxPort construct into the pKSac45 plasmid, necessary for double recombination in Rhodococcus. Cloning with SphI and EcoRI enzymes produced kanamycin-resistant E. coli colonies, but plasmid extraction (miniprep) gave almost no DNA. Colony PCR detected correct junctions in several colonies, especially colony 2, but sequencing showed that this clone only contained a self-ligated pKSac45 plasmid with a small deletion.

Figure 1 - Agarose gel electrophoresis of DNA extracts. The first two wells show abundance of plasmidic DNA, with very intense bands below 3000bp. Band shape is typical of coiled circular plasmids. The other wells all show a non-specific smear, probably derived from background nucleic acid extraction from E. coli.

Figure 2 - Snapgene screenshot showing colony 2 extract sequence (below) aligned on pKSac45 sequence. Results indicate a 50bp deletion over MCS region. Restriction enzymes used for cloning were SphI and EcoRI.

The combination of antibiotic resistance, lack of plasmid recovery, and inconsistent PCR results suggested that LoxPort in high-copy plasmids may cause unwanted Cre recombinase expression in E. coli DH5-α. This expression could trigger recombination and plasmid instability or even integration into the bacterial chromosome. In contrast, the construct remained stable when carried on a medium-copy plasmid, supporting the idea that copy number strongly affects plasmid stability and Cre activity.

Overall, these results suggest that GC content, secondary structures, and plasmid copy number are key factors for successful assembly and maintenance of Cre-lox–based constructs in E.coli. Future experiments should test LoxPort in medium-copy versions of pKSac45 and may include genome sequencing of resistant colonies to confirm whether plasmid integration has occurred.

Constitutive Promoter Characterization

To extend the available toolkit, several constitutive promoters were tested to compare their ability to drive sfGFP expression in Rhodococcus opacus PD630. The goal was to identify promoters with different expression strengths that could be implemented in the future molecular toolkit of Rhodococcus opacus PD630.

Promoters p2, pB2, pB3, pLac, and pNit were selected. pLac was obtained from a vector containing an IPTG-inducible system, while p2, pB2, and pB3 were amplified from the Rhodococcus jostii RHA1 genome. In contrast, PNit is part of the standard toolkit of Rhodococcus opacus PD630 and represents the most well-known constitutive promoter for this bacterium. All promoters were cloned into the pTip plasmid, replacing its native promoter and positioned upstream of the sfGFP gene. The sfGFP sequence was domesticated by removing unwanted restriction sites and adding compatible cloning ends.

Figure 3 - gel electrophoresis performed on enzymatic digestion of reporter plasmids used for Rhodococcus opacus PD630 transformation. pNit + sfGFP was digested with NcoI and NotI restriction enzymes, pTip-based reporter plasmids were cut with NcoI and BsrGI restriction enzymes. All bands are as expected.

The reporter plasmids were first assembled and verified in E. coli through heat-shock transformation and plasmid purification. After sequence validation, reporter plasmids were electroporated into Rhodococcus, plated on tetracycline selective media, and stable colonies were selected. For each promoter, four colonies were grown in liquid culture to an OD₆₀₀ of 0.5, and fluorescence intensity was measured to compare promoter strength.

Promoters p2, pB2, and pB3 successfully drove sfGFP expression in Rhodococcus. Among them, pB2 showed the highest activity, p2 produced intermediate fluorescence, and pB3 had the weakest expression. We aimed to study also p10 promoter, but it could not be tested due to primer design issues that prevented successful amplification of it and its optimizations. pLac promoter also showed measurable activity comparable to pNit, confirming its function in Rhodococcus.

We decided to include the pNit promoter in our study as a positive control. Originally described by Nakashima and Tamura (2004), pNit is a well-established constitutive promoter in Rhodococcus. Our results confirmed that pNit consistently drives strong sfGFP expression, in agreement with the published data. Therefore, in the table below, the fluorescence values of the other promoters were normalized to pNit activity to allow a reliable comparison of their relative strengths

Figure 4 – Bar graph showing constitutive promoter strength in Rhodococccus oapcus PD630.

Promoter Strength Data

Promoter	Strength (norm. pNit)
pB2	1.20
pNit	1.00
pLac	0.93
p2	0.60
pB3	0.24

Overall, this first characterization cycle confirmed that all the tested promoters function constitutively in Rhodococcus opacus PD630 at varying intensities, with pB2 emerging as the strongest among the new candidates. The experiment also highlighted areas for improvement, like redesigning primers for p10 and optimization of pB3 (which may have lost key regulatory sequences during cloning). These results provide a solid foundation for building a standardized library of Rhodococcus promoters based on normalized sfGFP expression data. The table on the right reports the relative fluorescence values normalized to the activity of the pNit promoter

References

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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
Round JW, Roccor R, Eltis LD. A biocatalyst for sustainable wax ester production: re-wiring lipid accumulation in Rhodococcus to yield high-value oleochemicals. Green Chemistry. 2019;21(23):6468-6482. doi: https://doi.org/10.1039/c9gc03228b

Biosensor Characterization

For the development and optimization of a colorimetric biosensor for monitoring lipase activity, the initial step focused on identifying the optimal buffer for enzyme solubilization and activity. Several buffers were tested, and their effects on enzyme stability and assay performance were evaluated; some buffers caused precipitation or inconsistent activity, while others maintained enzyme stability over time. The combination of 1.5% BSA with 1 M Tris-HCl was selected for subsequent assays as it provided complete enzyme solubilization and consistent activity measurements.

Lipase activity was assessed using the p-nitrophenyl palmitate (pNPP) assay, with enzyme concentrations ranging from 50 to 200 U/mL: absorbance was measured at 410 nm using a Varioskan spectrophotometer. The results showed a dose-response relationship, with maximal activity observed at 125 U/mL, which was established as the optimal working concentration; higher concentrations (>150 U/mL) displayed minor deviations from linearity, likely due to substrate saturation or aggregation. Replicate measurements confirmed the reliability of the results, with standard deviations within acceptable limits.

Lipase activity assay (Type II, ≥125 units/mg)

Lipase activity 125 U/mL - Control and optimized conditions

During optimization, initial enzyme preparations occasionally produced inconsistent activity readings: these inconsistencies were attributed to incomplete dissolution or buffer pH variations. Implementation of a pre-incubation step and fine-tuning of the buffer composition resolved these issues, resulting in reproducible measurements across independent experiments.

Under the optimized conditions, the biosensor generated stable and measurable colorimetric signals corresponding to lipase activity. The linear response observed in the pNPP assay indicated that the system could be reliably employed for enzyme monitoring. These results demonstrate that the biosensor design is robust and that methodological optimizations significantly improved performance and reproducibility.