Results

In our search for ways to improve the CADABRA enzyme cocktail, we obtained the following results

1) Designing, synthesizing, and cloning two new MBL coding sequences

We designed two new metallo-β-lactamase (MBL) coding sequences. The first one featured the E. coli codon-optimized version of the IMP-1 MBL (Q79MP6). The second construct represented an artificial fusion of the NDM-2 (F2YZ26) and NDM-5 (A0A2S1PIF3) variants, known to exhibit the highest meropenem hydrolyzing capabilities based on existing literature. In both coding sequences, C-terminal 6xHis tags were incorporated for subsequent purification purposes. The two sequences were synthesized as gBlock fragments by IDT.

Schematic overview of both gBlock fragments

Following the acquisition of the fragments, they were subjected to dilution and subsequently underwent PCR amplification using VF2 and VR primers. In this process, Phusion polymerase was utilized with a limited number of cycles to minimize the likelihood of occurrence of unintended mutations.

PCR-amplified MBL fragments

Subsequently, both PCR products underwent purification with the Zymo DNA Clean & Concentrator kit, followed by elution in 20 µl of nuclease-free H2O. These products were then digested using XbaI and PstI, followed by purification as previously described. Concurrently, plasmid DNA was obtained from part BBa_K608003, which contains a strong promoter and a medium RBS. This DNA was treated with SpeI and PstI, and afterward purified from the restriction endonucleases. The quantities of all digested DNA molecules were determined using Quantus (Promega), and ligations were conducted using a 3:1 insert-to-vector molar ratio. Positive transformants were chosen on dishes with chloramphenicol and ampicillin, as only the MBL-positive clones would be capable of eliminating the latter antibiotic.

2) Confirming that both new constructs express MBLs

To confirm that both new constructs produce functional MBLs, we prepared antibiograms according to the EUCAST guidelines, utilizing discs with imipenem, meropenem, and imipenem + EDTA. The distinction in zone diameters between imipenem and imipenem + EDTA (>> 8 mm) indicated a MBL production in our transformants.

Illustrative antibiogram for phenotypic MBL detection

3) Assessing the meropenem degradation capabilities of IMP-1 and NDM-2&5 cell free extracts

All cultures were established using E. coli DH10B cells containing the respective IMP-1 and NDM-2&5 expression constructs. Cultivation was conducted in LB medium supplemented with meropenem. Following inoculation, the cultures were incubated for a minimum of 16 hours at 37°C and 250 rpm on an orbital shaker. Post-cultivation, centrifugation was employed at 2700 g for 10 minutes, followed by filtration through a 0.22 µm filter to yield a cell-free supernatant.

Cell supernatant filtration through 0.22 µm filter

Following this step, we prepared Muller-Hinton broth, enriched with 50 µg/ml of meropenem, and subjected it to treatment with CADABRA. To assess the extent of antibiotic degradation achieved by our MBL enzyme, we created petri dishes filled with 0.7% LB agar medium and inoculated them with E. coli DH10B containing pSB1C3-amilGFP as an indicator strain. Once the agar had solidified, we created small wells in it and introduced serially 2-fold diluted portions from the treated sample into these wells. Before this procedure, we blocked MBL activity by adding EDTA.

All cultures were established using E. coli DH10B cells containing the respective IMP-1 and NDM-2&5 expression constructs. Cultivation was conducted in LB medium supplemented with meropenem. Following inoculation, the cultures were incubated for a minimum of 16 hours at 37°C and 250 rpm on an orbital shaker. Post-cultivation, centrifugation was employed at 2700 g for 10 minutes, followed by filtration through a 0.22 µm filter to yield a cell-free supernatant.

Preparation of Petri dishes for the agar well dilution method

Subsequently, the petri dishes were gently placed in an incubator and allowed to incubate overnight at 37°C. The following day, all the dishes were examined under blue light transillumination to check for the presence of growth inhibition zones. None were detected, indicating complete hydrolysis of the antibiotic by the corresponding MBLs.

Meropenem degradation by cell free extracts

4) Monitoring the meropenem degradation in real time

To monitor the meropenem degradation process, we utilized ultraviolet-visible (UV-Vis) spectroscopy. For this purpose, we first procured quartz cuvettes with appropriate volumes.

Quartz cuvettes

Next, we optimized the conditions for meropenem detection, looking for the corresponding peak at 298 nm

Monitoring of the meropenem concentrations in water samples via UV-Vis spectroscopy

We created a sample by combining the cell-free extract with meropenem inside a quartz cuvette and recorded absorbance measurements at 298 nm for a duration of 10 minutes using a JENWAY 6715 UV-Vis spectrophotometer. The slope of the resulting line was employed as an indicator of our MBL extract's enzymatic activity.

Illustrative curve of meropenem’s degradation monitored via UV-Vis spectroscopy

Results indicated that NDM-2&5 outperforms VIM-2 and IMP-1.

5) Assessing the MICs of meropenem for VIM-2 and NDM-2&5 producers.

A broth dilution assay was used to determine both MICs.

Broth dilution assay to find the MIC of meropenem for MBL producers

6) Optimization of the NDM-2&5 MBL via directed evolution

To further elevate the performance of the new CADABRA based on NDM MBL, we opted to employ directed evolution for the purpose of enhancing the enzyme's activity. To execute this, we cloned both the coding sequences of NDM-2&5 and IMP-1 into a plasmid featuring a T7 promoter and RBS (BBa_K525998) while adhering to the BioBrick RFC 10 assembly guidelines.

Schematic representation of the T7 constructs

All cloning procedures were carried out in E. coli KRX cells, which harbor the T7 RNA polymerase gene controlled by an L-rhamnose-inducible promoter. In this instance, identifying positive transformants through incubation on Petri dishes with β-lactam antibiotics was unfeasible, prompting us to resort to colony PCR for verification.

Identification of positive clones via colony PCR

Subsequently, we attempted to assess the functional performance of our constructs by generating antibiograms. In this instance, we cultivated the strains in MH liquid medium supplemented with L-rhamnose to induce the T7 promotor. The remaining part of the procedure was performed as usual. Regrettably, we did not observe any indications of MBL production in neither of our PCR-positive transformants.

All antibiograms showed no MBL activity

To address this challenge, we made the decision to design novel constructs in which we placed a J23101 constitutive Anderson promoter upstream of the T7-RBS-bla_NDM2&5 fragment. Our expectation was that this promoter would promote protein expression, allowing the T7 promoter to be dedicated to mutagenesis. To evaluate this concept, we constructed a hybrid promotor comprising J23101-T7-RBS, followed by the coding sequence of amilGFP (BBa_K592010).

Schematic representation of the j23101-T7 constructs

Upon examination of the transformed cells under blue light, conspicuous evidence of amilGFP expression became apparent.

The hybrid J23101-T7 promotor is functional as indicated by the amilGFP positive clones

After we confirmed the effectiveness of our approach, the next step involved subcloning the T7-RBS-bla_NDM2&5 5 fragment into the BBa_J23101 plasmid. Subsequently, both of our constructs were introduced into the low copy number SC101 ori-based vector, pSB4K5 (kanamycin-resistant), resulting in the final constructs required for in vivo mutagenesis.

The in vivo mutagenesis was performed using a dual gene-specific mutator system capable of introducing transition mutations at consistent frequencies within a target sequence in vivo that was available for ordering from Addgene. (Seo D, Koh B, Eom GE, Kim HW, Kim S. A dual gene-specific mutator system installs all transition mutations at similar frequencies in vivo. Nucleic Acids Res. 2023 Jun 9;51(10):e59. doi: 10.1093/nar/gkad266. PMID: 37070179; PMCID: PMC10250238.).

We performed 3 rounds of mutagenesis for the J23101-T7-RBS-amilGFP construct and ten rounds for the J23101- T7-RBS-bla_NDM2&5 sequence using DH10B cells containing pDae079.

Monitoring the mutagenesis efficiency at the different rounds via the number of colonies with altered colour, brightness, and/or size

We combined samples from the NDM-2&5 constructs taken from each mutagenesis round and incubated them in liquid LB medium with increased meropenem concentrations. In contrast to the unmodified version, which could only grow on medium with up to 200 µg/ml, we observed bacterial growth in cultures exposed to 600 µg/ml of meropenem after the mutagenesis.