To validate LANCET’s performance, Lambert iGEM conducted an intensive series of experiments to assess the performance of both the diagnostic and therapeutic components of our project.

Diagnostic Results

PDL + PCR

To evaluate the performance of our proximity-dependent ligation (PDL) assay under varying expected target CspZ protein biomarker concentrations, we used polymerase chain reaction (PCR). We visualized the PCR results using 1.5% agarose gel electrophoresis to confirm that ligation products continued to produce amplifiable DNA fragments over time at progressively lower CspZ concentrations.

Figure 1. ImageJ analysis of post-PDL PCR on agarose gel electrophoresis with protein concentrations from 2-150 days pi, quantitatively validating PDL viability until at least 100 days pi.

We then used ImageJ to quantify the standardized integrated density of the fluorescent DNA bands, which proved that PCR amplification of PDL remained viable up to 100 days post-infection (pi) (see Fig. 1). Although the signal magnitude dropped over the 150 day period, with a loss of assay activity after 150 pi, our results demonstrate that our PDL system is viable for extended periods of time.

PDL + RPA

Once we verified the ability for PCR to amplify the produced double stranded DNA (dsDNA) PDL product, we decided to ensure that the Recombinase Polymerase Amplification (RPA) step was compatible within our overall assay. To choose optimal primers, we used CASPER, the software generated by our team for RPA/CRISPR-Cas12a assays. CASPER generated a pair of forward and reverse primers for use in RPA.

We used protein concentrations that reflected CspZ levels over time and tested the conjugated PDL-RPA assay in spiked samples of biosafe simulated blood serum to assess LANCET’s performance across a greater time period in physiologically relevant samples (see Fig. 2) (see Blood Serum Testing).

Figure 2. Comparative ImageJ analysis of PDL-RPA on agarose gel electrophoresis in standard in vitro conditions and with simulated blood samples with protein concentrations from 2-300 days pi, quantitatively validating assay viability for at least 250 days pi.

Results from ImageJ show that post-PDL RPA amplification remains significantly higher than the negative controls until at least 250 days pi, which surpasses the capability of PCR amplification (see Fig. 2). Experimentation with simulated blood serum showed a reduction in assay activity for some time periods, but generally showed no significant difference between samples run with standard in vitro conditions (see Fig. 2) (see Recombinase Polymerase Amplification).

These findings demonstrate that our approach with RPA using primer sequences generated from our CASPER software are more sensitive and robust than traditional PCR amplification across various stages of Lyme even under simulated hematological conditions.

PDL + RPA + Cas12a

To ensure that our overall assay was highly specific in its recognition of CspZ, we also conjugated a Cas12a step alongside the PDL-RPA reaction.

Cas12a was integrated alongside the PDL-RPA reaction to confirm that the system could generate a measurable fluorescent signal following target amplification. CRISPR RNA (crRNA) sequences were created both using commercial software and CASPER. Successful cleavage confirmed that Cas12a functions effectively within LANCET’s diagnostic workflow, accurately detecting amplified targets.

Figure 3. Comparative results of Cas12a assay with CASPER-designed crRNA on post-RPA PDL product, showing strong fluorescence for our Cas12a reactions, while all negative controls remain at baseline.

Both Cas12a reactions generated a strong fluorescence signal comparable to the positive control, confirming successful collateral cleavage and accurate detection of the amplified PDL-RPA product. In comparison, the negative controls depicted minimal fluorescence. These results indicate that our crRNA sequence as generated by our software tool exhibited precise target recognition, responding only to the correct amplified sequence (see Fig. 3).

To visualize the results of our complete diagnostic workflow, we integrated the PDL-RPA-Cas12a reaction with a lateral flow assay (LFA). (see Fig. 4)

Figure 4. Experimental reaction LFA (top) and negative control reaction LFA (bottom) showing different results at the test line and control line.

When placing the PDL-RPA-Cas12a reaction on the LFA strip, we observed a visible band at the test line, showing a positive result. The negative control showed only the control line, confirming that the test responds only when the target is present (see Fig. 4).

Therapeutic Results

To assess the effectiveness of CRISPR interference (CRISPRi) for gene repression, we implemented our system following the protocol optimized by the SHIELD 2024 Lambert iGEM project.

After confirming robust expression from the Bb0250 target construct in TXTL, we evaluated CRISPRi knockdown using sgRNA60.5 under our standardized conditions. As expected, the positive control shows strong fluorescence, while the sgRNA60.5 sample remains consistently lower across the course of time, indicating successful repression. This corresponds to ~73% reduction at the 8-hour read, with the effect appearing within the first few hours and remaining stable for the rest of the assay (see Fig. 5).

Figure 5. Fluorescence intensity (RFU) over time for Bb0250 sgRNA60.5 reactions.

After validating Bb0250, we assessed a second target, Bb0841, using sgRNA54.3 under the same conditions. As with Bb0250, the positive control shows strong fluorescence, while the sgRNA54.3 sample also remains consistently lower across the time course, indicating successful repression. This corresponds to ~65% reduction at the 8-hour read, with the effect evident within the first few hours and stable for the remainder of the assay (see Fig. 6).

Figure 6. Fluorescence intensity (RFU) over time for Bb0841 sgRNA54.3 reactions.

To test simultaneous repression, we combined the Bb0250 and Bb0841 reactions in a single TXTL assay with dCas9 adjusted for multiplexing. Despite the higher overall signal of the combined positive control, the multiplexed CRISPRi condition remained clearly lower across the time course and achieved ~76% repression at 8 hours, indicating that both guides function effectively together without loss of performance (see Fig. 7).

Figure 7. Multiplexed CRISPRi reaction targeting Bb0250 and Bb0620 indicating successful combined downregulation

The ThermoFisher LIVE/DEAD™ BacLight™ Bacterial Viability Kits were used to quantify the in vivo effect of the CRISPRi system using red and green fluorescence. Using our SynTek plate reader, setting two reads at wavelengths 528/20 (green), 645/40 (red). From the data, we determined successful fatal effect of the CRISPRi and determined two conclusions:

1. The sample transformed with pdCas9sgRNA (dCas9-sgRNA) had a 9.53-fold decrease in viable biomass in comparison to the sample transformed with just pBbdCas9s (dCas9 alone), measured using the average value of green RFU in each sample (see Fig. 8). This shows how the presence of the full CRISPRi system prevented and slowed overall bacterial growth.

Figure 8. Average Green RFU for dCas9 alone was 1,005,200, and 105,466.67 for dCas9-sgRNA, showing a 9.53-fold decrease for the experimental group, and proving the preventative effect of the CRISPRi.

2. The sample transformed with pdCas9sgRNA also had a 14.26-fold increase in the proportion of dead to alive bacteria compared to the sample transformed with pBbdCas9. The full CRISPRi system successfully killed a comparably large portion of the sample, proving the system’s efficacy in vivo (see Fig. 9).

Figure 9. Average dead/alive ratio for dCas9 alone was 0.219, and 3.13 for dCas9-sgRNA, a 14.26-fold increase in experimental dead/alive bacteria. The dCas9-sgRNA group also showed the dead cell signal constituting 60.7% of the total, with dCas alone presenting just 17.7%