Results

The Culmination of All Our Hardwork

Results

CRISPRi

CRISPRi GFP

Lambert iGEM successfully completed experimentation by running a CRISPRi reaction to downregulate deGFP, following the protocol outlined by Marshall et al. (2020). Our objective was to achieve high fluorescence levels in positive controls and effective repression of deGFP production by the sgRNAs as expected. We managed to successfully repress deGFP production, with sgRNA6 achieving 54.8% downregulation and sgRNA9 achieving 59.8% downregulation (see Fig. 1). This concluded our GFP testing, validating the efficacy of our Sigma70 TXTL kits and the dCas9 protein we used, thereby confirming the success of our CRISPRi reaction.

Figure 1. Graphs of deGFP expression from our positive control (deGFP and Arbor Bioscience’s deGFP), negative control, sgRNA6 and sgRNA9 assays. The assays with sgRNA6 and sgRNA9 resulted in fluorescence levels higher than the negative control but lower than the positive control, indicating success in GFP CRISPRi.

CRISPRi M. tb

After confirming the success of our CRISPRi in downregulating deGFP and verifying the efficacy of our reagents and concentrations, we designed and tested sgRNAs targeting the inhA gene—a critical factor in M. Tuberculosis’s pathogenicity—as a proof of concept. We tested four inhA sgRNAs and found that sgRNA70 was effective when paired with an inhA target construct at a working concentration of 5nM and sgRNA70 at 24.75nM (see Fig. 2). We then followed guidance from Ms. Kathryn Eckartt, a PhD student specializing in CRISPRi research at Rockefeller University, to identify sgRNA70’s percent repression. She advised that a 50% reduction in fluorescence was necessary for functional repression of the inhA gene. Our calculations showed that sgRNA70 achieved a 60.4% decrease in fluorescence compared to the positive control, indicating effective downregulation of the inhA gene. This significant reduction indicates that sgRNA70 can effectively downregulate the inhA gene, disrupting the pathogenicity of M. tuberculosis and enhancing our ability to eliminate the bacteria.

Figure 2. Successful inhA CRISPRi using sgRNA70 (24.75nM working concentration) and inhA target construct (5nM working concentration) achieving 60.4% decrease in fluorescence compared to the positive control.

Toeholds

The use of both our CRISPRi and toehold systems has shown promising results when using the inhA gene as a proof of concept for combating the rise of antimicrobial resistance. We successfully characterized six toehold constructs, with inhA toehold 4 emerging as the most efficient design. This toehold demonstrated the highest GFP expression when paired with a trigger concentration of 15 nM, proving its potential as an effective biosensor. Although the expression levels were lower than our positive control, the clear separation between toehold 4 and other constructs supports the successful binding to the complementary trigger (see Fig, 3, 4, and 5). These findings provide a solid foundation for further development of our approach, potentially leading to new strategies for targeting critical genes in antibiotic-resistant bacteria without relying on conventional antibiotics. In the future we will focus on optimizing the toehold design for even greater sensitivity and exploring its application in conjunction with our CRISPRi system.

Figure 3. Characterization curve for inhA toehold 4 with 10 nM to 20 nM trigger concentration
Figure 4. Characterization curve for inhA toehold 5 with 10 nM to 20 nM trigger concentration
Figure 5. Characterization curve for inhA toehold 6 with 10 nM to 20 nM trigger concentration

Analysis of the concentration curves for our three toeholds reveals that inhA toehold 4 outperformed all other construct combinations. Specifically, at 1nM concentration with 15nM of trigger DNA, toehold 4 exhibited a significantly higher expression rate. While its peak expression of approximately 200 RFU falls short of the positive control values, the clear distinction between toehold 4 and its counterparts strongly suggests successful binding to its complementary trigger.

HP-Soil

Lambert iGEM collected soil samples from various locations in Georgia and tested them for AMR. With the help of MiniPCR’s Sampling Soil for Antibiotic Resistance kit, we were able to extract DNA from 15 soil samples and successfully test them with chosen primers. Our first set of experiments aimed to extract DNA from the soil. The extraction protocol (see Experiments) includes homogenizing soil samples, lysing cells, and purifying the DNA. The resulting DNA was assessed using a NanoDrop spectrophotometer, with concentration values ranging from 10 ng/μl - 150 ng/μl.

We compiled our data (see Table. 1) and created a map displaying the results. Our samples exhibited the widespread presence of the tetracycline resistance gene TetM across the state (see Fig. 6). Initially, there appears to be a correlation between positive Tet resistance and proximity to the Chattahoochee River, though more samples will be needed to confirm this observation. The detection of TetM resistance sequences across these sites suggests that soil can act as a reservoir for resistant bacteria, posing a potential public health risk. These findings underscore the importance of continued surveillance and proactive measures to address the spread of AMR in environmental settings.

Overall Tetracycline Results

Location:Tube Name:Volume of eDNAAMR-Tetracycline
Nichols Rd T1 -T2 / Lambert HS1A0Fail
Nichols Rd T3 /Lambert HS3F13.1 ng/ulTetB
Chatahochee pointe RiverCP13.0 ng/ulTetM
WindermereW13.7 ng/ulTetM
Chatahochee River National Recreation CenterNR14.0 ng/ulTetM
Vogel State ParkV16.4 ng/ulTetM
Nichols viewNV24.9 ng/ulBoth
River MistRM6.5 ng/ulNegative
Cauley Creek RiverCC18.3 ng/ulFail
Villa RicaS112.8ng/ulTetB
Fish Hatchery: RiversideFHR0.7 ng/ulNegative
Fish Hatchery: Mort BinFHM35.0ng/ulNegative
Site 2 FarmS244.6ng/ulFail
Wastewater treatment plantWT74.6ng/ulNegative
Swaunee Mountiain ReserveSM72.6ng/ulFail
Cumming Fair groundsCF110.2ng/ulNegative
West Bank LanierWB145.9ng/ulFail
Buford Dam ParkBD12.2ng/ulFail
Table 1. Compiled data from tetracycline testing
Figure 6. Map showing tetracycline resistance

The results from testing for the presence of integrons were invalid due to persistent primer dimerization during gel electrophoresis (see Notebook). With primer dimers persisting after multiple tests, our team believes that initial positive results may have been false positives. The absence of proper band lengths suggests the sampling area may have been too limited to capture a representative portion of the microbial population. This better aligns with our expectations, as integrons are relatively rare in soil environments. Statistically, it is expected that only around 15% of environmental samples would test positive for integrons (Byrne-Bailey et al., 2011), meaning a more extensive and geographically diverse sampling strategy may be required to detect integrons in this region (see Table. 2).

Integron Results

LocationTube NameRB 201/202RB 317/320Hep74/51HS 463a/464
WindermereWNegativeNegativeNegativeNegative
Chattahochee River National Recreation CenterNRNegativeNegativeNegativeNegative
Vogel State ParkVNegativeNegativeNegativeNegative
Nichols viewNVNegativeNegativeNegativeNegative
River MistRMNegativeNegativeNegativeNegative
Cauley Creek RiverCCNegativeNegativeNegativeNegative
Villa RicaS1NegativeNegativeNegativeNegative
Fish Hatchery (river)FHRNegativeNegativeNegativeNegative
Fish Hatchery (mort bin)FHMNegativeNegativeNegativeNegative
Site 2 farmS2NegativeNegativeNegativeNegative
Cumming FairgroundsCFNegativeNegativeNegativeNegative
Sawnee MountainSMNegativeNegativeNegativeNegative
West Bank LanierWBNegativeNegativeNegativeNegative
Buford Dam ParkBDNegativeNegativeNegativeNegative
Wastewater Treatment CenterWTNegativeNegativeNegativeNegative
Table 2. Compiled data from integron testing

Gel Results

After the extraction, the samples were run on a gel to determine what type of resistance was present in the sample. These samples were run on a 2% agarose gel along with a positive and negative control utilizing tetracycline primers, TetB and TetM (see Fig. 7). Well A consists of a 16s primer, wells B, D, & F consisting of TetB primer, and wells C, E, & G consisting of TetM primer (see Experimentation). The positive gel results displayed 4 positive TetM samples, 2 positive TetB samples and 1 sample carrying both TetB and TetM resistance.

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Figure 7. Slide 1: Chattahoochee Point (CP6) showing positive fluorescent band for TetM. Slide 2: Vogel State Park (V): showing positive fluorescent band for TetM. Slide 3: Chattahoochee River National Recreation Center (NR) showing positive fluorescent band for TetM. Slide 4: Windermere Park (W) showing positive fluorescent band for TetM. Slide 5: Lambert High School (3F) showing positive fluorescent band for TetB. Slide 6: Villa Rica (S1) showing positive fluorescent band for TetB. Slide 7: Nichols View (NV) showing positive fluorescent band for TetM and TetB.