To improve ADP1’s viability as a chassis organism, we knocked out pbpG and acrB, two intrinsic genes that contribute to intrinsic β-lactam antibiotic resistance. pbpG encodes a penicillin-binding protein involved in peptidoglycan synthesis, and acrB encodes proteins involved in efflux pumps [1]. After deleting these genes using our ADP1 Genetic Engineering protocol, we designed experiments to test whether ADP1 became more sensitive to β-lactam antibiotics, such as Carbenicillin and Ampicillin. We measured the minimum inhibitory concentration (MIC) of each antibiotic for our engineered ADP1 strains. Our results indicate that several different “Wild-Type” ADP1 strains are already sensitive to Carbenicillin and Ampicillin, but knocking out pbpG and acrB from these strains increased ADP1’s susceptibility to these antibiotics.
Fig. 1. ΔpbpG and ΔacrB Carbenicillin MIC Growth Assay. Plates were streaked out overnight. ΔpbpG and ΔacrB biological triplicates, ADP1-ISx biological duplicates, and one E. coli MG1655 colony were scraped and diluted in 1 mL saline. 2 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB and 2-fold concentrations of Carbenicillin, ranging from 2-128 μg/mL . Cultures were grown at 30°C for 24 hours. E. coli was used as a negative control.
ΔpbpG vs. ΔacrB Carbenicillin MIC
Figure 1 shows both ΔpbpG and ΔacrB have a Carbenicillin MIC of ~2 μg/mL, the lowest concentration at which ADP1 shows noticeable growth. An interesting finding was that ADP1-ISx has an MIC of 16 μg/mL since we expected ADP1-ISx to be intinsically resistant to Carbenicillin. Although deleting ΔpbpG and ΔacrB did make ADP1 more sensitive to Carbenicillin, these results made us think about whether this domesticated ADP1-ISx strain [2] had removed some sort of DNA sequence conferring Carbenicillin resistance. Next, we tested ADP1-ISx vs. ADP1-ISx's ancestral strain, named "ADP1-WT" for the purpose of these experiments.
Fig. 2. ADP1-WT and ADP1-ISx Carbenicillin MIC Growth Assay. Plates were streaked out overnight. ADP1-WT and ADP1-ISx biological triplicates, one ΔpbpG colony, and one E. coli MG1655 colony were scraped and diluted in 1 mL saline. 2 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB and 2-fold concentrations of Carbenicillin, ranging from 2-128 μg/mL . Cultures were grown at 30°C for 24 hours. E. coli was used as a negative control.
ADP1-WT vs. ADP1-ISx Carbenicillin MIC
Figure 2 shows both ADP1-WT and ADP1-ISx have a Carbenicillin MIC of ~8 μg/mL, showing no noticeable differences in MIC between the two ADP1 strains. As our results continued to show that ADP1-WT and ADP1-ISx were susceptible to Carbenicillin, we took a closer look at Gomez and Neyfakh's work [1] to try to replicate their experiment as closely as possible. We obtained a culture of BD413, which was the "Wild-Type" ADP1 strain Gomez and Neyfakh used to demonstrate ADP1's intrinsic resistance to β-lactam antibiotics. Another modification was inoculating fresh cells grown in LB overnight into LB cultures containing antibiotics, rather than directly inoculating diluted colony mixtures. Additionally, we decided to start using Ampicillin, rather than Carbenicillin.
Fig. 3. Ampcillin MIC Pilot Growth Assay. Plates were streaked out overnight. Individual colonies for each strain were scraped and diluted in 1 mL saline. 5 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB. Fresh cells were diluted 1000-fold into standard culture tubes containing LB and 2-fold concentrations of Ampicillin, ranging from 2-128 μg/mL. Cultures were grown at 30°C for 24 hours. E. coli was used as a negative control.
Ampicillin MIC Pilot
As a preliminary experiment, we tested a variety of ADP1 strains to test their resistance to Ampicillin. Figure 3 shows both the ADP1-WT and ADP1-ISx strains exhibited an MIC of about 2~4 μg/mL, but our knockout pbpG and acrB strains exhibited an increased susceptibility to Ampicillin, with an MIC < 2 μg/mL. Using the same Ampicillin stocks for the final MIC experiment, we included a positive control, pUC19 (carrying an ampR gene) expressed in E. coli, to confirm our Ampicillin concentrations were correct. Our last thought was to test whether growth temperature had an impact on ADP1 antibiotic resistance.
Fig. 4. BD413 30°C vs. 37°C Ampicillin MIC Growth Assay. Plates were streaked out overnight. Single BD413 and E. coli MG1655 colonies were scraped and diluted in 1 mL saline. 5 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB. Fresh cells were diluted 1000-fold into standard culture tubes containing LB and 2-fold concentrations of Ampicillin, ranging from 2-128 μg/mL. Cultures were grown at 30°C for 24 hours. E. coli was used as a negative control.
BD413 30°C vs. 37°C Ampicillin MIC
Figure 4 shows that BD413 had an MIC ~2 μg/mL in both 30°C and 37°C. Growth in our positive control confirmed correct Ampicillin concentrations.
Our data consistently shows that "Wild-Type" ADP1 strains, including ADP1-ISx, ADP1-WT, and BD413 do not have intrinsic resistance to β-lactam antibiotics, but future experiments involving replicates for each strain are necessary to confirm our findings. However, our findings show that knocking out genes that contribute to β-lactam antibiotic resistance can increase ADP1's susceptibility to β-lactams (Figure 1), providing an additional selection method for ADP1.
Before developing the Pseudogymnoscus destructans detector, it was important to first prove that ADP1 could be engineered to detect environmental DNA. To accomplish this, we created a novel biosensor for the detection of the TEM-1 antibiotic resistance gene and adapted the work done by De Vries et al. [3] to create a biosensor for the detection of the nptII antibiotic resistance gene. De Vries et al. [3] created a system to detect the nptII (Kanamycin resistance) gene by inserting a plasmid into ADP1 that contained a mutated, non-functional version of the gene. We adapted this system by integrating the nonfunctional gene into ADP1's genome as opposed to putting it on a plasmid. In this section, we will reiterate the results found on our Proof of Concept page and discuss additional experiments we performed to better characterize ADP1’s ability to detect environmental DNA.
Both the TEM-1 and the nptII biosensor were constructed by inserting a broken version of the target gene into ADP1’s genome. For an in-depth explanation of how these detectors were developed, see our Proof of Concept Page. We confirmed that both biosensors were engineered properly by amplifying the nptII and TEM-1 broken genes from purified genomic DNA of each biosensor. Figures 5a and 5b show that the ADP1 genomes of both biosensors contained the correct length of the non-functional gene, confirming that we properly engineered the detector. Nanopore sequencing provided additional confirmation that both detectors were successfully engineered.
Fig. 5. Confirmation of (A) TEM-1 and (B) nptII Detectors. (A) TEM-1 Detector PCR contains the TEM-1 Broken Gene with the expected length of 1133 bp. (B) nptII Detector PCR contains the nptII Broken Gene with the expected length of 1639 bp.
After confirming proper engineering, a growth assay was performed on each biosensor. The goal of this assay was to determine if the biosensors could detect the WT target gene while simultaneously confirming that the broken gene in the chromosome was nonfunctional.
For this growth assay, the detector strains were grown in three separate solutions: one with the target gene (+DNA), one with no DNA (-DNA), and one with an off-target gene (nptII for the TEM-1 detector and TEM-1 for the nptII detector). After an overnight incubation period, each culture was plated onto non-selective plates LB plates and selective LB-Amp or LB-Kan plates (Ampicillin for the TEM-1 detector and Kanamycin for the nptII detector). Figures 6 and 7 show that the TEM-1 and nptII biosensors were unable to grow on the selective plate when no DNA (-DNA) was added, proving that the mutated version of the TEM-1 and nptII genes inserted into ADP1’s chromosome were non-functional. Additionally, the plates highlighted in orange show that each strain grew successfully on the selective plate when the target gene was added, confirming that the ADP1 cells detected the WT target gene.
LB
Off-target DNA
TEM-1 Gene
-DNA
LB+Amp
Fig. 6. Detection of the TEM-1 Gene. Each column shows plates of the the TEM-1 detector strain grown in the presence of the following DNA sequences. From left to right: the target TEM-1 gene, no DNA, and off-target DNA (nptII gene PCR product from pKD13). The top row shows the biosensor's ability to grow on Ampicillin. The bottom row shows the biosensor's ability to grow on LB. The highlighted plate shows the expected result; The TEM-1 detector strain was able to grow on ampicillin when the WT target gene was added.
LB+Kan
LB
NptII Gene
-DNA
Off-target DNA
Fig. 7. Target Gene Detection. Each column shows plates of the the nptII detector strain grown in the presence of the following DNA sequences. From left to right: the target nptIIgene, no DNA (-DNA), and off-target DNA (TEM-1 PCR product from pBTK404). The top row of plates shows the biosensor's ability to grow on Kanamycin plates. The bottom row shows the biosensor's ability to grow on LB. The highlighted plate shows the expected result; The nptII detector strain was able to grow on Kanamycin when the target gene was added.
It is important to note that whenever the off-target gene was added to the nptII detector strain, ADP1 was unable to grow on selective plates, but when the off-target gene was added to the TEM-1 detector, ADP1 shows some growth on the selective plate. In Figure 7, the top right plate shows this result. The off-target DNA we used to test the TEM-1 detector was PCR product of the nptII gene. The template DNA for this PCR was the pKD13 plasmid, which happens to have the TEM-1 gene as its backbone. We believe that growth on the yellow highlighted plate could have occurred because our PCR cleanup failed to remove all of the template DNA. One could do DpnI digestion to completely remove all the template DNA.
To better characterize ADP1’s ability to detect environmental DNA, we tested the biosensor against target DNA concentrations (ng/mL) ranging from 0.0001 ng/mL to 100 ng/mL. Figure 8 shows that both detectors could detect the target gene at 0.001 ng/mL. Additional tests showed that both biosensors could not detect the target gene at .0001 ng/mL, suggesting our system's detection threshold to be ~0.001 ng/mL.
Fig. 8. Detection Threshold Determination. TEM-1 Detector (left), nptII detector (right).
We also calculated the transformation efficiencies for each concentration by dividing the selective CFU counts by the amount of DNA in the solution. Figure 9 graphs the transformation frequency as a function of the DNA concentration. The graphs for both detectors show that as the concentration increases ten-fold, transformation frequency also increases ten-fold. This relationship, however, seems to level off near the 10 and 100 ng/mL concentrations, suggesting that our detectors possess a maximum transformation frequency.
Fig. 9. Transformation Frequency Graphs. (A) TEM-1 Transformation Frequency and (B) nptII Transformation Frequency.
Fig. 10. Transformation Frequency in the Presence of Mixed DNA. (A) nptII Detector and (B) TEM-1 Detector parsing through 10-fold amounts of E. coli genomic DNA relative to WT Gene PCR Product. All samples had ~1 ng/mL of the WT Gene added to the transformation mixture.
As we continued to further characterize our antibiotic resistance detectors, one of our goals was to determine whether ADP1 could parse through mixtures of random DNA to detect the correct WT gene. Figure 10 shows that there is not a significant difference in Transformation Frequency, when adding up to 1000-fold more E. coli genomic DNA to the transformation culture. This shows how ADP1 has potential to function efficiently in more realistic environments to detect DNA sequences of interest. Future experiments could test >1000-fold amounts of random DNA, multiple types of random DNA, and DNA detection thresholds in co-cultures.
To summarize, the TEM-1 and nptII biosensors we developed were confirmed to have been engineered properly and proved that they could successfully detect the target gene in a lab setting. Furthermore, we determined that both biosensors were successful, provided that the WT target gene was present at a concentration of at least .001 ng/mL. The ADP1 transformation frequency data indicates that the calculated transformation frequency is proportional to the concentration of the target gene in the environment. Finally, ADP1 transformation frequency not being affected by random DNA demonstrates how ADP1 can efficiently detect the correct WT target gene in the presence of other DNA.
We designed a modular detection construct that utilizes a YFP gene and repressor system in conjunction with a tdk/kan cassette to detect environmental DNA from Pseudogymnoascus destructans. Figure 11 shows the tdk/kan cassette and cymR repressor between the P. destructans homology regions. Additionally, the cymR associated YFP gene is inserted into a completely seperate part of the chromosome. This YFP gene is repressed by the cymR repressor until the target P. destructans DNA sequence is identified in the environment. At this point, ADP1 homologous recombination occurs, removing the tdk/kan cassette and the cymR repressor gene. ADP1 cells that successfully knocked out the tdk/kan cassette can then selectively grow on AZT and produce a YFP signal.
Fig. 11. P. destructans Modular Construct Designed to Detect White Nose Syndrome.
First, we identified a YFP and repressor system that was compatible with ADP1. We tested the cymR, lacI, vanR, and betI YFP repressor systems designed by Meyer et al. [4]. The YFP genes for these systems are controlled by promoters specific to each repressor. For example, the cymR-compatible YFP gene is controlled by a cymR-specific promoter. We attempted to individually integrate all four YFP variatons into ADP1's chromosome. This was completed by replacing the ADP1 acrB gene with individual YFP genes. Figure 10 shows that the CymR, LacI and BetI YFP gene insertions integrated successfully, and the VanR insertion did not integrate successfully.
Fig. 12. YFP Insertions. From left to right: Ladder, Control, ADP1-ISx, cymR-YFP, vanR-YFP, lacI-YFP, and betI-YFP. Expected length for each YFP variation was ~3000 base pairs.
To determine which YFP variations were functional, the fluorescence intensity of each engineered YFP strain was measured with a plate reader using an excitation wavelength of 488 nm and emisssion wavelength of 530 nm. The YFP fluorescence measurements were compared to the ADP1-ISx strain. As seen in Figure 11, the cymR and lacI YFP genes had fluorescence readings greater than ISx’s basal fluorescence, signaling that these YFP genes were compatible in ADP1-ISx. This data led to our decision to use the cymR-YFP repressor system for the White Nose Syndrome detector construct.
Fig. 13. YFP Average Fluorescence.
After identifying the cymR-YFP repressor system, we began create the more complex White Nose Syndrome detector construct. To create this detector, we ran three different BsaI Golden Gate Assembly (GGA) reactions. Figure 13 shows how we used these three reactions to create the detector construct.
Fig. 14. Creating the P. destructans Detector Construct. Reaction 1 ligates the ACIAD2049 Upstream part with the P. destructans Upstream part. Reaction 2 ligates the cymR repressor, the P. destructans Downstream part and the ACIAD2049 Downstream part together. Reaction 3 ligates the Reaction 1 and Reaction 2 products with the tdk/kan cassette, creating the entire detector construct. All reactions used BsaI digestion.
After running the three GGA reactions, we succesfully produced the entire detector construct. Figure 15 shows a bright band ~7 kb, the expected size of Reaction 3. The intensity of this band indicates that the primary DNA sequence amplified in Reaction 3 is the correct White Nose Syndrome detector construct. This Golden Gate product was then transformed into ADP1's genome using Step One of the ADP1 Genetic Engineering protocol outlined on our Parts Page.
Fig. 15. Confirmation of P. destructans Detector Assembly. From left to right: Ladder, Reaction 3 GGA Product. Expected length of the White Nose Syndrome Detector is ~7 kb.
To integrate the P. destructans detector cassette into ADP1's genome, we added 15 μl of the above GGA reaction to a standard culture tube containing 500 μl of LB and 35 μl of an overnight culture of ADP1 cells with the cymR-controlled YFP gene. After overnight incubation (30°C, 200 rpm), we plated 100x diluted transformations onto LB-Kanamycin (Kan) plates to select for cells that integrated the construct. The plates in Figure 16 demonstrate that some cells successfully integrated the detection cassette.
Fig. 16. Inserting the White Nose Syndrome Detector Cassette into ADP1's Genome.
We picked 3 colonies from the highlighted LB-Kan plate to inoculate and make glycerol stocks. We attempted to test these WNS detector strains and their ability to detect the target P. destructans gene. However, all of the tests we ran proved to be inconclusive. Our results suggest the detector strains may not have been properly engineered. To test this, we ran a growth assay to determine if any of the P. destructans triplicates (034A, 034B, 034C) produced the expected growth pattern (Figure 17). We streaked ADP1-ISx, ADP1 containing an Integration tdk/kan cassette and the three P. destructans detector strains (034A, 034B, and 034C) onto both Kanamycin and AZT plates. A properly engineered WNS detector strain would show colony growth on the LB-Kan plates while showing no growth on the LB-AZT plates. The 034A and 034C strains did not show this growth pattern, but 034B proved to be promising. This strain was susceptible to AZT, and the zoomed in image on Figure 17 shows very small colonies, suggesting that the 034B strain may possess the correct White Nose Syndrome detector construct. We are planning to sequence the entire P. destructans Detector 034B strain to determine if the proper construct was integrated into the ADP1 genome. We hope to have these results by the Grand Jamboree.
Fig. 17. Streaks of Detector Strains on Kanamycin and AZT
This year, the 2022 UT Austin iGEM team improved ADP1 as a chassis organism for synthetic biology by successfully deleting the acrB, pbpG, ACIAD2049, and recJ genes. Additionally, we characterized ADP1’s susceptibility to β-lactam antibiotics and documented how deleting the pbpG and acrB genes increase β-lactam susceptibility. We also demonstrated how to engineer a ADP1-based biosensor by creating two antibiotic resistance biosensors. These biosensors were capable of detecting exogenous DNA sequences in a lab setting. Furthermore, we show that ADP1 can efficiently detect the target DNA sequence in the presence of other DNA sequences. Finally, we designed and successfully created the DNA sequence required to make the White Nose Syndrome Detector, but we were unable to confirm whether we successfully inserted the complex construct into ADP1’s chromosome. Future directions for this project include confirming the successful creation of the White Nose Syndrome Biosensor, testing our biosensors' abilties to detect both synthetic and environmental P. destructans DNA in realistic environments, and further characterizing ADP1's natural competence capabilities.
[1] Gomez, M. J., & Neyfakh, A. A. (2006). Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrobial agents and chemotherapy, 50(11), 3562-3567. https://doi.org/10.1128/AAC.00579-06
[2] Suárez, G. A., Dugan, K. R., Renda, B. A., Leonard, S. P., Gangavarapu, L. S., & Barrick, J. E. (2020). Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining. Nucleic acids research, 48(8), 4585-4600. https://doi.org/10.1093/nar/gkaa204
[3] De Vries, J., Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Molecular and General Genetics, 257, 606-613. https://doi.org/10.1007/s004380050688
[4] Meyer, A., Shegall-Shapiro, T., Glassey, E., Zhang, J., Voigt, C. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small molecule sensors. Nature Chemical Biology 15, 196-204 (2019). https://doi.org/10.1038/s41589-018-0168-3
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