Overview

Our ultimate goal was to detect environmental DNA (eDNA) from the organism Pseuodogymnoascus destructans , which causes White Nose Syndrome. To prove to the iGEM community that ADP1 could be engineered to detect such eDNA, we adapted an ADP1-based system created by De Vries et. al [1] to detect synthetic antibiotic resistance genes. Obtaining this proof of concept required us to design and engineer these biosensors and test their DNA detection capabilities in a controlled lab setting. The biosensors we developed were confirmed using nanopore sequencing.

Engineering The Detector

De Vrires et al. [1] created a system to detect the nptII (kanamycin resistance) gene by inserting a plasmid into ADP1 that contained a mutated nonfunctional version of the gene. We chose to adapt this system by putting the broken antibiotic resistance gene into ADP1's genome as opposed to putting it on a plasmid. As seen in figure 1, the non-functional gene was inserted into ADP1’s chromosome using a two-step genetic engineering protocol, which is discussed in-depth on our Engineering page. The broken gene we inserted into the chromosome with PCR and Golden Gate Assembley

Detector in Action

For this proof of concept 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. to create a biosensor for the detection of the nptII Antibiotic resistance gene. These genes confer resistance to β-lactam antibiotics–– such as ampicillin [1] or kanamycin [2], respectively. Figure 2 shows the biosensors detecting either the Wild-Type (WT) TEM-1 or nptII gene. When the WT target gene is detected, ADP1’s homologous recombination machinery “fixes” the broken gene by replacing it with the WT gene from the environment. Successful recombination with the WT gene results in resistance to the analogous antibiotic allowing ADP1 to grow on plates containing that antibiotic.

Engineering Confirmation

Before testing both detectors, we confirmed proper engineering of the nptII and TEM-1 biosensors. We amplified the nptII and TEM-1 broken genes from purified genomic DNA for each biosensor. The gels in Figure 3 show that these PCR products were the correct lengths confirming that the broken gene was inserted properly. Additionally, we used Nanopore Sequencing to confirm that the detectors were correctly made.

Testing & Results

To test the system, 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 selective plates and non-selective LB plates (See Figures 4 and 5). In both cases, the only sample that grew on selective plates was the one inoculated with the target gene in solution. The results of these experiments demonstrate that ADP1 can be engineered to detect exogenous DNA in the lab. Further experiments were run to determine the detection threshold and transformation frequency for both biosensors. The results for these experiments can be found on our Results page.

The antibiotic resistance gene detectors we developed expanded on the findings of De Vries and colleagues [1], by proving that ADP1's genome could be engineered to detect synthetic antibiotic resistance genes. Furthermore, these results demonstrate that Acinetobacter baylyi ADP1 is an ideal chassis organism for detecting exogenous DNA made in the lab. Both biosensors were analyzed to determine if they had been engineered properly, and we tested their ability to detect synthetic versions of the target WT gene. The results of these experiments were:

TEM-1 Detector

References