Biology subjects

Mishler, D. M.

Publications and source records attributed to Mishler, D. M..

5 recordsLinked to original sources

bioRxiv2026

Caffeinated Coli: Inspiring the Next Generation of Scientists through Synthetic Biology

The Caffeinated Coli Educational Module brings inquiry-driven learning to high schools, introducing students to scientific research, synthetic biology, and genetic engineering. The module focuses on the exploration of a genetically engineered strain of E. coli modified to grow exclusively on caffeine and, as such, can be used as a measurement device to determine the amount of caffeine in a liquid or beverage. Students conduct two bioassay experiments using these bacteria. During this process, they learn to create cultures and then measure bacterial growth followed by calculating the caffeine concentrations of unknown samples using their own data. This flexible module contains five weeks of original lectures and student learning materials that follow Next Generation Science Standards (NGSS), allowing the content to be adapted for basic, intermediate, or advanced biology courses. Upon completion of the module, students and teachers expressed that the most memorable aspects of the module include collaboration with peers, hands-on learning of content, and the opportunity to interact with the professor/mentors through office hours. Already implemented in 8 Texas high schools over the past two academic years, our module inspires STEM learning while bringing 21st century biology research to new audiences.

scientific communication and education↗

bioRxiv2026

Refactored genetic parts for modular assembly of the E. coli MccV type I secretion system used to screen class II microcin candidates from plant-associated bacteria

BackgroundMicrocins are small antibacterial proteins secreted by gram-negative bacteria. The activities of new microcins discovered using bioinformatic searches need to be validated and characterized to understand how they mediate competition in microbiomes and to evaluate their potential as new therapeutics for combating antibiotic resistance. Engineered plasmids containing the type I secretion system associated with Escherichia coli Microcin V (MccV) can secrete heterologous proteins, including other class II microcins, and this system functions in other bacterial hosts. However, existing microcin secretion constructs are not designed for easily swapping components -- such as origins of replication, resistance genes, promoters, and signal peptides -- that may need to be changed for compatibility with other chassis. ResultsWe refactored the E. coli MccV type I secretion system into genetic parts compatible with a modular Golden Gate assembly scheme and used these parts to construct two-plasmid microcin secretion systems. In our design, one plasmid encodes the type I secretion system proteins, and the other encodes a signal peptide fused to the cargo protein that will be secreted. We tested two versions of a system with inducible promoters separately controlling expression of the components on each plasmid. One used plasmids that replicate in E. coli and its close relatives. The other used broad-host-range plasmids. When induced to secrete MccV, both versions produced similar zones of inhibition against a susceptible strain of E. coli. Next, we identified putative class II microcins in genomes of bacteria from plant-associated genera (Pantoea, Erwinia, and Xanthomonas) using an existing bioinformatics pipeline. We screened 23 of these putative microcins for E. coli self-inhibition. Seven exhibited some inhibition, mostly later in growth curves, but none had effects that were comparable in strength to MccV. ConclusionsThe genetic parts we created can be assembled in various combinations into tailored systems for secreting small proteins from diverse bacterial chassis. These systems can be used to further characterize the targets of novel microcins and to secrete these or other small proteins for various applications. For example, beneficial bacteria used in crop protection could be engineered to secrete microcins that kill or inhibit plant pathogens to increase their efficacy.

synthetic biology↗

bioRxiv2025

Engineered Acinetobacter baylyi ADP1-ISx cells are sensitive DNA biosensors for antibiotic resistance genes and a fungal pathogen of bats

Naturally competent bacteria can be engineered into platforms for detecting environmental DNA. This capability could be used to monitor the spread of pathogens, invasive species, and resistance genes, among other applications. Here, we create Acinetobacter baylyi ADP1-ISx biosensors that detect specific target DNA sequences through natural transformation. We tested strains with DNA sensors that consisted of either a mutated antibiotic resistance gene (TEM-1 bla or nptII) or a counterselectable gene flanked by sequences from the fungus Pseudogymnoascus destructans, which causes white-nose syndrome in bats. Upon uptake of homologous DNA, recombination restored antibiotic resistance gene function or removed the counterselectable gene, enabling selection of cells that sensed the target DNA. The antibiotic resistance gene and P. destructans biosensors could detect as few as 3,000 or 5,000,000 molecules of their DNA targets, respectively, and their sensitivity was not affected by excess off-target DNA. These results demonstrate how A. baylyi can be reprogrammed into a modular platform for monitoring environmental DNA.

synthetic biology↗

bioRxiv2024

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

synthetic biology↗

bioRxiv2018

Synthetic genome defenses against selfish DNA elements stabilize engineered bacteria against evolutionary failure

Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of five, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.\n\nSignificanceEngineered cells often cease to function or lose productivity when mutations arise in their genomes. Mobile DNA elements, such as transposons, are major sources of these inactivating mutations. Eukaryotic genomes have evolved flexible epigenetic defenses against mobile DNA that help them to maintain genome integrity, but bacteria do not possess comparable silencing systems. We developed a synthetic control system based on CRISPR interference that can be used to give bacterial cells a reprogrammable line of defense against selfish DNA elements in their genomes. We show that this system effectively represses multicopy transposons and multiple families of transposons. Limiting selfish DNA proliferation within a genome in this way improves the reliability of genetically engineered functions in replicating bacterial cell populations.

evolutionary biology↗