Mycoplasma genitalium (MG) is a sexually transmitted bacterial pathogen commonly associated with urethritis in men and cervicitis, endometritis, pelvic inflammatory disease, infertility, and preterm birth among women as it invades the upper reproductive tract. Infections can persist for months to years without effective treatment due to antimicrobial resistance. Current first-line drug choices are only successful in less than half of all patients. Preliminary in vitro data suggests that MG is susceptible to tinidazole (Tdz) and may fill the need for additional treatment options for drug resistant infections as it is already FDA-approved for other indications. As strains can vary in their susceptibility to particular drugs, we aim to identify the minimum inhibitory concentration (MIC), the concentration that inhibits growth by 99%, of Tdz against 10 MG clinical isolates. This data will determine if these strains are susceptible to Tdz, define the range of MICs, and reveal whether current strains have already developed resistance. Twofold dilutions of Tdz and doxycycline (DOX) antibiotics are added to MG clinical strains in 48-well plates and incubated at 37 C/ 5% CO2 for 21-28 days. DOX is one of the first-line drug choices with a known MIC; it is used to confirm that assays are performed correctly and to compare the effectiveness of Tdz. Four of the wells have no drugs to serve as a control to compare the number of genomes against those in the Tdz wells to determine MG inhibition. MG growth in each Tdz dilution is quantified with qPCR by isolating DNA from the wells. Calculations of the percent inhibition will dictate which antibiotic concentration is useful for treating infected patients. As physicians are already beginning to treat MG patients with Tdz, data regarding susceptibility of multiple isolates is crucial in informing these treatment regimens.

Mycoplasma genitalium (MG) is a sexually transmitted bacteria that causes urethritis in men and cervicitis, pelvic inflammatory disease, and infertility in women. MG infections vary in length: some infections are cleared within a few weeks, while others last for years and are difficult to treat due to antimicrobial resistance observed in MG. Previous studies have determined that MG was susceptible to nitroimidazoles. Preliminary analysis of four resistant strains of the MG type strain G37 found mutations in the MG_342 gene which encodes an oxidoreductase hypothesized to react with nitroimidazoles and produce the toxic form of the drug. In order to determine the possible mechanism for nitroimidazole resistance we will amplify the MG_342 region of MG. As MG clinical strains require months to establish cultures in vitro we will develop a sensitive PCR assay so that resistance-associated mutations can be identified directly from patient specimens in future clinical trials. The PCR products will then be sequenced to determine if the MG clinical isolates have mutations that confer resistance to nitroimidazoles. Understanding how these mutations affect nitroimidazole resistance could allow for future studies on nitroimidazoles as a possible treatment for MG.

The Field Programmable Cellular Arrays (FPCA) project at the Molecular Information Systems Lab (MISL) aims to improve current biocomputing systems utilizing spatial organizations of cellular components for logical operations. This can open doors for computation to be done within biological systems where artificial computation has never before been possible. This project encompasses three aims: characterizing the properties of signal propagation within E. coli, constructing biological circuit components for spatial signal processing, and optimizing bioprinting methods for circuits. Signal propagation through molecular signaling is employed to communicate the presence or absence of a signal and truth values to specific cells. We are demonstrating logical states of "1," "0," and the absence of a signal, thereby enabling differentiation between a logical "0" and a lack of signal. Two strains of bacterial cells are capable of performing the logic of a traditional "wire" and a NOR gate. Consequently, by arranging strains in spatially organized layouts, we engineer cellular arrays capable of performing diverse complex logical functions. This research is still in progress and we are in the process of optimizing NOR gate and wire strains. My role explores bioprinting circuits into hydrogels, and I have built a bioprinter with dual extruders to bioprint biological substances into containing slurries. This required designing, printing, and assembling 3D-printed parts. I am now characterizing the behavior of 3D printed materials into various containing slurries. This requires testing the ability of different bioprinting inks to encapsulate bacteria, testing various slurry methodologies, and testing interactions between combinations of these materials over space and time. I am also computationally modeling FPCA circuits at various levels of abstraction. Computational modeling serves to further broader computational goals in this project to compile a logic circuit specification into bioprinter GCODE.

Our research aimed to investigate the microbial diversity in Pieris rapae larvae obtained from three distinct settings: wild-collected larvae, larvae fed with leaves in a laboratory setting, and larvae fed with a standard artificial diet. Additionally, this investigation sought to identify the factors contributing to developmental delays in artificial diet-fed larvae, while also investigating the underlying cause of increased cases of infection and mortality affecting the laboratory colony. Initial observations indicated that wild larvae exhibited the fastest development and appeared healthiest, followed by those fed with leaves, both settings showing the lowest instances of disease and mortality. Conversely, the artificial diet group experienced developmental delays and frequently succumbed to disease before pupation. Based on these observations, we hypothesized that variations in microbial composition within different diets and environments contributed to the disparate outcomes. Furthermore, we speculated the possible identification of bacterial strains with known associations with promoting or hindering larval success. To analyze the larval gut microbiome, a specialized contraption was designed for sterile frass (feces) collection. Frass, skin, artificial diet, and leaf surfaces were swabbed onto TSA media, enabling the isolation of distinct colonies and species. Morphological characteristics, gram staining, and Sanger sequencing data of the 16S rRNA gene were used to identify the bacterial colonies at the species level. The identification of six bacteria at the species level yielded intriguing results, with particular interest surrounding Bacillus subtilis and Bacillus thuringiensis. The presence of B. subtilis exclusively in the artificial diet group suggests its potential involvement in the developmental delay observed in the larval population fed by artificial diet. Conversely, the identification of B. thuringiensis implicates a potential bacterial infection, which could explain symptoms experienced by the colony. These findings have potential implications for enhancing larval outcomes in colonies or, in contrast, pesticidal applications in an agricultural setting. 

Cannibalism is a widespread phenomenon among arthropods with consequences for population dynamics, stability, pathogen transmission, and epidemiology; while it is common in carnivorous arthropods, incidents of cannibalism among non-carnivorous species are less frequent, and the mechanisms behind cannibalism in these species are still poorly understood. Laboratory observations of Pieris rapae (cabbage white) larvae have found that larvae will engage in cannibalistic behavior under certain conditions. The objective of our research was to better understand the conditions that may cause this herbivorous larva to become cannibalistic. Experiments were conducted on variables of density, food source, food scarcity, instar, instar gaps, sex, and prior experience as possible influencing factors in the likelihood that a larvae will engage in cannibalism. Experiments in density, food source, and food scarcity had groups of larvae monitored for 48 hours at differing densities with a collard leaf, artificial diet, or withheld food. For experiments in sex, instar, and instar gaps inexperienced larvae were monitored in sets of two for 72 hours for cannibalism. For prior experience experiments, larvae that had previously engaged in cannibalism were paired with inexperienced larvae and monitored for another 72 hours. We found that higher larvae densities had a greater percentage of cannibalization. Furthermore, a lack of food source produced significantly higher cannibalization than either artificial or leaf diets. A higher rate of cannibalism was also seen in early instars. Prior cannibalistic behavior increased the rate of new cannibalism events within the first 24 hours of observations.These findings provide insights into cannibalistic behavior in P. rapae, with potential applications to prevent cannibalism in laboratory settings and related disease transmission, understand fluctuations in wild populations, and in agricultural settings to leverage cannibalism as a form of natural population control. Future research aims to isolate other possible factors contributing to cannibalism in P. rapae.

Temperature has been shown to impact the physiology and performance of ectothermic organisms including metabolic rate with enzymatic activity. The majority of studies have focused on thermal performance at constant temperatures although organisms experience a range of fluctuating temperatures. Understanding which aspects of performance are affected by fluctuations in temperature can be crucial for predicting how ectothermic species will respond to changes in their environment. Research has shown that ectotherms are more vulnerable to climate change and their growth rates are often temperature-dependent. Previous studies on the cabbage white butterfly (cabbageworm), Pieris rapae, have shown that caterpillars maximize growth at higher temperatures even when higher temperatures are infrequent. Physiological response of P. rapae on fluctuating temperature change will provide an important insight into its ability to adapt and survive to changing temperature ranges. For our study eggs from a lab colony were reared at two different fluctuating temperature regimes (11-35℃ and 18-24℃) until pupation. Our studies measured the overall performance of P. rapae, including egg hatch percentage, growth rate of 4th instar caterpillars, development time from 4th instar to pupation, percent survival, and mass gain at two fluctuating temperature regimes of 11-35°C and 18-24°C. We found that the percent survival was significantly higher at 18-24°C. There was significant mass gain and shorter development time of 4th instar to pupation at 11-35°C. Our results differed from other studies that found growth rate to be greater at 11-35°C. Many studies have found a significant genetic variation in growth rate, development rate, and pupal mass. Future studies using a split sib-family design, may provide insight into the mechanisms of thermal performance in a fluctuating thermal environment. Understanding how fluctuating temperatures impact ectothermic organisms, such as the cabbageworm, can contribute to a deeper understanding of organismal responses to climate change.