Chelsea, SUNY New Paltz
Mentor: Pallavi Ghosh

Mycobacterium abscessus is an environmental mycobacterium that can be found in water, soil and dust. M. abscessus is an opportunistic pathogen capable of causing lung or skin infections in immunocompromised individuals. Due to the high level of antibiotic resistance, these infections are even more difficult to treat compared to other mycobacterial infections. In order to study an organism it is important to have reliable genetic tools. These tools are used at a molecular level to examine the phenotypes of genes of interest. Recombineering is the most commonly used genetic tool to create isogenic deletions in mycobacteria; however this process is highly inefficient in M. abscessus. In this study, we explored the effect of substrate length on recombineering efficiency in M. abscessus. We have used 500nt and 1000nt of homology on either end of Mab3028 and are presently screening for differences in recombineering efficiency.

Joe, Washington & Jefferson College
Mentor: Michael Koonce

Macroscopic motion exhibited by organisms begins at the cellular level with the work of various motor proteins: myosins, dyneins, and kinesins. Kinesin motor proteins act using microtubule filaments along which they move in order to perform tasks from transporting of vesicles, mitochondria, or other organelles, to directing mitosis. Due to their essential role in cellular processes, disruptions in normal kinesin functioning have been revealed to be involved in certain diseases. These motor proteins have been well documented in organisms at warm temperatures (over 40 kinesin proteins have been characterized in humans), yet there exists little investigation into these important proteins with regards to how they function in organisms adapted to the extreme cold. One such group of these organisms are the Foraminifera. These carnivorous protists have been found to exist in many of Earth’s marine ecosystems. Though largely recognized for the role their tests, or shells, play in in the fossil record and global carbon levels, Forams, particularly those found in the cold waters of Antarctica, have remained largely unstudied. Forams collected from sediment of Explorers Cove, Antarctica have been observed to exhibit robust organelle transport activity despite temperatures reaching as low as -1.8°C. Under similar conditions, activity in mammalian cells would grind to a halt and cause the cell to shrivel and die. To investigate whether there are structural modifications in the kinesins of these Forams which would confer the ability to function at such temperatures, samples of four species were transported to Albany, New York. The RNA of cells was collected, and used as the template to generate DNA through reverse transcription polymerase chain reaction (RT-PCR) using custom primers which were designed to target regions within the kinesin motor domain that have been documented as highly conserved motifs involved with nucleotide binding. DNA collected from the RT-PCR was inserted into vectors and used to clone into competent E. coli cells, interrupting the LacZ gene. Upon verification of vector and DNA insertion via restriction enzyme digest, the DNA was isolated to be sent out for sequencing. Initial analysis of sequences through nucleotide and protein Blasts revealed sequences of kinesin proteins. Upon further analysis, however, the samples were identified as fungal in origin; these characterizations may not be definitive, reflecting on the disparity of information available on Forams. Further sequence analysis revealed that the nucleotide and amino acid sequences across the samples and species were nearly identical. This, in addition to the presence of kinesin residues in water controls, suggest contamination, though contaminant presence was inconsistent.

Sarah, Russell Sage College
Mentor: Joseph Wade

CRISPR, or clustered regularly interspaced short palindromic repeats, is an adaptive immune system found in roughly 50% of bacteria and 90% of archaea. This system works through three stages: adaptation, biogenesis, and interference. Acquisition of new CRISPR immunity elements, known as spacers, occurs during adaptation. There are two types of spacer acquisition: naïve and priming, both of which require different protein elements. Naïve acquisition requires Cas, or CRISPR associated genes 1 and 2, whereas priming adaptation, the focus of this project, requires Cas 3. A variety of elements are necessary to make adaptation function; however, these requirements have only recently been explored.

Integration host factor, or IHF, has recently been identified as a protein element necessary for naïve adaptation (Nunez et al., 2016). In this study we investigated if IHF plays role in priming spacer acquisition, and also explored Hu as a potential protein element necessary to adaptation, as Hu is a homologue of IHF.

Additionally, recent studies have shown that the leader sequence is a necessary genetic element for spacer acquisition (Yosef et al., 2012). The leader sequence is a region of roughly 150 base pairs that separates the CRISPR array and Cas genes. A study performed in 2012 showed that the 40-60 bp region upstream from the first repeat in the CRISPR I array is important to spacer acquisition (Yosef et al.). In this project, 10 bp chunks were deleted from the 40-60 bp region via FRUIT, or flexible recombineering using the integration of ThyA. A region conserved across many Type I-E CRISPR systems was also deactivated through point mutations introduced via FRUIT. Priming assay results suggest that IHF and all three leader sequence elements described are necessary for priming spacer acquisition, while Hu is not.

Jen, The College of St. Rose
Mentor: Anil Ojha

We are using the model organism M. smegmatis to investigate biofilm-associated persistence. Biofilm persistence is indicated in the resistance of Mycobacterium tuberculosis (Mtb) to antibiotics, requiring a 6-month long antibiotic regimen in tuberculosis patients. This in vivo persistence can be compared to the persistence of mycobacterial biofilms in vitro, which undergo differentiation and experience heterogeneity. To better understand the long-term persistence of biofilms, we tested whether all of the cells in a differentiated biofilm were viable. Using FACS (fluorescence activated cell sorting) to separate active from inactive cells, we found that both populations were able to recover. Since we know the cells are alive, we can begin to study their genetic activity. A cluster of genes were identified in our lab which are approximately 1000 times more active in biofilms compared to planktonic (single cells), and expression of these genes increases tolerance to the antibiotic rifampicin. The main objective of this project was to design a GFP (green fluorescent protein) fusion under control of different genetic promoters. Once functional, these promoter fusions will allow us to visualize the population of cells expressing these genes and therefore acquiring tolerance.

Priscilla, University of Puerto Rico – Mayaguez & Julian, University at Albany
Mentor: Randall Morse

One essential factor in the eukaryotic pre-initiation complex is transcription factor II H (TFIIH), which phosphorylates the carboxy-terminal domain of RNA polymerase II to enable its release for transcription. An important subunit of TFIIH is coded in budding yeast (Saccharomyces cerevisiae) by KIN28, and is connected to Mediator function. KIN28 is an essential gene in yeast, and Kin28 inactivation results in loss of Pol II transcription. Interestingly, however, exceptions to this model have previously been observed, primarily upon heat shock induction. Unpublished data from our laboratory indicates that MET genes may also be inducible without the presence of essential pre-initiation factors. To expand on this study, we utilized kin28-anchor away mutants to investigate MET expression in vivo upon CdCl₂ induction. We observed that, in two independent studies analyzing RNA expression and chromatin immunoprecipitation of RNA polymerase II, MET genes were upregulated upon cadmium induction, with and without TFIIH intact. These studies complement previous findings, which challenge the notion that TFIIH is a universally required subunit for eukaryotic transcription.

Samantha, Brown University
Mentor: Sam Bowser

Antarctica is popularly thought to be a barren environment, but under the Antarctic sea ice, the ecosystem is actually very lush. In this harsh environment, as in all of nature, there are living inhabitants that interact in a variety of ways including as predator and prey. Understanding predator-prey relationships is important because these interactions direct how energy moves through the food web. Disruptions in any part of the food chain, even at the lowest levels, can have far-reaching effects for populations higher up on the trophic scale. One type of organism that lives beneath the Antarctic ice is a foraminifera (foram) called Pyrgo peruviana. Forams are single-celled organisms characterized by unique protective shells, called tests. Pyrgo peruviana, which inhabit the sediment of Explorers Cove (McMurdo Sound, Antarctica), were collected at various times from 1987 to 2006. To identify possible predators and determine the frequency of predation on these organisms, marks on the tests were documented using light and scanning electron microscopy. Four mark types were identified: two that were likely due to predator activity, one caused by errors during calcification, and one caused by mechanical damage during specimen handling. Although the foram predator remains unknown, we found significant inter-annual variation on the extent of predation in this environment.

Michelle, William Smith College
Mentors: Nilesh Banavali, Janice Pata

DNA polymerases help synthesize new DNA strands using complementarity with existing template strands. Their ability to do this without errors varies depending on the polymerase type. Next Generation Sequencing (NGS) allows billions of DNA strands to be sequenced in one experiment. Here, we analyzed DNA polymerase errors made by different types of polymerases (dpo4, KAPA, gkPolc, and saPolc) using a novel NGS-based DNA polymerase assay. Since NGS techniques have sizable error rates, this novel assay uses an enhanced accuracy NGS technique adapted from the duplex sequencing method. We used Du Novo, a pipeline to process raw duplex sequencing data, to analyze and correct millions of reads into a smaller set of single-stranded consensus sequences. A designed De Bruijn sequence containing all possible 5-mers was used as the template to get full monomer error spectra for each DNA polymerase, and the specific differences between polymerases that were identified are discussed.

Jennifer, Case Western Reserve University
Mentor: Todd Gray

Many bacteria spread antibiotic resistance through a process called conjugation, where bacteria transfer genes to each other in the form of a plasmid. In contrast, a different type of DNA transfer, called Distributed Conjugal Transfer (DCT) occurs in Mycobacterium smegmatis, a non-pathogenic relative of Mycobacterium tuberculosis (Mtb). DCT generates complex, randomly mixed transconjugant genomes with donor genes comprising up to a quarter of the genome. It is known that DCT involves the type VII secretion systems and, very recently, the ESX-1 secretion system was found to be involved in activating expression of ESX-4 during mating. If ESX-1 or ESX-4 are knocked out in the recipient strain, DCT does not occur. In Mtb, ESX-4 gene expression is attributed to the transcription sigma factor, SigM. We hypothesized that in DCT, ESX-1 might be activating ESX-4 expression by regulating the M. smegmatis SigM.  Current models suggest that SigM protein is normally sequestered by its membrane-bound anti-sigma factor, and released as cells enter stationary phase. We knocked out the sigM gene (6931) and the anti-sigM gene (6932) separately or together in the M. smegmatis recipient strain. Complemented strains of these knockouts were also created. These knockout recipient strains were mated with wild type donor strains to see if DCT could occur. We also created a fluorescent protein reporter for ESX-4 transcription.  Collectively, these strains will provide an initial assessment of communication networks linking ESX-1, ESX-4, SigM, and DCT in M. smegmatis. In the future, this may help characterize similar networks in other mycobacteria.

Maddie, Washington College
Mentor: Laura Kramer

Although first discovered in 1947 in the Zika Forest in Uganda, the Zika virus (ZIKV) had remained relatively absent from the attention of researchers and media alike until the 2015-2016 ZIKV outbreak in the Americas. In February 2016, the World Health Organization declared a Public Health Emergency of International Concern in regards to growing concern of a connection between ZIKV and microcephaly and Guillain-Barré syndrome. In the Americas, ZIKV is spread primarily by Aedes aegypti and Aedes albopictus mosquitoes, who range into the northern United States, and through sexual, perinatal, and blood transfusion transmission. This investigation aimed to characterize ZIKV replication in cell culture of Puerto Rican (PR), African (YMP) (highly passaged in vertebrate cells), Honduran (HON), and Cambodian (CAM) ZIKV strains in Aedes albopictus, Aedes aegypti, Green Monkey Kidney, Human Embryonic Kidney, Human Lung Epithelial, and Neuroblastoma cell lines. Growth curves were generated by infecting each cell line in triplicate at 0.01 MOI and harvesting samples at 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours post infection. Viral titers at each time point were calculated by plaque assay on Vero cells and were analyzed using a One-Way ANOVA and Tukey's Multiple Comparison test and T-tests. In the invertebrate cells, all ZIKV strains reached the highest titers in Aedes albopictus, as the cells lack an intact RNAi; all strains exhibited competent replication in Aedes aegypti cells, supporting that both species are competent vectors. PR and HON replication was significantly lower than CAM and YMP in Human Embyonic Kidney cells, Human Lung Epithelial and Neuroblastomas, despite the fact that the PR and HON strains are associated with significant disease. PR and HON did not replicate in Neuroblastomas. Further investigation should be conducted to determine PR and HON infection and pathogenicity in vertebrate cells.

Matt, Siena College
Mentor: Michael Koonce

Much like a vertebrates skeleton, the interphase microtubule arrays found within eukaryotic cells have long been known  for their roles in providing both structural support, as well as motility through  directing  vesicular transport and organelle trafficking. While there is continuing investigation into the effector proteins underlying construction and coordination of the morphologically diverse interphase microtubule arrays present within cells, little effort has been put towards the standardization or objectification of the techniques used to investigate genes and/or proteins believed to act within these processes. Here we present evidence demonstrating the efficacy of a procedure for the confinement of Dictyostelium discoideum cells within PDMS microwells, as a means to manipulate and standardize cell size and shape during experimentation. This method potentially allows for expedited, statistical comparison of knockout mutants for various novel effector proteins of interest. In testing this method, an attempt was made to objectively replicate the findings of Tikhonenko et al. who reported a decrease in centrosomal separation within Ase1 knockout mutants. While preliminary, our results were concurrent with these findings, providing support for the identification of Ase1 as a microtubule cross linker which acts within a robust mechanism to prevent the interdigitation of separate microtubule arrays within binucleate (dicentrosomal) cells.

Zuri, Agnes Scott College
Mentor: Todd Gray

Our lab studies Mycobacterium smegmatis (or M. smegmatis), a fast-growing, non-pathogenic model organism related to M. tuberculosis, the causative agent of tuberculosis. The function of many M. smegmatis genes remain unknown. If the genes are vital (necessary for growth and development, metabolic pathways, or cell-cell communication), they are considered essential genes. Other genes, called non-essential genes, are not necessary for the bacterium to grow and develop but may aid in other important functions that may not be as easy to identify. Synthetic genetic array, or SGA, allows us to make connections in the missing functional links in M. smegmatis. This is done by using known mutations, DMS_5447 and DMS_0732 (conserved genes) and combining them with unknown mutations in search for difference phenotyples between single mutants and double mutants. Specifically, we are looking for signs of synthetic lethality, characterized by cell death from inactivity of two non-essential genes. Characteristics that may be worth analyzing include colony size, morphology, or vitality. We can infer that genes whose mutations together result in synthetic lethality are somehow connected; and when a known gene paired with an unknown gene results in synthetic lethality, we can assume that the unknown gene’s function correlates with the known gene’s function. In this way, we will better define the function of genes in M. smegmatis, and if they are conserved, we will gain a better understanding of the genetics of M. tuberculosis.

Kim Hayden, Hoosick Falls Jr./Sr. High School
Mentor: Janice Pata

Science education in America has never been more important for the entire population.  The world becomes more complex every day and science knowledge and quality of thought are key to making informed decisions about healthcare, using technology, and understanding current events. Science in its purest form is the perfect framework to develop the ability to think and science educators must systematically cultivate the process of inquiry and investigation in their curricula to improve quality of thought. Despite understanding the importance of Science, Technology, Engineering and Mathematics (STEM) talent in driving society forward, “only 16 percent of American high school seniors are proficient in mathematics and interested in a STEM career.” Through a pedagogical change, science educators can improve interest and retention in STEM fields as well as develop critical thinkers. Students are fascinated by mutations therefore I am developing an inquiry-based experience for my high school biology labs using a modified Ames assay to detect mutagenic compounds found in household products.

Benjamin Long, New Lebanon Jr./Sr. High School
Mentors: Ellen Braun-Howland

Cyanobacteria, sometimes referred to as blue-green algae, are a phylum of bacteria that obtain their energy through photosynthesis. Some cyanobacteria have the ability to produce toxins called cyanotoxins. Cyanobacteria are capable of thriving in almost all environments. Recent changes in climatic conditions due to increased human activities favor the occurrence and severity of harmful cyanobacterial blooms all over the world. Knowledge of the regulation of cyanotoxins by various environmental factors is essential for effective management of toxic cyanobacterial blooms. Generally as light intensity increases so does toxin production. What is unknown is whether or not toxin production is linked to a specific wavelength of light. This study’s aim was to reveal a possible relationship between light wavelength and production of a cyanotoxin. We exposed the cyanobacteria Aphanizomenon flos-aquae to 3 different wavelengths of light (520 nm, 550 nm, 640 nm) to see if there were any differences in the production of the toxin cylindrospermopsin. We will use RT-qPCR of RNA to measure the amount of transcription taking place to produce the toxin. In addition we will run ELISA tests to detect the presence of cylindrospermopsin. Using this experiment and my experience in the lab I have developed lessons to bring back to my classroom in the fall.