This project focused on creating viable drosophila models that could be used to evaluate and understand the significance of the N-tarp portion in the effector genes of Chlamydia Trachomatis. During our project we performed multiple DNA clean ups, PCR\u27s, gel electrophoresis, double digestions, and DNA ligations. In this we learned not only the methodological importance within these laboratory techniques but that a great deal of learning occurs in being able to evaluate and analyze ways to troubleshoot when intended results from an experiment are not observed.https://stars.library.ucf.edu/hip-2023fall/1045/thumbnail.jp

Davis, JaDaya V

Patel, Om

Velez, Sebastian

English

University of Central Florida (UCF): STARS (Showcase of Text, Archives, Research & Scholarship)

University of Central Florida STARS High Impact Practices Student Showcase Fall 2023 High Impact Practices Student Showcase Fall 2023 Examining the Role of CtEG using Drosophila JaDaya V. Davis Ja889449@ucf.edu Om Patel ompatel@ucf.edu Sebastian Velez sebastianvelez@ucf.edu Find similar works at: https://stars.library.ucf.edu/hip-2023fall University of Central Florida Libraries http://library.ucf.edu This Research-Intensive is brought to you for free and open access by the High Impact Practices Student Showcase at STARS. It has been accepted for inclusion in High Impact Practices Student Showcase Fall 2023 by an authorized administrator of STARS. For more information, please contact STARS@ucf.edu. Recommended Citation Davis, JaDaya V.; Patel, Om; and Velez, Sebastian, "Examining the Role of CtEG using Drosophila" (2023). High Impact Practices Student Showcase Fall 2023. 26. https://stars.library.ucf.edu/hip-2023fall/26 University of Central Florida STARS High Impact Practices Student Showcase Fall 2023 High Impact Practices Student Showcase Fall 2023 Examining the Role of CtEG using Drosophila JaDaya V. Davis Om Patel Sebastian Velez Find similar works at: https://stars.library.ucf.edu/hip-2023fall University of Central Florida Libraries http://library.ucf.edu Examining the Role of CtEG using Drosophila presented by GEAR 2023Project SummaryWithin our GEAR course the project was centered around setting up viable models using Drosophila to evaluate the relevance of the N terminus portion of translocating actin recruiting phosphoprotein (N-tarp), in the effector genes of Chlamydia trachomatis (C. trachomatis). One of the leading inclinations that  encouraged interest in this portion of the gene is that it is found to be conserved among multiple, if not all, pathogenic chlamydial species. The significance in conservation is well known in the world of science and an indication of possible importance to a species life cycle and molecular maintenance. There are multiple effector genes involved in the molecular functions of C. trachomatis with prominent studies primarily focusing on the C terminal end however  there isn’t remotely any regarding the molecular value of the N terminus of tarp. So, to address the unknown surrounding N-tarp our lab began with developing the plasmid containing an assigned gene of interest and vector; this was done through series of plate isolations, PCR’s, gel electrophoresis chromatography's, and quantifications using nano-drop technique. The gene of interest in our case was the effector gene ctEG that was inserted into E. coli in order to be cloned via polymerase chain reactions, then confirmed using gel electrophoresis and quantified using nano-drop technique. Similar process was done to isolate vector pUAST; pUAST is a vector system that is well studied and effective in generating transgenic flies and controlling transgene expression. After ctEG and pUAST were properly isolated and confirmed, DNA clean ups were performed using mini-prep protocol and then each component underwent a double digestion using NotI and KpnI. By double digesting with unique restriction enzymes, it ensured that pUAST and ctEG was ligated together in correct orientation; From there a confirmational screening was run on E. coli transformants, by doing serial dilutions that allowed for isolated colonies to be sampled. Transformants of at least 8 colonies were separated into samples that were amplified using PCR, then ran on an agarose gel to confirm transformants with ctEG (gene of interest/effector gene) aptly within the pUAST vector. The identified E. coli transformants were then sent to a laboratory where they will be inserted into Drosophila allowing for N-tarp to be observed through physical characteristics and molecular evaluations. From this research, future studies regarding the relevance and effects that the N-tarp portion of the effector genes has on C. trachomatis bacterial cycle, survival, proliferation, and ability to alter its host cell are now possible.Image 1: Desired DNA fragment purified from gel electrophoresis in UV boxImage 2: Laboratory notebook on CtEG gel electrophoresis and pUAST quantificationImage 3: Laboratory notebook colony PCR of E. coli transformantsImage 4: Results from nano-drop quantification of pUAST vectorImage 5: Drosophila melanogaster  (N.d.). photograph. Retrieved from https://www.pghr.org/post/the-impact-of-inhaled-pollution-on-health-outcomes-in-drosophila-melanogaster. Community ImpactThis research on the N terminus of the translocating actin recruiting phosphoprotein (N-tarp) within Chlamydia trachomatis offers pivotal insights with profound community implications. As this gene segment is conserved across various pathogenic chlamydial species, exploring its molecular relevance could revolutionize disease management strategies. Chlamydia trachomatis infections pose significant public health challenges globally, making the investigation into its effector genes, particularly the overlooked N terminus of tarp, a vital endeavor. By unraveling the molecular significance of this gene segment, this research might pave the way for innovative therapeutic interventions or preventative measures against Chlamydia trachomatis (Pais). The project’s meticulous methodology, involving plasmid creation and genetic manipulation in Drosophila, sets a precedent for rigorous scientific exploration. This not only advances our understanding within the scientific community but also showcases a template for precision and methodology that can inspire future research endeavors, potentially reshaping approaches to combatting chlamydial infections and impacting broader scientific domains.Course Connection   Research on the N terminus of the translocating actin recruiting phosphoprotein (N-tarp) within Chlamydia trachomatis perfectly mirrors the ethos of UCF's GEAR course. GEAR, known for its collaborative, interdisciplinary approach, finds resonance in this study's fusion of molecular biology, genetics, and microbiology. This project, nestled within the faculty instructor's program, embodies GEAR's focus on hands-on, team-oriented projects aligned with concept-driven lectures.  The research methodology, from plasmid creation to utilizing the Drosophila model, echoes the structured and practical approach advocated by GEAR. Exploring uncharted territories within Chlamydia trachomatis effector genes, the project embodies the curiosity, collaboration, and interdisciplinary spirit central to GEAR. It provides students with real-world research experiences while emphasizing teamwork and diverse skill integration, core tenets of the GEAR curriculum.(N.d.-a). photograph. Retrieved from https://economictimes.indiatimes.com/magazines/panache/pills-or-injections-pharmaceutical-scientist-explains-science-behind-medication-intake/articleshow/92303954.cms?from=mdr. (N.d.-a). photograph. Retrieved from https://www.freepik.com/premium-vector/graph-with-decrease-report-diagram-with-recession-bankruptcy-progress-business-finance-vector-illustration_21721517.htm. Civic Responsibility The project in this course allowed for greater knowledge of Chlamydia trachomatis, as well as learning how important effector genes of C. trachomatis work. This can help to expand our knowledge of the disease, and furthermore pave the way for discovery into its more complex mechanisms and physiology. Studying C. trachomatis helped us learn many important biomedical laboratory techniques that allowed for purification and amplification of the effector gene DNA. It allowed us to become more informed members of our community to spread greater awareness on how this disease works. In this way, patients and scientists alike will be able to use this work as a framework from which to branch off to study a variety of different illnesses.ReflectionThis project showcases the approach to biomedical research. Proper methodology is critical when working through the various experiments that were completed with the effector genes. We learned important problem-solving skills that allowed us to think of how to proceed with our experiments in the midst of setbacks. For example, when our PCR product did not work as anticipated, we troubleshooted our problem by modifying the annealing temperature to allow for the expression of our desired PCR product. Overall, these skills allowed us to become better-prepared scientists in an environment that constantly requires change and modification to ensure success.Expand your knowledge: (N.d.-a). photograph. Retrieved from https://www.theodysseyonline.com/expand-your-knowledge. Project SummaryCourse ConnectionCommunity ImpactThis research on the N terminus of the translocating actin recruiting phosphoprotein (N-tarp) within Chlamydia trachomatis offers pivotal insights with profound community implications. As this gene segment is conserved across various pathogenic chlamydial species, exploring its molecular relevance could revolutionize disease management strategies. Chlamydia trachomatis infections pose significant public health challenges globally, making the investigation into its effector genes, particularly the overlooked N terminus of tarp, a vital endeavor. By unraveling the molecular significance of this gene segment, this research might pave the way for innovative therapeutic interventions or preventative measures against Chlamydia trachomatis (Pais et al.). The project’s meticulous methodology, involving plasmid creation and genetic manipulation in Drosophila, sets a precedent for rigorous scientific exploration. This not only advances our understanding within the scientific community but also showcases a template for precision and methodology that can inspire future research endeavors, potentially reshaping approaches to combatting chlamydial infections and impacting broader scientific domains.ReflectionThis project showcases the approach to biomedical research. Proper methodology is critical when working through the various experiments that were completed with the effector genes. We learned important problem-solving skills that allowed us to think of how to proceed with our experiments in the midst of setbacks. For example, when our PCR product did not work as anticipated, we troubleshooted our problem by modifying the annealing temperature to allow for the expression of our desired PCR product. Overall, these skills allowed us to become better-prepared scientists in an environment that constantly requires change and modification to ensure success.Civic Responsibility The project in this course allowed for greater knowledge of Chlamydia trachomatis, as well as learning how important effector genes of C. trachomatis work. This can help to expand our knowledge of the disease, and furthermore pave the way for discovery into its more complex mechanisms and physiology. Studying C. trachomatis helped us learn many important biomedical laboratory techniques that allowed for purification and amplification of the effector gene DNA. It allowed us to become more informed members of our community to spread greater awareness on how this disease works. In this way, patients and scientists alike will be able to use this work as a framework from which to branch off to study a variety of different illnesses.Within our GEAR course the project was centered around setting up viable models using Drosophila to evaluate the relevance of the N terminus portion of translocating actin recruiting phosphoprotein (N-tarp), in the effector genes of Chlamydia trachomatis (C. trachomatis) (Pais et al). One of the leading inclinations that  encouraged interest in this portion of the gene is that it is found to be conserved among multiple, if not all, pathogenic chlamydial species. The significance in conservation is well known in the world of science and an indication of possible importance to a species life cycle and molecular maintenance. There are multiple effector genes involved in the molecular functions of C. trachomatis with prominent studies primarily focusing on the C terminal end however  there isn’t remotely any regarding the molecular value of the N terminus of tarp. So, to address the unknown surrounding N-tarp our lab began with developing the plasmid containing an assigned gene of interest and vector; this was done through series of plate isolations, PCR’s, gel electrophoresis chromatography's, and quantifications using nano-drop technique.  The gene of interest in our case was the effector gene ctEG that was inserted into E. coli in order to be cloned via polymerase chain reactions, then confirmed using gel electrophoresis and quantified using nano-drop technique. Similar process was done to isolate vector pUAST; pUAST is a vector system that is well studied and effective in generating transgenic flies and controlling transgene expression. After ctEG and pUAST were properly isolated and confirmed, DNA clean ups were performed using mini-prep protocol and then each component underwent a double digestion using NotI and KpnI. By double digesting with unique restriction enzymes, it ensured that pUAST and ctEG was ligated together in correct orientation; From there a confirmational screening was run on E. coli transformants, by doing serial dilutions that allowed for isolated colonies to be sampled. Transformants of at least 8 colonies were separated into samples that were amplified using PCR, then ran on an agarose gel to confirm transformants with ctEG (gene of interest/effector gene) aptly within the pUAST vector. The identified E. coli transformants were then sent to a laboratory where they will be inserted into Drosophila allowing for N-tarp to be observed through physical characteristics and molecular evaluations. From this research, future studies regarding the relevance and effects that the N-tarp portion of the effector genes has on C. trachomatis bacterial cycle, survival, proliferation, and ability to alter its host cell are now possible.ReferencesPais, Sara V et al. “CteG is a Chlamydia trachomatis effector protein that associates with the Golgi complex of infected host cells.” Scientific reports vol. 9,1 6133. 16 Apr. 2019, doi:10.1038/s41598-019-42647-3AcknowledgementsSpecial thanks to Dr. George Aranjuez, instructor of the course, for his effort and dedication in ensuring each concept and experiment was understood by all while sharing his passion and dedication for his work.Research on the N terminus of the translocating actin recruiting phosphoprotein (N-tarp) within Chlamydia trachomatis perfectly mirrors the ethos of UCF's GEAR course. GEAR, known for its collaborative, interdisciplinary approach, finds resonance in this study's fusion of molecular biology, genetics, and microbiology. This project, nestled within the faculty instructor's program, embodies GEAR's focus on hands-on, team-oriented projects aligned with concept-driven lectures.The research methodology, from plasmid creation to utilizing the Drosophila model, echoes the structured and practical approach advocated by GEAR. Exploring uncharted territories within Chlamydia trachomatis effector genes, the project embodies the curiosity, collaboration, and interdisciplinary spirit central to GEAR. It provides students with real-world research experiences while emphasizing teamwork and diverse skill integration, core tenets of the GEAR curriculum.Image 1: Desired DNA fragment purified from gel electrophoresis in UV boxImage 1

Examining the Role of CtEG using Drosophila

https://stars.library.ucf.edu/cgi/viewcontent.cgi?article=1045&context=hip-2023fall

Examining the Role of CtEG using Drosophila

Abstract

Similar works

Full text

Available Versions

University of Central Florida (UCF): STARS (Showcase of Text, Archives, Research & Scholarship)