Student Team Members Attributions:
This season, Beteel served as one of the team’s co-captains. She helped to provide training for the wetlab team throughout the summer. She also finalized the in vivo circuit design for circuits measuring transcriptional (WM21_011 and WM21_012) and translational burden (WM21_013 and WM21_016), and tested both designs of the translational burden sensors, one of which (WM21_016) was designed by Ceroni et al., 2015. She collected cell samples containing circuit pBbB8k-csg-amylase (Birnbaum et al., 2021) for RNA-sequencing using the curli fiber spin down assay protocol from Harvard iGEM 2017. She also led the Collaborations Committee for this year’s team. As the head of the Collaborations Committee, she helped to organize the 2021 MidAtlantic Meetup in hybrid format, which was attended by Baltimore Biocrew iGEM, Gaston Day School iGEM, and iGEM teams from the University of Virginia, the University of Maryland, Johns Hopkins University, and the University of Pittsburgh. She also reviewed the design for the protease sensor circuits incorporating the lon, hslVU, and clpB promoters, and reviewed the design for the in vitro sensor circuits (WM21_025, WM21_026, WM21_027, and WM21_028). Beteel also assisted with IHP, participating in the interviews with John Marken, Dr. Gale Smith from Novavax and Dr. Brian Renda from Ginkgo Bioworks.
This season, Avery served as co-captain of the team. Avery designed and tested the PTM sensor circuits (WM21_014 and WM21_015), which sense changes in phosphorylation rates using the OmpR and EnvZ system, along with assisting in the development of the other central dogma sensor circuits. In addition, she also designed and tested the in vitro circuit system (WM21_025, WM21_026, WM21_027, and WM21_028). The in vitro circuits test for host-circuit interactions in the transcription, translation, and post-translational modification portions of the central dogma. To assist with collecting RNA-seq data, Avery took RNA samples of the toggle switch construct developed by Barbier et. al, 2020, following the protocol included in this paper. In addition to wet lab work, Avery acted as a wet lab liaison with the math modeling team, offering advice when needed, and helped review the parameters for accuracy, replacing parameters as needed. Avery also was the head of our team’s education committee. As the head, Avery was responsible for reaching out to retirement homes and scheduling presentations with them. She also created the education powerpoint and spoke with representatives from a retirement home to improve it. She wrote the team’s “Synthetic Biology for Retirement Homes” pamphlet and the design, education, and inclusivity wiki pages, as well as writing the overview portions of many wiki pages. Avery also reviewed other team members' pages. She assisted with IHP, interviewing John Marken, Aqib Hasnain, and Dr. Jeffery Barrick.
Matt confirmed the presence and functionality of the pDawn-sfGFP circuit, compiled a database for orthogonality in 2019 iGEM wikis, worked on the design of the transcriptional and translational sensor circuits, designed and tested the glycosylation sensor circuit, conducted human practices interviews with commercial leaders in synthetic biology, researched and implemented genome integration of the sensor circuits, coded the wiki, and wrote the safety page of the wiki.
Justin developed concepts for in vivo and in vitro sensor circuits to measure transcriptional and translational burden. He also performed a literature analysis and contacted researchers to decide on an optimal aptamer for circuit design, and he helped lead further efforts to design the transcriptional sensor circuits. Additionally, he designed four in vivo circuits incorporating the lon, hslVU, and clpB promoters. He tested two of them and analyzed data for the three lon and clpB circuits. Justin also worked to research, confirm the sequence of, and functionally confirm plasmids pEvol-pCNF, pET-GFPY151X, and pET-GFPWT, although they were not ultimately used for RNA-seq data. He also helped create and present a project pitch at the Mid-Atlantic Meetup. Additionally, Justin helped build databases describing how 2019 iGEM teams and 2020 ACS Synthetic Biology article authors addressed orthogonality. Furthermore, he helped identify and organize differentially expressed genes upon circuit implementation from the literature for integration into the Venn diagrams. Justin also automated manipulation and graphing of wet lab data, and he detailed parts used by the team on the iGEM Registry. Finally, he created the Proposed Implementation and Parts wiki pages, along with contributing to and coding various other parts of the wiki.
KC reviewed the plastic degradation literature and contributed to the PETase parts page with a theoretical circuit design for cell membrane display of PETase and MHETase enhanced by rhamnolipid secretion. She also analyzed all RNA-sequencing data done before and after circuit implementation or induction in the literature to identify the most common differentially expressed genes to inform our circuits and models. KC wrote pseudocode to automate this method so that new RNA sequencing data could be quickly analyzed and the following could be established: a hierarchy of common DE genes, a hierarchy of circuit specific DE genes (for plasmid copy number and E. coli strain), and GO molecular function/biological process commonalities. Furthermore, KC conducted differential expression analysis on our team’s own RNA sequencing data. She also gathered parameters on common DE genes for inclusion in our models, participated in the development of the ACS Synbio 2020 orthogonality database, assisted wet lab in circuit testing, and partook in the writing and coding of our wiki.
For drylab, Julia created graphics for the team promotional video and wiki, led an animation workshop at the MidAtlantic Meetup as well as assisted with organizing and planning the event, participated in the SDG Impact Challenge collaboration for our team, and helped create a database of mentions of relevant terms in ACS Synbio papers. For wetlab, she worked to functionally confirm the Repressilator 2.0 circuit, although this ended up being extraneous to our project. Julia also helped in designing the transcriptional circuit, and designed and tested the secretion circuit.
Ubaid's primary role was in analyzing the current mathematical classification methods being used in systems biology, bioinformatics, and the computational methods currently being used to understand the relationship between the host and the circuit from a mathematical standpoint. From this basis, the modeling team developed methods to integrate behavior from RNA-seq data into mechanistic models and transform them into tools that can be used to assess orthogonality.
Linda synthesized a dynamical model of host-circuit interactions with regard to burden from the literature, while reviewing modeling techniques for quantifying orthogonality. In addition, she worked on integrating classical mechanistic models of burden with data driven methods derived from high throughput data, such as RNA-sequencing, and making those modifications compatible with measurements taken by the sensor circuits developed in wetlab. Finally, she extended the model to include aspects of orthogonality not covered by burden, such as toxicity and post-translational modifications.
In wetlab, Alana confirmed the functionality of the pDawn-Ag43 circuit and conducted repeated experiments to collect cell samples for RNA-seq. She also tested one of the heat-shock sensing circuits. In drylab, she helped complete and review a database of orthogonality and relevant terms in ACS SynBio papers, worked on the design of the in vivo transcriptional and translational sensor circuit, and wrote the Description wiki page. As a part of the education committee, she gave a presentation of synthetic biology to retirement home residents and planned community outreach at a local arts festival. As a part of the design team, Alana created and animated graphics for the promotional video, presentations, and the team Wiki, and helped plan an animation workshop for the MidAtlantic Meetup.
In the wetlab, Pinar conducted preliminary research for the in vivo and in vitro diagnostic circuits for detection of burden at the three levels of the central dogma. She designed the dnaKJ heat-shock sensing circuit and was in charge of functionally testing it. Though unsuccessful, she also worked to recreate the original Repressilator and Repressilator 2.0 experiments with the goal of collecting RNA-seq samples. In the drylab, Pinar performed literature analysis to analyze the state of the field in assessing orthogonality and its related concepts. She also assisted with the analysis of the RNA-seq data to develop a hierarchy of common DE genes that would serve as candidates for assessing orthogonality. As part of the education committee, she presented informative slides on synthetic biology to the elderly and helped with putting together the pamphlet for future teams to use when communicating with the elderly community about synthetic biology. Lastly, along with the design team, Pinar worked on making animations, edited the video and audio for our promotional video.
PI Team Member Attributions:
Margaret Saha served as the PI for the team. She was responsible for overseeing all aspects of the administration of the project (funding, orders) and served as the liaison between the team and administrative offices. She was also responsible for the safety of the team in all respects as well as ensuring that the team followed best practices in terms of scientific conduct (good, accurate record keeping). She provided both lab safety training and training in the practical aspects of labwork. She vetted protocols used by the team and was involved in troubleshooting when procedures or protocols did not work. She met with the team twice per week in formal meetings, participated in weekly math meetings, and met with team members on a nearly daily basis to discuss progress and problems and offer advice and guidance.
Mainak Patel served as a co-PI and provided the expertise with the math modeling aspect of the project. The team met weekly with Dr. Patel throughout the project during which he would provide guidance and answer questions. He met even more frequently with the math team and provided guidance on all aspects of the mathematical modeling.
Eric Bradley served as co-PI and was responsible for overseeing safety and matters related to infrastructure and space. He ensured that equipment was working properly and responded promptly to any and all equipment failures, often fixing the equipment himself.
Other Individuals Who Contributed to Our Project
Elias provided assistance to our team by reviewing our wetlab protocols and offering general wetlab troubleshooting advice.
Taiana provided invaluable assistance, expertise, and effort with RNA-Seq analysis, including the Galaxy pipeline of running mapping, feature counts, and DeSeq2 programs.
Bryan provided assistance with Galaxy and uploading files to Galaxy for RNA-Seq analysis.
Justin provided assistance with using the 3D printer in the William and Mary MakerSpacer for our Gaston Day School collaboration.
Acknowledgements and Appreciation
FundingWe would like to thank Vice Provost Dennis Manos, who has championed William & Mary iGEM since its inception in 2014. His support has been invaluable. The financial support of Vice Provost Manos (Vice Provost for Research and Graduate/Professional Studies), Dean Maria Donoghue Velleca, Dean Teresa Longo (Dean for Interdisciplinary Studies and Director of the Charles Center), Dr. Dan Cristol (Charles Center, Undergraduate Research Director), and the Charles Center made our participation in iGEM possible. We are extremely grateful.
SponsorsThank you to the iGEM Foundation, the entire iGEM staff for their continuing helpfulness and support, and Integrated DNA Technologies, and Twist Bioscience for their extraordinary support and gifts of DNA synthesis. This has allowed so many teams such as ours to participate in iGEM.
Thank You to ...We would like to thank Dr. Jennifer Stevens, Chair of the Protection of Human Subjects Committee at William & Mary. Her guidance in revising and submitting our PHSC protocol made our work in integrated human practices possible.
We would like to thank Renee Peace for overseeing financial records and ordering.
We would like to thank Charnel Thornton and Jessica Shelton for their help with placing orders.
We would like to thank Mr. Sudip Paudel for his help with RNA sample extraction.
We would like to thank Elias Nazfiger for his assistance with circuit design.
We would like to thank Taiana James and Bryan Weselman for their assistance with RNA-Seq analysis.
We would like to thank Justin Cartailler for his assistance with 3D printing.
IHP IntervieweesWe would like to thank the following people for their guidance concerning aptamers and the transcriptional sensor plasmid of our central dogma circuit.
Dr. Aaron Engelhart
Dr. Keith Gagnon
Dr. Elisa Franco
Dr. Xianjun Chen
Dr. Donald Burke-Aguero
Dr. Paul Gardner
For our integrated human practices this year, we consulted experts in orthogonality related fields, such as burden and toxicity. We are so grateful to these individuals for giving us their valuable advice and for their willingness to discuss our project with us.
Dr. Jeffrey Barrick
Mr. John Marken
Mr. Aqib Hasnain
Dr. Gale Smith
Dr. Brian Renda
Mr. Paul Maschhoff
Mr. Luis Ortiz