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Escherichia coli were engineered to enable programmed motility, sensing and phenotypic response to the density of epidermal growth factor receptor expressed on the surface of cancer cells.

There have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenic Escherichia coli strains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq). E. coli BL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2’s relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.

In order to carry out innovative complex, multistep synthetic biology functions, members of a cell population often must communicate with one another to coordinate processes in a programmed manner. It therefore follows that native microbial communication systems are a conspicuous target for developing engineered populations and networks. Quorum sensing (QS) is a highly conserved mechanism of bacterial cell-cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the E. coli QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an "enhanced" repression and derepression eLsrR and a reversed repression/derepression function "activator" aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the lsr promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products.

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OBJECTIVES/GOALS: a) Explore topics related to AMC CRP job titles, descriptions, and pre-requisites for hire b) Describe impact of COVID-19 on the AMC CRP workforce c) Discuss opportunities for improving diversity in the CRP workforce d) Discuss opportunities to enhance institutional staffing culture to retain CRP workforce METHODS/STUDY POPULATION: Qualitative data from a series of workshop breakout sessions and open-text survey materials focusing on AMC CRP recruitment, retention and diversity were analyzed to inform content and recommendations for clinical research job titles and descriptions, pre-requisites, diversity, and current needs. RESULTS/ANTICIPATED RESULTS: While certain institutions have established competency-based frameworks for job descriptions and career ladders, standardization remains generally lacking across CTSA hubs. Significant hiring needs have reached exponential proportions across hubs, unable to meet current and projected clinical research goals. Data confirmed an urgent need for closing gaps in clinical research workforce at AMCs, especially for improving diversity and equity of personnel. Improved collaboration with human resource departments, engagement with principal investigators, and overcoming both organizational and resource challenges were suggested strategies, as well as pipeline development via outreach to universities, community colleges, and high schools to raise awareness of the professional pathways for CRPs. DISCUSSION/SIGNIFICANCE: Based on input from 130 CRP leaders at 38 CTSA hubs and 4 IDeA sites evaluating data from 23 breakout transcripts and ~92 surveys from the Collaborative Conversations Unmeeting, new opportunities emerged during the analysis. The findings will be summarized in a 2022 Synergy manuscript including best practice benchmarking recommendations.

OBJECTIVES/GOALS: We conducted a review of CTSA websites to understand the current landscape for CRP institutional professional development and training revealed in the CTSA hub websites. METHODS/STUDY POPULATION: We accessed and reviewed 59 currently funded CTSA hub websites for evidence of CRP training opportunities. Parameters reviewed included: 1) opportunities were specified for CRPs versus K and T trainees; 2) mandated training; 3) leveling; 4) delivery methods/resources; 5) public accessibility; 6) unique features. The website reviews informed a REDCap survey sent to the CTSA Administrators (n = 149) and the Coordinator Taskforce (n = 105) listservs to gain additional knowledge of CRP training available at the institution. A subsequent repeat review of the CTSA hub websites will be conducted to determine evolving trends. RESULTS/ANTICIPATED RESULTS: A total of 40 responded to the survey from 59 CTSA hubs. Survey results are being analyzed. Website review data are being tabulated and the subsequent review of websites will be collected in February. Those findings are pending and will include a comparison of prior findings. 42% of CRP hubs list CRP training within the CTSA hub website. Required onboarding training (beyond CITI certificates) is revealed for some hubs (15%). DISCUSSION/SIGNIFICANCE OF IMPACT: On our initial website review less than half of the CTSA hub websites list specific CRP training on their website. Many were hidden behind firewalls and could not be reviewed for content. The REDCap Survey will provide more granular descriptions of programs. Data from a second website review will be collected for comparison. Based on a preliminary re-review of sites, there is a suggestion of increasing CRP workforce development information. CTSAs are well-positioned to be a central hub for promoting educational excellence of the institutional workforce, for medical centers and in other venues where clinical research is performed.