Abstract
Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.
Highlights
Scientific research has recently come under fire for what is being dubbed a crisis of reproducibility
Imaging MCPs using negative-stain transmission electron microscopy (TEM) is a standard technique used by the MCP field that has been widely adopted since Sinha and colleagues first described a method for MCP purification [40]
Our results suggest that the technique used to visualize and measure MCPs can alter how we interpret our experimental results
Summary
Scientific research has recently come under fire for what is being dubbed a crisis of reproducibility. Current studies estimate that 75–90% of findings in high-profile journals are not reproducible [1]. The issue has seeped into fields across every domain of scientific inquiry [2]. While the cause of any given irreproducible result will vary from case to case, a lack of technique standardization across studies can lead to artefactual results or false conclusions [3]. In fields in which different techniques are employed to test similar hypotheses, it is important to place results into the proper context and understand the limitations of each technique. We provide guidelines for technique standardization and result interpretation in the bacterial microcompartment engineering field
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