The use of primary literature can be an effective way to expose students to the scientific research process. At the same time, primary literature can be intimidating to many students, particularly those with limited exposure to science research. The CREATE (consider, read, elucidate hypotheses, analyze and interpret the data, and think of the next experiment) method is an evidence-based teaching strategy that uses a structured series of pedagogical tools to allow students to perform intensive analysis of primary literature. The use of CREATE in biology classes increased student self-rated attitudes, confidence, and understanding of science. On the basis of these prior reports, we implemented CREATE in a one-semester, stand-alone, undergraduate chemistry course in which students analyzed a linked series of primary literature articles tracking the trajectory of a specific area of chemistry research. The course was aimed at second-year students, and our goals were to increase student confidence in engaging scientific literature, demystify the scientific process, and help students see themselves as having a role in that process. Pre- and post-course surveys indicated significant increases in understanding of the scientific process, along with gains in self-rated skills and attitudes related to science. Students also demonstrated gains in course content knowledge as assessed by a pre- and post-course concept mapping exercise. Thus, the CREATE method has been implemented in a chemistry teaching context that resulted in increased student knowledge.

A recent push toward addressing antiracism in science reveals a need to integrate discussions of racism, social justice, and equity into undergraduate STEM classes. Toward this end, in Fall 2020, a theme of "Racism is a Public Health Emergency" was incorporated as an overlay for an existing undergraduate biochemistry curriculum. This theme was used throughout the entire semester and involved discussions of racism, social justice, and equity that were directly integrated with the science content of the course. Students enrolled in the class were surveyed at the end of the semester regarding their perceptions of integrating discussions of racism, social justice, and/or equity into chemistry courses. Students indicated that the inclusion of the overlay increased the relevance of the course material, indicated a high level of understanding of the impact of bias and race on the field of biochemistry, and expressed overwhelmingly positive views regarding future inclusion of similar themes of equity and antiracism in chemistry courses. Additionally, student scores and answers on common final exam questions were compared to previous semesters, revealing that the inclusion of this overlay did not negatively impact coverage of biochemistry content.

The Gram-negative bacterium Vibrio cholerae adapts to changes in the environment by selectively producing the necessary machinery to take up and metabolize available carbohydrates. The import of fructose by the fructose-specific phosphoenolpyruvate (PEP) phosphotransferase system (PTS) is of particular interest because of its putative connection to cholera pathogenesis and persistence. Here, we describe the expression and regulation of fruB, which encodes an EIIA-FPr fusion protein as part of the fructose-specific PTS in V. cholerae Using a series of transcriptional reporter fusions and additional biochemical and genetic assays, we identified Cra (catabolite repressor/activator) and cAMP receptor protein (CRP) as regulators of fruB expression and determined that this regulation is dependent upon the presence or absence of PTS sugars. Cra functions as a repressor, downregulating fruB expression in the absence of fructose when components of PTSFru are not needed. CRP functions as an activator of fruB expression. We also report that Cra and CRP can affect fruB expression independently; however, CRP can modulate cra expression in the presence of fructose and glucose. Evidence from this work provides the foundation for continued investigations into PTSFru and its relationship to the V. cholerae life cycle.IMPORTANCEVibrio cholerae is the causative agent of cholera disease. While current treatments of care are accessible, we still lack an understanding of the molecular mechanisms that allow V. cholerae to survive in both aquatic reservoirs and the human small intestine, where pathogenesis occurs. Central to V. cholerae's survival is its ability to use available carbon sources. Here, we investigate the regulation of fruB, which encodes a protein central to the import and metabolism of fructose. We show that fruB expression is controlled by the transcriptional regulators Cra and CRP. This work contributes toward a clearer understanding of how carbon source availability impacts the physiology and, potentially, the persistence of the pathogen.

A strategy for addressing racism and promoting social justice was incorporated into an undergraduate biochemistry curriculum. The impetus for this change was to demonstrate solidarity with the Black Lives Matter movement and to support students traditionally excluded from STEM because of their ethnicity or race. At the same time, we hypothesized that inclusion of the theme, Racism is a Public Health Emergency, would make the material more relevant to students, without negatively impacting content coverage and learning. In the Fall 2020 semester, students engaged in seven activities related to the anti-racism overlay, spaced over the course of the semester. While 95% of the students in the course had previously encountered discussions about equity in previous classes, only one of them indicated that the material was covered in a chemistry course. The majority of the students (81%), moreover, agreed or strongly agreed that the inclusion of the anti-racism overlay made the course material more relevant to them. In addition, analysis of answers to final exam questions suggests student achievement of learning outcomes remained similar to previous iterations of the course. The activities detailed here, therefore, addresses an important need in biochemistry curricula to incorporate issues of social justice and could be adapted to a variety of other related fields.

Using 2-dimensional (2D) images in order to teach about 3 dimensional (3D) molecules can limit the ability of students to grasp key visuospatial elements of 3D macromolecular structures, including depth perception and a sense of scale. The lack of simple, accessible, and easy-to-use teaching tools for visualizing and interacting with (i.e., rotating, translating, zooming) 3D virtual representations of macromolecular structure also continues to be a limitation for biochemistry instructors interested in incorporating more 3D visualizations in their classrooms. To address this current gap in available instructional tools, a novel app utilizing augmented reality (AR) technology to visualize macromolecular structures is described. The app allows users to easily visualize and manipulate the 3D structure of the potassium channel and is accompanied by a set of curricular materials to facilitate app implementation during a discussion on membrane transport. The app is free to download on both iOS and Android systems, is suitable for use on mobile and tablet systems, and is aimed for target audiences ranging from introductory to advanced undergraduate levels.

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA's target gene.IMPORTANCEVibrio cholerae is a bacterial pathogen that relies on genetic tools, such as regulatory RNAs, to adapt to changing extracellular conditions. While many studies have focused on how these regulatory RNAs function, fewer have focused on how they are themselves modulated. V. cholerae expresses the noncoding RNA MtlS, which can regulate mannitol transport and use, and here we demonstrate that MtlS levels are controlled by the level of transcription occurring in the antisense direction. Our findings provide a model of regulation describing how bacteria like V. cholerae can modulate the levels of an important regulatory RNA. Our work contributes to knowledge of how bacteria deploy regulatory RNAs as an adaptive mechanism to buffer against environmental flux.

In vitro diagnostics (IVD) have become increasingly popular tool for tackling global health concerns, but conventional IVD tests, such as microscopy and nucleic‐acid amplification methods, are often expensive and complex to use, limiting their use in low‐resource settings. Whole‐cell biosensors in bacterial systems offer an attractive solution as an easy‐to‐use, portable device that non‐specialists could use for clinical purposes outside clinical locations. However, a strategy is needed to engineer ligand specific sensors for new targets. An appealing candidate for a sensing module is the riboswitch. Genetic RNA switches, termed riboswitches, are genetic regulatory elements found in the 5′‐UTR (untranslated region) of some prokaryotic mRNA and contain two functional components: an aptamer binding domain and an expression platform that modulates downstream gene expression. We developed an engineering platform coupling genetic selections and Fluorescence‐Activated Cell Sorting (FACS) screens to identify novel riboswitches from a 108 random‐sequence library in which we replaced the aptamer sequence of the ThiM#2 riboswitch with a degenerate 40‐nucleotide sequence. In a proof‐of‐principle validation of the platform, we identified novel riboswitches for the small molecule theophylline, which are distinct from the existing synthetic theophylline switches. Our best riboswitch hit displays a 2.3‐fold activation in response to theophylline and does not respond to the structurally unrelated molecule thiamine. We performed directed evolution on this riboswitch hit by mutagenizing its sequence and performing additional rounds of selections and screens, resulting in a variant with a 3.6‐fold activation. Current efforts are focused on assessing the efficacy of the selection platform when isolating riboswitches for dopamine. Furthermore, a variant of the engineering platform is being used side‐by‐side to compare if a dopamine riboswitch can be selected from a library composed of an in vitro selected aptamer coupled with a 15‐nucleotide degenerate sequence. We anticipate that these efforts will provide general guidelines toward the development of novel riboswitches for a variety of natural and non‐natural ligands.Support or Funding InformationNSF CBET 1258307Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In the facultative pathogen Vibrio cholerae, MtlS is a small RNA (sRNA) transcribed antisense to the 5′ untranslated region of mtlA, which codes for the transporter of the sugar alcohol mannitol. MtlS works in cis as a translational repressor of mtlA and it is postulated that the sRNA allows the bacteria to respond rapidly to changes in environmental carbon sources. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. Previous work in the lab used RNA target‐prediction programs and mass spectrometry‐based proteomics to identify additional MtlS targets. The results identified 6 potential targets: VCA1045 (mtlA), VCA0820, VCA1046, VC1560, VC1899, and VC2683. The objective of this study was to determine if the putative targets were post‐transcriptionally regulated by MtlS. We constructed gfp translational reporter fusions of the candidate genes in a V. cholerae ΔmtlS strain carrying either a control vector or a plasmid ectopically expressing mtlS. An mtlA‐GFP fusion served as a positive control and exhibited the expected down‐regulation of gfp expression when mtlS was expressed. We have tested reporter fusions to all the candidate genes for regulation by MtlS by monitoring total cell fluorescence and western blot analysis. In an experiment done in duplicate in the presence of MtlS, during stationary phase growth we observed 20% down‐regulation of VC1899, encoding a protein with homology to nucleic acid‐binding proteins. In an experiment done in duplicate, we have also observed 30% logarithmic and stationary phase downregulation of VCA1046, encoding the mannitol dehydrogenase mtlD, in the presence of MtlS. Results based on another experiment conducted in duplicate indicate 20% logarithmic and stationary phase down‐regulation of VC1560, encoding a protein with homology to a catalase‐peroxidase, in the presence of MtlS. Tests with VC2683, encoding a putative cystathionine gamma‐synthase, have been inconclusive, as both down‐regulation and up‐regulation by MtlS has been observed; further experiments will need to be conducted with the VC2683‐gfp fusion. The VCA0820‐gfp fusion does not exhibit any fluorescence, in the presence or absence of MtlS. This work positively contributes to the notion that a single sRNA can have multiple regulatory targets. Ultimately, this work will expand our knowledge on MtlS's role in helping V. cholerae adapt quickly to changes in environmental carbon sources and on sRNAs in general.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3-5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3-5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

Cholera disease, a gastrointestinal ailment caused by the Gram‐negative bacterium Vibrio cholerae, remains a global health burden affecting an estimated 3–5 million people annually. V. cholerae's capacity to persist in different environments stems from its adaptability to a variety of sugar sources, including the six‐carbon sugar alcohol mannitol. Produced by algae metabolism, mannitol is transported into V. cholerae through the mannitol transporter MtlA, whose production is downregulated at the post‐transcriptional level by the small RNA (sRNA) MtlS. Currently, little is known surrounding what governs the transcription of mtlS, other than that mtlS expression is equally robust in non‐mannitol sugars. We examined the hypothesis that mtlS expression is subject to transcriptional interference from the divergently transcribed mtlA. Because MtlS is a cis‐encoded sRNA transcribed directly antisense to mtlA, transcriptional activity from mtlA could pose a hindrance to mtlS transcription occurring in the opposite direction. To test this model, we grew V. cholerae in different sugar conditions and stimulated mtlA expression by knocking out mtlR (a transcriptional repressor of mtlA) or by adding exogenous mannitol. We then measured MtlS sRNA levels by northern blot analysis. Our results indicate a strict inverse relationship between mtlA expression and mtlS expression, whereby the degree to which mtlA expression increases correlates with the extent to which mtlS expression decreases. In glucose, knocking out mtlR restores MtlA levels to 30% of maximum expression (n=3, p<0.05) and decreases MtlS levels by 33% (n=2, p=0.1). In non‐mannitol, non‐glucose sugars such as mannose, knocking out mtlR restores MtlA levels to 50% of maximum expression (n=3, p<0.05) and decreases MtlS levels by nearly 95% (n=3, p<0.01). These findings support a transcriptional regulation model regarding cis‐antisense RNAs, known as the transcriptional interference model, for which there exist few characterized examples. Ultimately, this project has contributed to our understanding of how V. cholerae deploy sRNAs as an adaptive mechanism to buffer against environmental flux.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The phosphoenopyruvate:carbohydrate phosphotransferase system (PTS) enables Vibrio cholerae - and other bacteria - to recognize and transport exogenous carbon sources for energy, including the six-carbon sugar alcohol, mannitol. The mannitol-specific PTS transporter is encoded by mtlA and its expression is expected to be regulated by the putative repressor encoded by the mtlR gene. Here, we show that mtlR overexpression inhibits V. cholerae growth in medium supplied with mannitol as the sole carbon source and represses MtlA-mediated biofilm formation. We demonstrate that when V. cholerae is grown in non-mannitol medium, knocking out mtlR leads to both increased MtlA protein and mtlA mRNA levels, with these increases being especially pronounced in non-glucose sugars. We propose that in non-mannitol, non-glucose growth conditions, MtlR is a major regulator of mtlA transcription. Surprisingly, with regard to mtlR expression, transcript and protein levels are highest in mannitol medium, conditions where mtlA expression should not be repressed. We further show that MtlR levels increase during growth of the bacteria and linger in cells switched from mannitol to non-mannitol medium. Our data suggests an expression paradigm for mtlA where MtlR acts as a transcriptional repressor responsible for calibrating MtlA levels during environmental transitions.

The use of tandem mass tags (TMT) as an isobaric labeling strategy is a powerful method for quantitative proteomics, yet its accuracy has traditionally suffered from interference. This interference can be largely overcome by selecting MS(2) fragment precursor ions for high-energy collision induced dissociation (HCD) MS(3) analysis in an Orbitrap scan. While this approach minimizes the interference effect, sensitivity suffers due to the high AGC targets and long acquisition times associated with MS(3) Orbitrap detection. We investigated whether acquiring the MS(3) scan in a linear ion trap with its lower AGC target would increase overall quantification levels with a minimal effect on precision and accuracy. Trypsin-digested proteins from Saccharomyces cerevisiae were tagged with 6-plex TMT reagents. The sample was subjected to replicate analyses using either the Orbitrap or the linear ion trap for the HCD MS(3) scan. HCD MS(3) detection in the linear ion trap vs Orbitrap increased protein identification by 66% with minor loss in precision and accuracy. Thus, the use of a linear ion trap-HCD MS(3) scan during a 6-plex TMT experiment can improve overall identification levels while maintaining the power of multiplexed quantitative analysis.

Cholera, caused by the gram‐negative, halophilic bacterial pathogen Vibrio cholerae, remains a world health problem, causing millionsof deaths each year. Throughout its life‐cycle, V. cholerae is able to adapt to avariety of environments, from aquatic reservoirs to the human small intestine. This remarkable adaptability is likely due in part to gene regulation by small regulatory RNAs. We are examining one such regulatory pathway by studying the proteolysis of MtlA, the mannitol‐specific phosphotransferase system transporter. In the absence of environmental mannitol, the small RNA MtlS down‐regulates mtlA expression at the translational and post‐translational level. However, the mechanism by which MtlS post‐translationally directs MtlA proteolysis is currently unknown. Our lab has shown that MtlS induction significantly affects the expression of various proteins, which we hypothesize may be involved in MtlA proteolysis. Specifically, proteins up‐regulated by MtlS induction may act as proteases or protease adaptors. Proteins down‐regulated by MtlS induction may normally act to stabilize MtlA, such that when they are suppressed, MtlA degradation can occur. Currently, we are studying the potential role of three proteins—VC1899, GroL2, and VC1872—in MtlA proteolysis. VC1899 and GroL2 are both up‐regulated by MtlS induction, while VC1872 is down‐regulated. We created strains that overexpress or have knocked out one of each of these proteins, and analyzed the affect that these proteins have on MtlA levels over time in mannitol(MtlS‐repressing) and mannitol‐free (MtlS‐inducing) conditions. Additionally, we created knockout strains containing an arabinose‐inducible plasmid bearing MtlS, as an alternative method to analyze MtlA levels over time upon MtlS induction. Preliminary results suggest that VC1899, an uncharacterized protein thought to be involved in nucleic acid binding, represses MtlA protein levels. Single‐knockout mutants of either GroL2, an ortholog to the essential chaperone GroEL, or VC1872, a nun characterized protein with homology to serine protein kinases, do not display significant phenotypes with regard to cell growth or MtlA proteolysis. However, we are in the process of making double knockout mutant strains in order to test whether GroL2 and VC1872 may work in concert. Ultimately, gaining a better understanding of the mechanism of MtlA proteolysis could help to illuminate other regulatory systems in V. cholerae and other pathogens.Support or Funding InformationNIH grant R15AI090606 to J.M.L.

Bisphenol A (BPA) is a known endocrine disruptor and potential carcinogen found in polycarbonate plastic products, food packaging, and drinking water supplies. A quick and affordable detector, such as a whole‐cell biosensor that measures the presence of BPA, would greatly benefit safety and health. We set out to develop such a method by using riboswitch‐based biosensors. Riboswitches are regions of mRNAs that control downstream gene expression and can be used as genetic sensors to create whole‐cell biosensors. Although there are no naturally occurring BPA‐riboswitches, we reasoned that we could use the power of genetic selections to generate one. Starting from a known riboswitch that regulates expression of the downstream selection marker tetA linked to the reporter gene gfp, we used PCR or Gibson Assembly to replace the aptamer domain of the switch with 40 random bases, generating plasmid libraries. We then transformed Escherichia coli with these plasmids such that each individual bacterium harbored a unique member of the library. Libraries made by Gibson Assembly resulted in 108 unique members, while those made by PCR were generally on the order of 106 members. With libraries in hand, the dualistic nature of TetA allowed us to apply dual genetic selection to identify riboswitches that express tetA‐gfp only in response to BPA. TetA is a transporter protein that pumps the antibiotic tetracycline out of the cell, rendering cells tetracycline resistant. TetA also allows toxic metals, such as Ni2+, to enter the cell, inhibiting growth. The E. coli library was grown in the presence of BPA and tetracycline. Surviving cells were switched to medium with Ni2+ and no BPA. Only those cells that express tetA‐gfp exclusively in the presence of BPA were expected to survive both positive and negative selection. We succeeded in isolating unique riboswitches that converged to single sequences from starting libraries by bringing each library through three rounds of selection with varying tetracycline and Ni2+ conditions. Ultimately, our hits proved largely unsuccessful in producing a significant increase in fluorescence when incubated with BPA for either 6 or 16 hours compared to no‐ligand controls. In order to improve the ON/OFF ratio of our riboswitches, we integrated fluorescence‐activated cell sorting into our riboswitch‐development platform, allowing us to both select for survival through dual genetic selection and screen for maximum or minimum fluorescence depending on presence or absence of the ligand of interest. Further studies will include optimizing the obtained riboswitches by mutagenesis and directed evolution.Support or Funding InformationResearch funding is provided by the NSF.

This laboratory experiment provides undergraduate students enrolled in organic chemistry the opportunity to design and synthesize their own peptide, which is then tested for antimicrobial activity. After reading a primary scientific paper on antimicrobial peptides, students design and synthesize their own hexapeptide that they hypothesize will have antimicrobial activity. The students characterize their products by analyzing liquid chromatography-mass spectrometry and antimicrobial bioassay data for their synthesized peptide. The students are able to complete the synthesis and prepare their samples for analysis in three 3-4 h lab periods; instructors perform LC-MS and a bioassay with the peptides and provide data to students for analysis. This exercise is flexible and can be altered to include students performing the bioassay, or to meet different time constraints or target student populations. This experiment allows students to increase their knowledge of solid phase peptide chemistry and gain experience with developing and testing hypotheses through experimental design.

As with all facultative pathogens, Vibrio cholerae must optimize its cellular processes to adapt to different environments with varying carbon sources and to environmental stresses. More specifically, in order to metabolize mannitol, V. cholerae must regulate the synthesis of MtlA, a mannitol transporter protein produced exclusively in the presence of mannitol. We previously showed that a cis-acting small RNA (sRNA) expressed by V. cholerae, MtlS, appears to post-transcriptionally downregulate the expression of mtlA and is produced in the absence of mannitol. We hypothesized that since it is complementary to the 50 untranslated region (UTR) of mtlA mRNA, MtlS may affect synthesis of MtlA by forming an mtlA-MtlS complex that blocks translation of the mRNA through occlusion of its ribosome binding site. To test this hypothesis, we used in vitro translation assays in order to examine the role MtlS plays in mtlA regulation and found that MtlS is sufficient to suppress translation of transcripts harboring the 50 UTR of mtlA. However, in a cellular context, the 50 UTR of mtlA is not sufficient for targeted repression by endogenous MtlS; additional segments from the coding region of mtlA play a role in the ability of the sRNA to regulate translation of mtlA mRNA. Additionally, proximity of transcription sites between the sRNA and mRNA significantly affects the efficacy of MtlS.

As with all facultative pathogens, Vibrio cholerae must optimize its cellular processes to adapt to different environments with varying carbon sources and to environmental stresses. More specifically, in order to metabolize mannitol, V. cholerae must regulate the synthesis of MtlA, a mannitol transporter protein produced exclusively in the presence of mannitol. We previously showed that a cis-acting small RNA (sRNA) expressed by V. cholerae, MtlS, appears to post-transcriptionally downregulate the expression of mtlA and is produced in the absence of mannitol. We hypothesized that since it is complementary to the 5′ untranslated region (UTR) of mtlA mRNA, MtlS may affect synthesis of MtlA by forming an mtlA-MtlS complex that blocks translation of the mRNA through occlusion of its ribosome binding site. To test this hypothesis, we used in vitro translation assays in order to examine the role MtlS plays in mtlA regulation and found that MtlS is sufficient to suppress translation of transcripts harboring the 5′ UTR of mtlA. However, in a cellular context, the 5′ UTR of mtlA is not sufficient for targeted repression by endogenous MtlS; additional segments from the coding region of mtlA play a role in the ability of the sRNA to regulate translation of mtlA mRNA. Additionally, proximity of transcription sites between the sRNA and mRNA significantly affects the efficacy of MtlS.