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Journal of Microbiology & Biology... Apr 2023Undergraduate students often struggle to understand the basics of bacterial gene regulation, a key concept in microbiology. They find it hard to visualize the...
Undergraduate students often struggle to understand the basics of bacterial gene regulation, a key concept in microbiology. They find it hard to visualize the architecture of a bacterial operon or how the gene, RNA, and protein components interact with each other to regulate the operon. To better visualize the molecular interactions, students engaged in a role-playing exercise on bacterial gene regulation in the classroom. Before beginning the activity, they received a shortened, traditional lecture on the architecture and function of the operon under "on" and "off" conditions. Students chose one or more placards detailing a molecular role (such as promoter, repressor, RNA polymerase, gene X, gene Y, etc.). Upon receiving instructor prompts, they assembled in linear order to mimic correct genomic locations of genes and regulatory elements on the operon. When given a prompt for "operon on" or "operon off" condition, students identified all the necessary components (roles) for that condition, assembled in the correct order, and then moved through the assembled operon to mimic what happens inside the cell under that condition. Students were tested before and after the activity using a set of eight multiple-choice questions. Students showed significant gains in their ability to answer these questions correctly immediately after the activity. More importantly, the improved understanding was also reflected in a high median score on summative assessments given a few weeks after the completion of the activity. This activity can also be readily adapted to online or a hybrid mode of teaching to benefit larger student populations.
PubMed: 37089225
DOI: 10.1128/jmbe.00006-23 -
Methods in Molecular Biology (Clifton,... 2021The lacZ gene and corresponding β-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze...
The lacZ gene and corresponding β-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of β-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.
Topics: Bacterial Proteins; Bacterial Toxins; Base Composition; Codon; Escherichia coli; Gene Expression; Genes, Reporter; Hemolysin Proteins; High-Throughput Screening Assays; Lac Operon; Promoter Regions, Genetic; Staphylococcus aureus; beta-Galactosidase
PubMed: 34264459
DOI: 10.1007/978-1-0716-1550-8_6 -
Mutation Research. Genetic Toxicology... Nov 2019Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets... (Review)
Review
Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.
Topics: Animals; Animals, Genetically Modified; Biotransformation; Cell Division; Cell Line; Escherichia coli Proteins; Genes, Reporter; Genetic Vectors; Humans; In Vitro Techniques; Lac Operon; Mutagenicity Tests; Pentosyltransferases; Reproducibility of Results; Research Design; Rodentia; Transgenes; Validation Studies as Topic
PubMed: 31699347
DOI: 10.1016/j.mrgentox.2019.04.002 -
International Journal of Biochemistry... 2019Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the...
Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the culture. We utilized a well-characterized repression loop model assembled from elements of the operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.
PubMed: 31523479
DOI: No ID Found -
BioRxiv : the Preprint Server For... Jan 2023As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of...
As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of . We introduce activities and supporting curricula to teach the structure and function of the operon, dose-responsive behavior, considerations for biosensor outputs, and a 'build-your-own' activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms - and at home - without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.
PubMed: 36711593
DOI: 10.1101/2023.01.09.523248 -
Nucleic Acids Research Apr 2021Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there...
Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this 'loopometer' assay to measure DNA looping for a variety of bacterial and phage proteins.
Topics: Bacteriophage lambda; Binding Sites; Chemistry Techniques, Analytical; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Lac Operon; Lac Repressors; Nucleic Acid Conformation; Operator Regions, Genetic; Protein Binding; Repressor Proteins; Viral Regulatory and Accessory Proteins
PubMed: 33511418
DOI: 10.1093/nar/gkaa1284 -
Scientific Reports Jan 2021FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161a, lacking the major exon #3...
FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161a, lacking the major exon #3 which was replaced by a construct that include LacZ under the expression of the Fam161a promoter. LacZ staining was evident in ganglion cells, inner and outer nuclear layers and inner and outer-segments of photoreceptors in KO mice. No immunofluorescence staining of Fam161a was evident in the KO retina. Visual acuity and electroretinographic (ERG) responses showed a gradual decrease between the ages of 1 and 8 months. Optical coherence tomography (OCT) showed thinning of the whole retina. Hypoautofluorescence and hyperautofluorescence pigments was observed in retinas of older mice. Histological analysis revealed a progressive degeneration of photoreceptors along time and high-resolution transmission electron microscopy (TEM) analysis showed that photoreceptor outer segment disks were disorganized in a perpendicular orientation and outer segment base was wider and shorter than in WT mice. Molecular degenerative markers, such as microglia and CALPAIN-2, appear already in a 1-month old KO retina. These results indicate that a homozygous Fam161a frameshift mutation affects retinal function and causes retinal degeneration. This model will be used for gene therapy treatment in the future.
Topics: Animals; Calpain; Disease Models, Animal; Electroretinography; Eye Proteins; Frameshift Mutation; Humans; Lac Operon; Mice; Mice, Knockout; Retina; Retinal Degeneration; Retinitis Pigmentosa; Tomography, Optical Coherence; Visual Acuity
PubMed: 33479377
DOI: 10.1038/s41598-021-81414-1 -
Microbial Cell Factories Jan 2022The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in...
BACKGROUND
The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host.
RESULTS
Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels.
CONCLUSIONS
The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.
Topics: Bacteriophage T7; Cloning, Molecular; Culture Media; Escherichia coli; Genetic Vectors; Lac Operon; Lac Repressors; Peptones; Plant Proteins; Promoter Regions, Genetic; Recombinant Proteins
PubMed: 35090462
DOI: 10.1186/s12934-022-01740-5 -
The Journal of Comparative Neurology Feb 2021We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local...
We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.
Topics: Animals; Animals, Newborn; Chromosome Mapping; Female; Gene Expression; Homeodomain Proteins; Lac Operon; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pregnancy; Thalamus; Zebrafish
PubMed: 32420617
DOI: 10.1002/cne.24952 -
Nucleic Acids Research Mar 2022Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase...
Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.
Topics: DNA; DNA, Bacterial; DNA, Superhelical; DNA-Directed RNA Polymerases; Escherichia coli; Lac Operon; Lac Repressors; Nucleic Acid Conformation
PubMed: 35188572
DOI: 10.1093/nar/gkac093