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Escherichia coli: A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (Gram-negative facultatively anaerobic rods) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce Diarrhea and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (Enterotoxigenic escherichia coli), etc.

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota

JoVE 50762


Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

JoVE 10515

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan State University

Bacteria have the ability to exchange genetic material (DeoxyriboNucleic Acid, DNA) in a process known as horizontal gene transfer. Incorporating exogenous DNA…


Microscopy and Staining: Gram, Capsule, and Endospore Staining

JoVE 10513

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America

Bacteria are diverse microorganisms found nearly everywhere on Earth. Many properties help distinguish them from…


Replication in Prokaryotes

JoVE 10788

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell’s circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from two replication forks, resulting in two DNA molecules.

Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands. Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. DNA can only be synthesized in the 5’ to 3’ direction, so one strand of the template—the leading strand—is elongated continuously, while the other strand—the lagging strand—is synthesized in shorter pieces of 1000-2000 base pairs called Okazaki fragments. Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli, a commonly-used model organism. E. coli has 5 DNA polymerases: Pol I, II, III, IV, and V. Pol III is responsible for the majority of DN

 Core: DNA Structure and Function

Lysogenic Cycle of Bacteriophages

JoVE 10824

In contrast to the lytic cycle, phages infecting bacteria via the lysogenic cycle do not immediately kill their host cell. Instead, they combine their genome with the host genome, allowing the bacteria to replicate the phage DNA along with the bacterial genome. The incorporated copy of the phage genome is called the prophage. Some prophages can re-activate and enter the lytic cycle. This often occurs in response to a perturbation, such as DNA damage, but can also transpire in the absence of external cues. In some cases, the genes encoded by prophages can alter the phenotype of the infected bacterium, a process known as lysogenic conversion. Some phages encode proteins or toxins called virulence factors that can facilitate bacterial infections. Through lysogenic conversion, normally non-pathogenic bacteria can become highly virulent via infection by a phage carrying virulence factors. For example, such phages are largely responsible for the pathogenicity of the bacterial species that cause botulism (Clostridium botulinum), diphtheria (Corynebacterium diphtheriae), and cholera (Vibrio cholerae). Without lysogenic conversion, these bacteria do not usually cause disease. A particularly well-studied example of lysogenic conversion is that of the Escherichia coli strain O157:H7. Several massive food recalls have stemmed

 Core: Viruses

Mismatch Repair

JoVE 10791

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.

The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic information needs to be replicated. Despite the proofreading ability of the DNA polymerase, a copying error occurs approximately every 1 million base pairs. One type of error is the mismatch of nucleotides, for example, the pairing of A with G or T with C. Such mismatches are detected and repaired by the Mutator protein family. These proteins were first described in the bacteria Escherichia coli (E. coli), but homologs appear throughout prokaryotes and eukaryotes. Mutator S (MutS) initiates the mismatch repair (MMR) by identifying and binding to the mismatch. Subsequently, MutL identifies which strand is the new copy. Only the new strand requires fixing while the template strand needs to remain intact. How can the molecular machinery identify the newly synthesized strand of DNA? In many organisms, cytosine and adenine bases of the new strand receive a methyl group some time after replication. Therefore,

 Core: DNA Structure and Function

Binary Fission

JoVE 10759

Fission is the division of a single entity into two or more parts, which regenerate into separate entities that resemble the original. Organisms in the Archaea and Bacteria domains reproduce using binary fission, in which a parent cell splits into two parts that can each grow to the size of the original parent cell. This asexual method of reproduction produces cells that are all genetically identical. Though its speed varies among species, binary fission is generally rapid and can yield staggering growth. In the amount of time it takes bacterial cells to undergo binary fission, the number of cells in the bacterial culture doubles. Thus, this period is the doubling time. For example, Escherichia coli cells typically divide every 20 minutes. Bacterial growth, however, is limited by factors including nutrient and space availability. Thus, binary fission occurs at much lower rates in bacterial cultures that have encountered a growth-limiting factor (i.e., entered a stationary growth phase). In addition to organisms in the Archaea and Bacteria domains, some organelles in eukaryotic cells also reproduce via binary fission. Mitochondria, for example, divide by prokaryotic binary fission. This process requires the division of mitochondrial proteins and DNA.

 Core: Cell Cycle and Division

Prokaryotic Cells

JoVE 10690

Prokaryotes are small unicellular organisms in the domains Archaea and Bacteria. Bacteria include many common organisms such as Salmonella and Escherichia coli, while the Archaea include extremophiles that live in harsh environments, such as volcanic springs.

Like eukaryotic cells, all prokaryotic cells are surrounded by a plasma membrane and have DNA that contains the genetic instructions, cytoplasm that fills the interior of the cell, and ribosomes that synthesize proteins. However, unlike eukaryotic cells, prokaryotes lack a nucleus or other membrane-bound intracellular organelles. Their cellular components generally float freely within the cytoplasm, although their DNA—usually consisting of a single, circular chromosome—is clustered within a region called the nucleoid. Inside the cytoplasm, many prokaryotes have small circular pieces of DNA called plasmids. These are distinct from the chromosomal DNA in the nucleoid and tend to have just a few genes—such as genes for antibiotic resistance. Plasmids are self-replicating and can be transmitted between prokaryotes. Most prokaryotes have a cell wall made of peptidoglycan that lies outside of their plasma membrane, which physically protects the cell and helps it maintain osmotic pressure in different environments. Many prokaryotes also have a sticky capsule layer that covers

 Core: Cell Structure and Function

Population Growth- Concept

JoVE 10605

Population Models

An ecological population is a group of individuals of a single species living in an area at the same time. To persist, a population must either grow or maintain its size. Population ecology is the study of how population size and age distribution change over time through interactions with other species and the environment as well as with individuals of their own…

 Lab Bio

Tandem Mass Spectrometry

JoVE 5690

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a…


Water Quality Analysis via Indicator Organisms

JoVE 10025

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

Water quality analysis monitors anthropogenic influences such as pollutants, nutrients, pathogens, and any other constituent that can impact the water’s integrity as a resource. Fecal contamination contributes…

 Environmental Microbiology

Bacterial Growth Curve Analysis and its Environmental Applications

JoVE 10100

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

Bacteria are among the most abundant life forms on Earth. They are found in every ecosystem and are vital for everyday life. For example, bacteria affect what people eat, drink, and breathe, and there are actually more…

 Environmental Microbiology

Gram Staining of Bacteria from Environmental Sources

JoVE 10092

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

The spectrum of research in environmental microbiology is broad in scope and application potential. Whether the work is bench-scale with known bacterial isolates, or in the field collecting soil or water samples…

 Environmental Microbiology

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

1Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin, 2Department of Bioenergetics, Institute of Chemistry, Technische Universität Berlin, 3Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Department of Molecular Genetics, University of Groningen

JoVE 57551


Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

1College of Bioscience and Biotechnology, Yangzhou University, 2Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University, 3Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, 4Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 5The Testing Center, Yangzhou University

JoVE 59336

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