Prepare a simple flowchart of the experimental procedures for Bacteria Preparation and Transformationthe notes are attached below The bacterium Echerichia coli (E. coli) is used extensively in molecular biology for recombinant DNAresearch. E. coli is a normal inhabitant of the human colon and is a common soil bacterium. There areseveral characteristics of E. coli that makes them extremely useful. The strains used in the lab are non-pathogenic and, hence, pose a minimal risk to lab personnel. They grow at an optimal temperature of 37°C with no special requirements for nitrogen, oxygen or light. They undergo direct cell division, with oneparental cell producing two identical cells and will continue this process given enough nutrients and roomfor growth. On semi-solid media, an individual bacterium will continue to divide until it forms a colony ofbacteria with each member of the colony essentially a clone of the others.E. coli has a single, circular chromosome of approx. 5,000,000 base pairs of DNA, which is less than 0.1% of the DNA content of a typical human cell. E. coli is easily grown in the lab, either in suspension in aliquid media that provides nutrients such as Luria Broth (LB) or on semi-solid media containing nutrientssuch as LB agar. E. coli can also contain extrachromosomal, circular, double stranded DNA moleculesknown as plasmids that typically vary between 1kilobase pairs (kb) to more than 200 kb. Depending onthe type of plasmid, they may be present in the bacteria in low copy number or high copy number, andthey may replicate autonomously (the majority) or when the bacterium replicates its genomic DNA. Mostplasmids have a narrow host range and can only be maintained in a limited number of closely relatedspecies. Plasmids are dependent on the enzymes and proteins encoded by their host for their replicationand transcription. Over the last 40 years, molecular biologists have learned how to manipulate theseplasmids to change the bacteria such that the plasmids carry novel genetic information or foreign DNAthat alters the phenotype of the bacteria. The process known as "bacterial transformation" allows one totransmit the recombinant DNA molecules into the bacteria. Transformation can also be used to introduceuseful new genes from other species into a species that normally doesn't have those genes. For example,plants and animals can be made resistant to certain diseases by this process.E. coli cannot normally grow in the presence of the antibiotic ampicillin. However, the DNA plasmidPUC19 (see Fig.1) which will be put into the bacteria, contains the gene that encodes ampicillinresistance (ampr). After undergoing transformation, bacteria which successfully take up new DNA will beidentified by their ability to grow in the presence of ampicillin. They will form colonies on LB + Ampicillinagar plates that are essentially clones of the original bacterium that was transformed. Even under thebest conditions, transformation is inefficient, and plasmids only become established in a minority of the bacterial population. So, the amp gene acts as a selectable marker that allows these rare transformantsto be easily isolated, by killing all the bacteria that were not transformed with the plasmid. In everyexperiment, it is also essential to include negative controls to eliminate the possibility of contamination.In most cases, "transformation" with the buffer used to resuspend the plasmid DNA is sufficient i.e. noDNA is added to the competent bacteria in the control experiment. No bacteria should grow on theselective plate.Nucleic acids do not enter bacteria such as E. coli without help getting through the outer and inner cellmembranes and cell wall. Only once inside the cell can they be expressed and replicate. This process,known as competency, involves a change in the structure and permeability of the cell membrane thatallows the plasmid to enter the cell and can be achieved by the treatment with chemicals, such as CaCl2,followed by heat and cold treatments (as first described by Mandel and Higa, 1970), or by physicaltreatment, such as the use of electric pulses (electroporation). The chemical competency method issimple, rapid and usually generates transformation efficiencies of between 105 and 107 transformedcolonies per ug of plasmid DNA. Competent cells can be preserved at -70 °C, although there may besome reduction in their efficiency of transformation during prolonged storage.Your instructor will show you the basics of working with E. coli, sterile technique and plating bacteria.Sterile technique is essential to prevent contaminating bacteria or fungi from growing on your petri plate.Never assume a surface is sterile unless it has been specially prepared (i.e. wiped down with ethanol).Similarly, all glassware should be sterilized by autoclaving or by passing through a flame.Figure 2-1. „Map of the pUC19 plasmid. Glick& Pastemak.lacZ' geneAmp"Multiple cloning sequencegenePUC19lacI geneOrigin of replication

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Answered: Prepare a simple flowchart of the…

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Prepare a simple flowchart of the experimental procedures for Bacteria Preparation and Transformation

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

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