Chapter 18 - Biotechnology 

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  • Biotechnology tools are being used for a variety of purposes, including cancer therapies, increased agricultural yields, gene therapies for genetic disorders, de-extinction projects (such as the one to reintroduce the woolly mammoth), and extinction projects (such as attempting to eliminate pathogens that cause human disease), to name a few.
    • Understanding the fundamentals of how these strategies function, as well as their potential applications and misuses, is critical.
    • Bacterial transformation involves the introduction of foreign DNA into bacterial cells.
    • The foreign DNA, which is often a tiny, circular bit of DNA known as a plasmid, can either integrate into the host cell's chromosome or remain separate from the host cell DNA in the cell's cytoplasm.
  • Heat shock is one way of converting bacteria in which bacterial cells are combined with foreign DNA and then exposed to heat.
    • This heat shock opens up transient tiny holes via which foreign DNA can enter certain bacterial cells.
    • A selectable marker is required for the foreign plasmid DNA so that cells that have integrated the plasmid DNA can be identified.
    • %%Typically, the selectable marker is an antibiotic resistance gene that is not present in nontransformed bacterial cells.%%
    • By cultivating the transformed bacteria on an antibiotic-containing agar plate, one may select for bacteria that absorbed the plasmid and are now expressing the genes from the plasmid.
    • ^^It is referred to as recombinant DNA if the plasmid contains a gene from another creature. Simply put, recombinant DNA is DNA that has been recombined from different source species.^^
    • Using restriction endonucleases (also known as restriction enzymes), DNA may be sliced at particular sequences.
  • Using restriction endonucleases (also known as restriction enzymes), DNA may be cut at particular sequences and then recombined and linked with DNA ligases.
    • Some recombinant plasmids may contain both the selectable marker and a gene encoding a desired protein product.
    • Bacteria that consume the recombinant plasmid would be capable of manufacturing the product encoded by the plasmid's gene.
    • Bacteria currently create a considerable proportion of several medicinal compounds (such as insulin).
    • Gel electrophoresis is a method for separating DNA fragments based on size and charge.
    • Treatment of a DNA sample with restriction enzymes, which cleave DNA at specified base pair sequences, can result in the formation of DNA fragments.
  • %%Because of genetic code redundancy, even creatures with the same protein sequences will have slightly different DNA sequences.%%
    • When these distinct DNA sequences are treated with restriction enzymes, they are cut at different sites, resulting in varying fragment sizes.
    • The DNA double helix's backbone is made up of five-carbon deoxyribose sugars and phosphate groups.
    • Phosphate groups are somewhat negatively charged.
    • An electric current is supplied to the gel after DNA samples are put into wells at the top of the gel.
  • ^^A positive cathode is attached to the gel's bottom, while a negative anode is attached to the gel's top.^^
    • Because of the abundance of phosphate groups in DNA molecules, they carry a net negative charge.
    • The gel is typically constructed of agarose and has minute holes through which the DNA fragments flow.
    • ^^Shorter pieces will be able to travel faster via these pores, whereas larger fragments would take longer.^^
    • Asaresult,shorterfragmentswillbediscoverednearthebottomofthegel,farthestfromthewellintowhichtheDNAsamplewasinserted,whilelongerfragmentswillbelocatedclosertothetopofthegel.As a result, shorter fragments will be discovered near the bottom of the gel, farthest from the well into which the DNA sample was inserted, while longer fragments will be located closer to the top of the gel.
    • To amplify particular DNA fragments, the polymerase chain reaction (PCR) is performed.
    • PCR may be used to replicate a particular DNA segment millions of times.
    • PCR includes cycles of DNA replication utilizing primers specific to the beginning and end of the fragment of the DNA sequence to be amplified.
  • In nature, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system serves as an adaptive immune system in bacteria.
    • Bacteriophages are viruses that may infect bacteria.
    • Whenabacteriophageinfectsabacterium,theviralDNAisbrokenupandinsertedbetweenshortpalindromicrepeatsinthebacteria′sDNA.When a bacteriophage infects a bacterium, the viral DNA is broken up and inserted between short palindromic repeats in the bacteria's DNA.
    • This enables the bacterium to keep track of which viruses have already invaded its cell.
  • When a new virus infects the bacteria, the bacteria compare the DNA sequences from the new virus to those stored in the bacterium.
    • If it detects a match, it uses the Cas9 enzyme to cut the virus's DNA.
    • CRISPR has the ability to modify DNA sequences.
  • To determine where to cut DNA, the Cas9 enzyme employs a bit of guide RNA.
    • The Cas9 enzyme may be directed to cut at a specific DNA sequence by employing synthesized guide RNA bits that correspond to the intended site of the cut to be produced.
    • Ifthiscutoccursinthemidstofageneoroneofitsregulatoryregions,itmightresultina"knockout"ofthegene,renderingitinoperable.If this cut occurs in the midst of a gene or one of its regulatory regions, it might result in a "knockout" of the gene, rendering it inoperable.
    • Observing the results of this deletion can help scientists learn more about the gene's function.
    • A new DNA sequence at the location cut by the Cas9 enzyme is often desired instead of a knockout.