MIC 205A III

Scientific Nomenclature

Carlos Linnaeus, system gives every organism 2 names which include the genus and the species

CRISPR

Cas9 (an endonuclease used for gene editing Uses single-stranded RNA (gRNA) to recognize specific DNA sequence Makes double-stranded cut at that DNA sequence DNA at that site can be altered by manipulating DNA repair mechanisms Disable genes (imprecise repairs) Edit genes (homology-directed repair)

culture characteristics

Colony morphology can give initial clues to the identity of an organism Includes: Colony size Colony colour Colony odour Differential media aids in identification

dideoxy chain termination

DNA sequencing technique that allows you to determine the sequence of bases in a piece of DNA

Pyrosequencing

DNA sequencing technology that is based on the generation and detection of a pyrophosphate group liberated from a nucleotide triphosphate

Recombinant DNA

DNA that has been formed artificially by combining constituents from different organisms.

Genotypic characteristics

Differences in DNA sequences can be used to determine the point in time at which two organisms diverged from a common ancestor

Taxonomic Hierarchy

Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species

Genetically Engineered Eukaryotes

Engineered yeast for research and commercial products Transgenic plants have been engineered to resist pests and herbicides, have improved nutritional value, and function as edible vaccines.

Techniques of biotechnology

Enzymes: Restriction enzymes, ligases, DNA polymerases, reverse transcriptases, etc Vectors: used in cloning Transformation DNA/Protein gel electrophoresis Polymerase chain reactions RNAi CRISPR/Cas9

food production

GMO crops/microorganisms

Application of Biotechnology

Gene Editing Genetic Engineering Food production Diagnostic tests Gene therapies Precision/personized Medicine: Vaccines/Therapeutic proteins:

Culture

Grown in laboratory media

genetic engineering application

Health and agriculture, study expressions of particular genes, select traits that might benefit humans, study development of certain disease

Prokaryotic species

-Cells that share physical characteristics and have at least 70% DNA similarity -At least 97% identical 16S rRNA sequence similarity

Examples of Protein Production

-Human insulin expressed from bacteria -Proteins needed for vaccine production -Chymosin (rennin) for cheese production -Synthetic bacteria may express valuable products in future

cDNA libraries

-contain smaller fragments of DNA, and only include exons of genes expressed by the sample tissue -can be used to make recombinant proteins or for gene therapy

Number of archaeal phyla

26

Type of ribosome

70s for archaea and bacteria 80s for eukaryotes

Number of bacterial phyla

92

Biotechnology

A form of technology that uses living organisms, usually genes, to modify products, to make or modify plants and animals, or to develop other microorganisms for specific purposes.

whole genome sequencing

A method of genome sequencing that selects clones at random from a genomic library and after sequencing them, assembles the genome sequence by using software analysis

Sanger sequencing

A procedure in which chemical termination of daughter strands help in determining the DNA sequence.

Gram staining

A process by which components of bacterial cell walls are bound to Gram's stain. Depending on the amount of peptidoglycan in their cell walls, bacteria stain differently and are classified as Gram-negative or Gram-positive.

Cloning

A process in which a cell, cell product, or organism is copied from an original source.

isolate

A sample obtained from an infected individual, a specific site, etc

strain

A sub-population of a species that shares some properties absent in the rest of the species

variant

A sub-population that contains a number of nucleotide changes, may/may not have any change in properties

polymerase chain reaction (PCR)

A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.

RNA interference (RNAi)

A technique used to silence the expression of selected genes. RNAi uses synthetic double-stranded RNA molecules that match the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA.

genetic engineering

A technology that includes the process of manipulating or altering the genetic material of a cell resulting in desirable functions or outcomes that would not occur naturally.

Roche 454 Pyrosequencing

A type of "next generation sequencing": Questions of microfluidics, moving fluids around through small columns • Attach fragmented strands of DNA to little beads (certain fragment per bead, use primers to attach) and put in little holes and run fluids through them, producing lights that can sequence DNA • Similar to in vitro process of oligonucleotide synthesis - adds dNTPs one at a time, light will be produced when added - cheap, short, and reads fair amount of bases - read length = 500 bases, runtime (days per gigabase) = 2, cost per 1000 bases - $0.02

Intron presence

Absent in bacteria Present in eukaryotes Sometimes present in archaea

reverse transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

Ligase

An enzyme that connects two fragments of DNA to make a single fragment

clone

An organism that is genetically identical to the organism from which it was produced

genetically modified organisms

An organism whose genetic material has been altered through some genetic engineering technology or technique.

Cytoplasmic membrane lipids

Archaea - Hydrocarbons linked to glycerol by linkage Bacteria and Eukaryote- Fatty acids linked to glycerol by ester linkage

Nanopore sequencing

Involves passing a DNA sequence through a small nanopore and measuring the change in current (shape).

informal grouping of microbes

Lactic acid bacteria Anoxygenic phototrophs Endospore-formers Sulfate reducers

Identification

Matching characteristics of an "unknown" organism to lists of known organisms. Clinical lab identification, more practical Based on microscopic examination, culture characteristics, biochemical tests, nucleic acid analysis, or symptoms

PCR amplification

Melt: Highest temp, separates strands Anneal: Lowest temp, primers attach Extend: Medium temp, DNA replicates (Polymerase) Final Extension: Ten minutes at medium temp, ensure DNA has finished replicating

Ion semiconductor sequencing

Monitors H+ given off during addition of nucleotide to growing DNA molecule

What vectors contain

Multiple cloning sites Antibiotic resistance A secondary selectable marker

detecting specific nucleotide sequences

Nucleic acid probes and nucleic acid amplification tests can be used to identify prokaryotes grown in culture. In some cases, the method is sensitive enough to detect the organism directly in a specimen

Membrane bound nucleus

Only in Eukaryotes

Classification

Placing organisms in groups of related species. Lists of characteristics of known organisms, sequence similarities Bergey's Manual of Systematic of Archaea and Bacteria

peptoglycan cell wall

Present in bacteria and eukaryotes Absent in archaea

gel electrophoresis

Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

genetic engineering

Process of making changes in the DNA code of living organisms

Five Kingdoms

Prokaryotae, protoctista, fungi, plantae, animalia

Next Generation Sequencing Strategies

Pyrosequencing Sequencing by synthesis Sequencing by ligation Ion semiconductor sequencing Nanopore sequencing

antibiotic resistance

Resistance evolving rapidly in many species of prokaryotes due to overuse of antibiotics, especially in agriculture.

Vector

Self-replicating DNA used to carry the desired gene to a new cell (plasmids, viruses, etc)

SOLiD Sequencing (NextGen)

Sequencing by ligation. A pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. The oligonucleotides are annealed and ligated, the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR and the resulting beads (each containing single copies of the same DNA) are deposited on the glass slide for sequencing.

Fluorescence in situ hybridization (FISH)

Technique used to detect a given nucleotide sequence within intact cells on a microscope slide

colony blotting

Technique used to determine which colonies on an agar plate contain a given nucleotide sequence

sequence assembly

The process in which short nucleotide sequences of a long DNA molecule are arranged in the correct order to generate the complete sequence.

Taxonomy

The scientific study of how living things are classified

Sequencing rRNA genes

This requires amplifying and then sequencing rRNA genes, but it can be used to identify organisms that have not yet been grown in culture

acid fast stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol

gene editing

a highly precise type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases

Metabolic capabilities

a set of biochemical tests can be used to identify a microorganism

Cassava Brown Streak Virus

a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. Requires the interaction between a viral-genome- linked protein (Vpg) and eIF4E for expression of viral genome Engineer eIF4E so that it no longer interacts with eIF4E, creating host immunity

protein profile

a technique for visualizing the proteins contained in a cell; obtained by the use of polyacrylamide gel electrophoresis

Ion Torrent

add each base one at a time and monitor release of proteins cross of PCR machine and micro-pH meter cheaper and no need to separate reaction products

Illumina sequencing

allows you to get billions of bases on something the size of a microscope slide

restriction enzymes

an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases. Name of the enzyme represents the bacterium the enzyme was isolated from Most restrictions sites range from 4 to 8 nucleotides Palindromes: the same on both strands when read in 5′ to 3′ direction

Bioformatics

application of mathematics and computer science to store, retrieve, and analyze biological data

dCas9

can be used along with a gRNA to silence a gene by blocking transcription

serological testing

can differentiate not only among microbial species, but also among strains within species

RNAseq

cellular RNA is isolated, converted to cDNA, and sequenced, providing information on the RNA transcripts present in a cell

DNA libraries

collection of clones containing all the DNA fragments from one source (tissue type, cell type, or single individual)

gene therapies

designed to treat or cure a variety of diseases by inserting a functional gene into an individual's cells to replace a defective version of the gene

Next generation sequencing

entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences. Highly automated; fast, lower costs; analyze millions to billions of bases

Number of eukaryote kingdoms

five

microscopic morphology

fresh or stained microorganisms from specimen; shape, size, stain reaction, cell structures Quickly determine size, shape, and staining characteristics More useful to diagnose eukaryotic infections

Eukaryotic species

group of similar organisms that can sexually reproduce together

Diagnostics Tests

identify pathogens/diseases

Deoxy-chain termination sequencing

is based on the principle that during DNA synthesis, addition of a nucleotide triphosphate requires a free hydroxyl group on the 3′ carbon of the sugar of the last nucleotide of the growing DNA strand

PacBio sequencing

longest 10,000-70,000 nucleotides, 10-20% error, more expensive than Illuminia, more throughput than Sanger

Phenotypic characteristics

microscopic morphology, culture characteristics, metabolic capabilities, serology, fatty acid analysis

sequencing by synthesis

monitors identify which of 4 nucleotides is being added to complementary strand

viral species

population of viruses with similar characteristics that occupies a particular ecological niche

single cell genomics

refers to isolating individual cells, amplifying their genomes and sequencing these individual genomes one by one

Serotype

strain of bacteria that carries a set of similar antigens on its cell surface, often many in a bacterial species

Genomics

study of whole genomes, including genes and their functions

Phylogenetics

the analysis of evolutionary, or ancestral, relationships between taxa

Metagenomics

the study of genetic material recovered directly from environmental samples

transcriptiomics

the study of total metabolites produced

DNA Microarrays

tools that allow scientists to study many genes, and their expression, at once

Decoding Genomes

unlock the secrete of genes and genomes revolutionize our understanding of genetics, phylogeny Sequencing techniques change rapidly Human genome project

Sequencing by ligation

uses short fluorescently labeled oligomers that hybridize to complementary DNA template, flourescently labeled bases are used to ligate the sequence and are detected and the olignonucleotide is cleaved.

Other Applications of CRISPR Technology

• "Dead" or dCas9 does not cut DNA, but still uses RNA guide to bind to specific DNA sequence ◦ Tagged DNA modifying enzymes to modify specific nucleotides (editing) ◦ Tagged transcriptional factors promote gene expression ◦ May block RNA polymerase, shut off expression ◦ May deliver molecules to certain chromosome locations ‣ Fluorescent marker to locate a gene • CRISPR-based diagnostic tests: ◦ COVID-19 tests ◦ Cancer diagnosis

X-gal blue and white selection for recombinant DNA clones

• After transformation of bacteria with vectors carrying recombinant DNA, two layered selections allow for identification of recombinant bacterial clones

Vectors

• Independently replicate in an organism (replicon) • Can be plasmids, viruses, or artificial chromosomes • Carry new DNA to desired cell • Shuttle vectors can replicate in several different species • Ti (Tumor-inducing) plasmids of Agrobacterium tumefaciens insert DNA fragments to plant genome

Bacterial Defenses Against Invading DNA

• Restriction-Modification Systems • Evolved as a defense mechanism in bacteria • Restriction enzymes cut specific DNA sequences • Destroy bacteriophage DNA in bacterial cells • Modification enzyme protects cell's own DNA by adding methyl groups • Cannot digest (host) DNA with methylated cytosines

Hybridization with a probe

• The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe • Gene probe is a short segments of DNA of a known sequence • A probe carries a radioactive label

Concerns for GMOs and other Biotechnologies

• Valid concerns: ◦ Allergens ◦ Spread of herbicide resistant gene to weeds ◦ Spread of BT toxin genes may harm non-targeted insects ◦ Misuse of personal DNA data (the Genetic Information Nondiscrimination Act) ◦ Creating a deadly pathogen (bioterrorism)? • Unfounded concerns: ◦ Causing cancer, liver damages ◦ Contain DNA ◦ Poke holes in guts • Antivaccine: ◦ contain tiny chips to control people ◦ Autisms