UF MCB3020 EXAM 3 Bacusmo

Biotechnology

use of organisms to form useful products

Genetic engineering

The deliberate modification of an organisms genome sequence

Recombinant DNA technology

procedures used to carry out genetic engineering

Cloning

generation of a large number of genetically identical DNA molecules

What are the 5 basic steps of cloning?

1. Isolate DNA to be cloned

2. Use restriction enzymes to "chop up" the DNA into fragments

3. Insert the fragments into a cloning vector, creating recombinant DNA

4. Insert the recombinant DNA into a new host, such as E. coli

5. Culture host cell, growing many identical copies.

What are restriction enzymes?

A type of endonuclease that allows scientists to cut up DNA at specific sites.

True or False: Restriction enzymes sometimes make staggered cuts, which produce single-stranded DNA sequences known as "sticky ends".

TRUE

What enzyme forms covalent bonds between the cloned gene and the plasmid, creating recombinant DNA?

Ligase

What is reverse transcriptase?

When double-stranded DNA is created by a single-stranded RNA.

What is the process or constructing cDNA? What is the goal of this process?

Goal: make DNA from RNA template

1. Apply a short poly- T primer to RNA template

2. Add reverse transcriptase and the 4 nucleotides

3. Add RNaseH to cut up the RNA and regenerate the RNA primers.

4. Add DNA polymerase and DNA ligase to synthesize the new strand

What is gel electrophoresis? Why is it important?

A technique that separates nucleic acids and proteins on the basis of their size and electrical charge.

It is important because it determines which fragments are larger than others and tells us the approximate size for each fragment.

Nucleic acids have ____________ charge due to their _______________ _____________, so they migrate through the electric field towards the _____________ electrode.

negative; Phosphate backbones ; positive

True or False: Shorter DNA will travel farther in gel electrophoresis because smaller restriction fragments move faster.

TRUE

What dye is used to stain DNA?

Ethidium bromide

What is PCR?

Polymerase Chain Reaction; A technique for quickly and easily making many copies of even a very small amount of DNA.

Where does Taq polymerase come from?

Thermophilic bacterium found in hydrothermal vents (Thermus aquaticus)

What are the 4 things required in PCR?

•primers

•target DNA

•thermostable DNA polymerase (e.g. Taq polymerase)

*each of the four deoxyribonucleotide triphosphates

True or False: In PCR, hydrogen bonds of double-stranded DNA molecules are broken by subjecting the DNA to high levels of radiation.

False; under high levels of HEAT

If you perform 6 cycles of PCR on a single double-stranded DNA molecule. How many copies will you have?

64 double-stranded copies (Each cycle the DNA content doubles)

What is a cloning vector?

Provides means for transferring a gene of interest to a host organism during the cloning process

What are the 3 things every "good" cloning vector must have?

•an origin of replication

•a selectable marker

•a multicloning site (MCS)or polylinker

Plasmids

self-replicating piece of extrachromosomal DNA

KNOW: this is the most commonly used & it's only in prokaryotes

Bacteriophages are

A virus that infects bacteria

Cosmids

A hybrid between a plasmid and a phage

artificial chromosomes

Synthetic chromosomes that contain fragments of DNA integrated into a host chromosome

What is a genomic library?

a collection of the total genomic DNA from a single organism

How are cloning vector DNA introduced into bacterial hosts?

Transformation: the uptake of naked DNA from the environment

electroporation

(Related to Transformation) A technique that makes a cell competent to pick up DNA from the environment by applying an electrical shock to it

What are 2 techniques that scientists use to isolate or visualize the protein product of a cloned gene?

Polyhistidine Tagging & Fluorescent labeling

What is genomics?

Study of the organization of genomes, the information they store, and the gene products they code for.

Describe the Sanger sequencing method

One of the most commonly used methods.

Requires DNA to be in single-stranded form

Primer has a sequence that is complementary to 3' end of the region to be copied

During Sanger sequencing, the addition of dideoxyribonucleotide to the growing strand causes DNA replication to STOP

If you run the products of the reaction on an electrophoretic gel and read from the bottom to the top you will get the DNA sequence

Describe Next-generation sequencing technologies

Involves breaking the DNA into pieces. And attaching oligonucleotides to the ends

These are cheaper and faster methods that produce shorter reads.

Which method is used and why?

Next-generation because they are cheaper and faster

What does functional genomics tell us?

The study of the function of genes

Transcriptome

entire set of mRNA transcripts

Transcriptomics

Study of the transcriptome

Proteome

Entire set of proteins

Proteomics

Study of proteomes

Metagenomics

Study of metagenomes, genetic material recovered from environmental samples

Metatranscriptomics

Study of RNA found in environmental samples

Metabolomics

Study of metabolome, entire set of small-molecule metabolites

lipidomics

Study of the lipidome, entire set of lipid profile

glycomics

Study of the glycome, entire set of sugars.

What is DNA microarray analysis?

A tool that allows scientists to observe the pattern of DNA expression for thousands of genes at a time

What are the 3 main issues with microarray analysis?

1. Only identifies mRNA molecules

2. Produces some false positives

3. Hard to detect small changes in gene expression

What method was developed to overcome these issues?

RNA-Seq Method

What is the name of the project that sequences the genomes of a wide variety of cultured microorganisms in order to improve the reference database?

The Genomic Encyclopedia of Bacteria and Archaea Project

What is bioinformatics?

The study of the genome using computers

What is the process annotation?

Process that locates genes in the genome map

_______________ are two or more genes that have similar nucleotide sequences and are found in the __________________ genome. They usually arise due to _________________________ ___________________ events.

Paralogs; Same; Gene Duplication

____________________ are two or more genes that have similar nucleotide sequences and are found in _________________ organisms. They usually arise due to _______________ events.

Orthologs; different; Speciation

What is BLAST?

A publicly available computer program that compares two or more gene sequences and identifies regions of similarity.

Describe systems biology.

An interdisciplinary field that engages in the holistic study of cells to describe their impact on catabolic, anabolic, regulatory, and behavioral pathways.

What is comparative genomics?

The study of similarities between the nucleotide and amino acid sequences among organisms in order to make inferences about gene function and evolution.

A pan-genome consists of the __________________ ____________________ of genes found in all strains in a species, while a core genome represents the minimal number of genes needed for __________________.

Entire set; Survival

True or False: In general, the metabolic & morphological complexity of the cell decreases as genome size increases.

False; it increases alongside genomic size increase

Define a genomic island and a pathogenicity island.

Genomic Island: a mobile gene element that has been permanently integrated into a bacterial genome

Pathogenicity island: When the genomic islands encode for proteins that make the microbe virulent.

What is synteny?

Study of the order of orthologous genes in the genome

Taxonomy

science of biological classification

Classification

•arrangement of organisms into groups (taxa; s., taxon)

nomenclature

•assignment of names to taxa

Identification

•determination of taxon to which an isolate belongs

What scientists developed the first formal system of classification in the 1700s?

•Carolus Linnaeus

What are the 3 forms of classification? Briefly describe each.

1. Phenetic: Involves arranging organisms into groups based on similar phenotypes

2. Genotypic: Involves comparing the genetic similarity between two organisms, either by looking at a single gene sequence or the entire genome

3. Phylogenetic: Based on the evolutionary relationship between organisms

List the hierarchical Classification System (highest to lowest)

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

Define species

collection of strains that share many stable properties and differ significantly from other groups of strains

Define a Strain and the 3 types of strains.

A population descending from a single organism or pure culture isolate

1. Biovars: same species with biochemical and physiological differences

2. Morphovars: same species with morphological differences

3. Serovars: same species with different antigenic properties

What is the type strain?

The strain that is usually one of the first strains discovered and is often the most fully characterized and described, holds the species name.

Describe the Linnaeus binomial system.

Gives each organism a scientific name based on its genus and species. Genus is capitalized, species is not (both italicized).

What are some examples of morphological features used to classify and identify organisms?

Cell shape, cell size, colonial morphology, staining behavior, colony color, mechanism of motility, and the presence or absence of cilia and flagella.

What are some examples of Physiological and Biochemical features used to classify and identify organisms?

Carbon and nitrogen sources, cell wall constituents, nutritional type, motility, oxygen relationships, photosynthetic pigments, fermentation products, and optimum pH

What are some examples of Ecological features used to classify and identify organisms?

•Life-cycle patterns

•Symbiotic relationships

•Ability to cause disease

•Habitat preferences

•Growth requirements and pathogenicity

Why are molecular methods important?

They provide the most robust analysis of microbial evolution

True or False: If the variation in the G + C content of DNA exceeds 10% in two organisms, then they are not the same species.

TRUE

If you have 30% of guanine, how much % of C do you have? % of A? % of T?

30% C, 20% A, 20% T

What is DNA melting temperature?

Temperature at which half the strands of double-stranded DNA separate from one another

What is DNA-DNA hybridization?

Involves heating up the DNA strands of 2 microbial isolates so they become single-stranded DNA, then bringing temperature back down so the single-stranded DNA from the different isolates complementary base pair.

True or False: In general, the standard for DNA-DNA hybridization is that 2 microbes should be considered the same species when they have 70% or more whole genome similarity.

True

What is genetic fingerprinting?

Allows scientists to analyze genetic relatedness by looking at DNA fragmentation patterns resulting from restriction endonuclease cleavage.

What is a phylogenetic tree? What do nodes and branches represent?

Shows the inferred evolutionary relationship among a number of branching lineages.

Nodes represent divergence events, while branches represent lineages.

NOTE: Longer branches indicate there have been more molecular changes in that lineage

____________ gene transfer between domains complicates the construction of phylogenetic trees.

Horizontal

What domain was the LUCA (Last Universal Common Ancestor) a member of?

bacteria

What characteristics of mitochondria and chloroplasts provides evidence that supports the endosymbiotic hypothesis?

Single circular chromosome and 70S ribosomes

What have Mitochondrion arisen from through endosymbiosis:

Rickettsiae bacteria

What have Hydrogenosomes arisen from through endosymbiosis:

mitochondrial ancestor

What have Chloroplasts arisen from through endosymbiosis:

cyanobacteria

What are the recommended 4 criteria to meet a "gold standard" for the assignment of 2 organisms by the ICSP?

1. Organisms must be phenotypically similar

2. DNA-DNA hybridization must show whole-genome similarity of at least 70%

3. G + C content of the DNA must be such that the melting temperature of DNA is within 5 degrees Celsius between the individuals

4. rRNA sequence must have less than 3% divergence

What is genetic drift?

Small, random genetic changes that can lead to microevolution over many generations

What is the most accurate classification scheme for prokaryotes?

Bergey's Manual of Systematic Bacteriology

What is symbiosis?

Any stable association of one organism with another

Mutualism

Both organisms benefit and depend on each other for growth and survival

Cooperation

Both organisms benefit but do not have to interact for survival

Commensalism

One individual gains from the association, but the other is neither harmed nor benefited

Predation

One organism (predator) attacks and kills another (prey)

Parasitism

One organism (parasite) benefits from the other (host)

Amensalism

One organism has negative impact on another organism

Competition

two organisms attempt to use the same resource

What is coral bleaching?

A condition in which the coral either expels its endosymbiont or the endosymbiont loses its photosynthetic pigment

True or False: Temperature increases of 2 degrees Celsius can cause coral bleaching.

True

What microbial interaction are the following examples: Tapeworms infesting a canine.

Parasitism