UF MCB 3020 Exam 3

biotechnology

refers to the use of living organisms to create products that are useful to humans

Recombinant DNA

DNA from two or more sources...allows for creation of DNA sequences not otherwise found in biological organisms

Basic Mechanism of gene cloning

1. isolate plasmid DNA from bacterial cell

2. isolate DNA of interest

3. Insert the gene of interest into plasmid DNA...forms recombinant DNA

4. insert the recombinant DNA into a bacterium

5. culture and grow the bacteria

Restriction Enzymes

a type of endonuclease that cuts up DNA

*normally leave "sticky ends" of DNA

Restrictions sites (recognition sequences)

short nucleotide sequences that are recognized by restriction enzymes as the place to cut DNA

normally palindromes

sticky ends

cut DNA that is left with short, single stranded sequences on both sides of the fragments

DNA ligase

seals the two strands of "sticky ended" DNA together by catalyzing phosphodiester bonds between the single-stranded breaks.

**results in recombinant DNA

Electroporation

makes a cell competent to pick up DNA from the environment by applying an electrical shock to it, which increases membrane permeability.

RecA proteins

competent cells are engineered to lack these proteins, which may try to otherwise destroy the inserted sequence or attempt to correct the recombinant DNA

Reverse Transcriptase

turns single-stranded RNA into double-stranded DNA

it is an RNA-dependednt DNA polymerase

Complementary DNA (cDNA)

the DNA produced by reverse transcriptase and the RNA template

constructing cDNA

1. apply s short poly-T primer to single-stranded RNA

2. add reverse transcriptase and 4 nucleotides

3. add RNaseH enzyme to cut up the RNA and regenerate the RNA primers

4. add DNA polymerase and DNA ligase to synthesize a new strand

cloning vector

provides a means for transferring a gene of interest to a host organism

"good" cloning vectors include

an origin of replication

a selectable marker

unique restriction sites (polylinkers or multicloning sites)

Plasmid

most common cloning vector

a self-replicating piece of extrachromosomal DNA found in prokaryotes

-mid-sized range (20,000 base pairs or less)

pBR322 and pUC19

bacteriophage

virus that infects bacteria

carry smaller fragments between 9,000-25,00 base pairs

cosmid

a hybrid between a plasmid and a phage

carry large fragments between 30,00 and 47,000

artificial chromosomes

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

1. P1 artificial chromosomes (PACs)

2. Bacterial artificial chromosomes (BACs)

3. yeast artificial chromosomes (YACs)

**carry up to 1 MILLION bp

DNA analysis

used to determine the location of genes, differences in genes, and where and when genes are expressed

Includes...

1. PCR

2. gel electrophorese

3. restriction fragment analysis

4. blotting techniques

Polymerase chain reaction (PCR)

quickly and easily makes copies of even a very small amount of DNA.

targets the DNA with the following:

1. Primers

2. thermostable DNA polymerase (taq polymerase)

3.deoxyribonucleotide triphostates (dATP, dCTP, dGTP, dTTP)

**the solution is heated to 90 to 50 to 70 and then again

Gel electrophoresis

a technique that separates nucleic acids and proteins on the basis of their size and electrical charge, using a gel that is a polymer

then stained with ethidium bromide for DNA to be visible

Restriction fragment length polymorphism (RELP)

a tool for genetic fingerprinting, genome mapping, localization of the genes, paternity testing, and microbial classification.

STR analysis

compares genomes based on the number of short tandem repeats

Ribotyping

variation of RELP that compared ribosomal RNA

Blotting

technique that combines electrophoresis and hybridization to detect specific DNA fragments

Southern Blotting

uses radioactive DNA hybridization probes and autoradiography to identify the presence or absence of a specific gene

Autoradiography

method for detecting radioactively labeled molecules

genomic library

a collection of an organism's entire genomic DNA, which is stored in a population of identical vectors, each of which has a fragment of the DNA

site-directed mutagenesis

a technique to introduce a mutation in one or more of the genes in a genome (target sequence "swaps" for the complementary sequence that was introduced)

protoplast fusion

enzymes and other means are used to remove the cell walls of multiple cells, creating protoplasts. These are then fused together, which is a large scale method of change to the genome.

*mainly with fungi*

interferons

the body's "antiviral" molecule

genetic engineering has allowed for the creation of bacteria that specialize in producing alpha, beta, and gamma interferons

Gene therapy

the treatment of disease by inserting "therapeutic DNA" into a patient's cells

hope that specialized transduction can be used for this one day

Primary metabolite

essential for proper growth, development, and reproduction

immediate removal of a primary metabolite will impact the microbe's ability to survive and reproduce

Secondary metabolite

not directly involved in growth, development, or reproductions

removal will not immediately impact the microbe's ability to survive and reproduce

**exogenous even typically triggers the production of this

Antibiotics as a secondary metabolite

many spies of fungi and bacteria naturally produce antibiotics as a means of "chemical warfare", i.e. in the presence of another species of bacteria that is a strong competitor for resources.

semisynthetic penicillin

a modified form of penicillin created in a lab that is more effective

Tumor-inducing (Ti) plasmid

scientists have found a way to take out the tumor-inducing genes and other non-essential regions, which allows the plasmid to be used as a cloning vector for DNA of interest. allows for GMO

Biopesticides

naturally produced agents that kill pests. i.e. BT toxin

chemostat

a bioreactor in which the volume is kept constant b continuously adding fresh medium which removing the culture liquid

Fermentation

mass culture of microorganisms

Biopolymers

serve as gelling agents and modify the flow of liquids

dextran and PHB

biosurfactant

emulsifies and solubilizes another substance, such as oil

bioremediation

using organisms to remove pollutants from a contaminated site

cellulose

a polymer that consists of only 6 carbon glucose molecultes

hemicellulose

a polymer of both 6 carbon sugars and 5 carbon sugars

Dr. Ingram

patented a bacterium that produces alcohol using both 5 carbon AND 6 carbon sugars

bioconversion (biotransformation)

use of live organisms to facilitate a chemical reaction that converts a substance to a chemically modified form

microbial biosensor

uses microorganisms, enzymes, or organelles and a physical transducer to detect the presence of a substance.

vertical gene transfer

the transmission of genetic information from parent to offspring in sexual or asexual reproduction

horizontal gene transfer

the transmission of genetic info between organisms in a manner other then traditional sexual/asexual reproduction

genetic recombination

creation of new combinations of alleles through the exchange of genetic material

Conjugation

involves the transfer of genetic material between bacteria through direct contact

pilus

a bridgelike structure that is a part of the Type IV secretion system.

allows for direct transfer of plasmid

F factor

a special piece of DNA that can exist either as a segment of DNA or as a plasmid, which gives the bacterial cell the ability to form a sex pilus and encode DNA

donors

F+ cells

recipients

F- cells

F factor in the plasmid

it can simply be transferred from the F+ cell to the F- cell and the entire plasmid is transferred

F factor in the chromosome

rolling-circle replication happens and the recombination results in the integration of the Hfr genome into the acceptor cell genome.

the sex pilus is fragile, so only part of the Hfr cell's bacterial chromosome is transferred

High frequency of recombination cell (Hfr)

the male cell when the F factor is located in the actual chromosome

transformation

involves the direct uptake of genetic material from a bacterium's surroundings

"naked DNA"

when a bacterium dies and its pieces disassemble, bits of "naked DNA" are released into the surrounding and are able to be taken up through transformation

Where does transformation happen

rare in nature but commonly used in the lab

bacterium become more receptive to taking up genes from the environment when they undergo significant stress/environmental change

Competent

when cell is receptive to taking up DNA from the environment

transduction

sometimes a phage that is released after the lytic cycle carries with it pieces of its host's genome that were not originally part of the virus, when it infers another bacterium it will transduce that genetic info

specialized transduction

involves the transfer of a restricted set of only a few, specific bacterial genes to another bacterium via a phage that has become a part of the host chromosome.

generalize transduction

involves the transfer of any bacterial gene to a recipient bacterium using a phage

R plasmids

convey antibiotic resistance

Transposons/transposable elements/jumping genes

are able to "jump" from plasmid to the bacterial chromosome and from the bacterial chromosome to the plasmid.

bacteriophage

a virus that infects a bacterium

Lytic cycle

a type of page replication cycle resulting in the release of new phages by lysis (and death) of the host cell

lysogenic cycle

a phage replication cycle in which the viral genome becomes incorporated into the bacterial host chromosome as a prophage and does not kill the host

induction

the process in which the environmental cue causes the prophage to begin synthesizing new phage particles, which will eventually enter the lytic cycle

temperate phage

a phage that is capable of establishing lysogeny and integrating into the bacterial host

lysogen

a bacterium that contains a virus

**can actually help bacteria in some cases

prophage

an integrated phage that can stay integrated in the host's DNA for many generations

structural genomics

the study of the physical nature of genomes

i.e. the Human Genome Project

bioinformatics

the study of genome using computers

annotation

a computer process that determines where genes start and end, which allows them to locate genes on the genome

Open Reading Frames (ORFs)

a long sequence of codons not interrupted by a stop codon. used in annotation

functional genomics

the study of how a genome works

coding sequences

open reading frames presumes to encode proteins

Paralogs

two genes that consist of similar sequences and are found in the same genome. they have a similar function

usually arise due to gene duplication events

orthologs

two genes that consist of similar sequences and are found in different organisms. they likely have the same function

motifs

short patterns of amino acids that are known to have a specific function

in silico analysis

analysis carried out by a computer

DNA microarray analysis

allows scientists to observe the pattern of DNA expression for thousand of genes at a time

proteomics

the study of the entire collections of proteins produced by an organism

functional proteomics

the study of protein function that determines what actually occurs in the cell

structural proteomics

the study of 3D structure of proteins

two-dimensional gel electrophoresis

separates proteins based on charge and molecular mass

metabolomics

the study of metabolome, or the entire set of a small-molucules metabolites present which in the cell at a given time

lipidomics

the study of lipide, or the entire lipid profile of a cell

comparative genomics

the study of the differences among genomes of different organisms.

given researchers insights into what kind of vaccines or therapies may be useful to fight against virulent pathogens

metagenomics

the study of the metagenomes, or the genetic material recovered from environmental samples.

Dr Pandey

published a rough draft of the human proteome, or the complement of proteins produced by different organs throughout the body.

virus

an obligate, intracellular parasite that is sub-microscopic and has a wide variety of hosts

viroid

an inert version of the host that exists outside the cell

Viral Structures

Mandatory:

Nucleic acid (single/double DNA/RNA)

capsid

Optional:

envelope

spike

capsid

the outer protein cover that protects the virus' genetic material and is made of capsomeres which automatically self-assemble about the nucleic acid

**determines the shape, protects, interacts with receptors on host cell, ad facilitates the penetration

icosahedron shape

unique to viruses, a polyhedron with 20 triangular faces, 30 edges, and 12 vectors