MCB3020 EXAM 3 :(

the longer the DNA...

the short the distance towards the positive electrode the DNA will migrate (smaller restriction fragments move faster than larger ones) migration rate is inversely proportional to the log if its molecular weight

restriction fragment length polymorphism (RFLP)

tool for genetic fingerprinting, genome mapping, localization of genes, paternity testing, and microbial classification; uses the fact that that highly conserved and repetitive DNA sequences are present in nearly every genome

how to make host E. coli cells competent to take up naked DNA from the environment via transformation

treating them with calcium chloride and then applying a heat shock- electroporation - increases permeability of cell membrane

two-dimensional gel electrophoresis

uses an electrophoretic gel to separate proteins based on 2 dimensions: charge and molecular mass; pH gradient separates proteins by charge using isoelectric focusing , adn gravity is used to separate proteins based on molecular mass; involved in proteomics; proteins move through the gel until they reach a point where their net charge is 0; then turned on its side and proteins will move from the top of the gel to the bottom based on their molecular mass (small proteins will migrate further down the gel); final product is an audioradiograph

southern bottling

uses radioactive DNA hybridization probe and autoradiography to identify the presence or absence of a specific gene autoradiogrpahy- method for detecting radioactively labelled molecules

Immunotherapy

using genetic engineering in cancer therapy altering the genetic composition of immune system cells

bacteriophages

virus that infects bacteria; vector fragments of 9000 to 25000 base pairs

applications of genetic engineering of microbes

-production of biopolymers -production of biosurfactant -production of biofuels -bioconversion -biosensors -prodoction of biopesticides -reverse vaccinology

biopesticides

naturalled produced agents that kill pests; example: Bacillus thuringiesis (Bt) is a gram positive bacterium that produces an intracellular protien toxin crystal called parasporal body during sporulation, the body kills specific groups if insects; Bt toxin does not accumulate in the body

secondary metabolite

not directly involved in growth, development, or reproduction; removal of a secondary metabolite will not immediately impact the microbe's to survive and reproduce; triggered by an exogenous event (such as exposure to a pathogen)

viral structure

nucleic acid (double or single stranded RNA or DNA) and capsid (outer protein colver that protects the virus's genetic material

examples of commercially available plasmids

pBR322 and PUC19

cloning vector

provides a means for transferring a gene of interest to a host organism during the cloning process; have cahracteristics that allow genes to be inserted into the vector to create recombinant DNA; recombinat DNA can be transferred to a host organisms, which will naturally make copies of it during standard DNA replication

PCR

technique for quickly and easily making copies of even a very small amount of DNA; used in lab settings to amplify pieces of DNA ranging in size from 100 base pairs to several thousand

blotting

technique that combines electrophoresis and hybridization to detect specific DNA fragments

DNA shuffling

technique used to propagate beneficial mutations and increase the size of a DNA library by randomly fragmenting a pool of related genes and reassembling those fragments

gene therapy

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

bioconversion

use of live organisms to facilitate a chemical reaction htat converts a substance to a chemically modified form Rhizopus migricans facilitates the conversion of progesterone to 11-a-hydroprogesterone--step in teh industrial production of cortisone microorganisms are acting as biocatalysts-- facilitating changes to a substrate molecule, but they are not used up as a result of the reaction; can produce the exact sterior isomer needed

biotechnology

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

whole genome shuffling

use of protoplast fusion to introduce recombination throughout the entire genome; after these techniques have been applied, organisms with the desired phenotype can be selected

DNA analysis

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

microbial biosensor

uses microorganisms, enzymes, or organelles and a physical transducer to detect the presence of a substance; converts products of a biological reaction into electrical currents, which can be easily read and analyzed ; can be used to cell where cotnaminants are in a river, or used to detect arsenic in drinking water ________ act as bioreporters

reverse transcriptase

when scientists are cloning DNA fragments sometimes it is easy to isolate RNA fragments but not DNA fragments. when this is the case they do __________ to turn single stranded RNA into double stranded DNA;____________ is an RNA -dependent DNA polymerase, meaning that is synthesizes DNA based on an RNA template; produced complementary DNA (cDNA)

characteristics of a good cloning vector

-origin of replication (DNA polymerase begins DNA replciation at the origin of replication) -selectable marker (problem is identifying the cells that have a vector [and thus the gene of interest] and those that do not. thus a vector uses a marker (an antibiotic resistance gene or a marker that causes a cell to turn a certain color) - unique restriction sites (vector must allow the insertion of the gene of interest, requires unique restriction sites called polylinkers or multicloning sites [MCSs])

medical applications of genetic manipulation or microbes

-production of desirable proteins -gene therapy

use of microorganisms in agriculture

-production of plant hormones (can be manipulated to mass produce gibberellins [a plant hormone]) - production of food additives (like AAs, organic acids, nucleotides, vitamins, and polysaccharides, MSG, citric acid, lactic acid, acetic acid) - production of genetically modified plants (can be used to make plants with desirable characteristic [tumor inducing plasmids]) - production of biopesticides

field of genomics subcategories

-structural genomics -functional genomics -comparative genomics

which direction to nucleic acids migrate

DNA and RNA have a negative charge due to their phosphate backbones, so they migrate through the electric field produced in a thin slab of gel towards the positive electrode

methods of DNA analysis

PCr, gel electrophoresis, restriction fragment analysis, and blotting techniques

variations of RFLP

STR analysis and ribotyping

virus

obligate intracellular parasite - cannot grow or produce energy on their own, require a host; require the genetic machinery and has a specific range of hosts that is sub-microscopic - too small to be seen with a light microscope, only can be seen with an electron microscope; nanometers; filterable agents bc filters can catch bacteria but not viruses host and tissue specific - such as bacteriophages; spcificity derives from the fact that they interact with specific cell surface receptors

tumor inducing plasmid

often genes are inserted into plant genomes using teh _____________________ found in Agrobacterium tumefaciens; induces formation of tumors in hundreds of plant species; this plasmid can be used as a cloning vector that carries the DNA of interest and a selectable marker; can create plants with desirable charactersitics

scale-up

often techniques in the lab do not translate perfectly to large, industrial environments, so microbiologists use this process; the modification of teh growth process so that it can work as efficiently in large fermenters as it does in a small test tube; requires careful control of environmental conditions

exampples of coding sequences

paralogs- two genes are paralogs if they consist of similar sequences and are found in the same genome; because they consist of similar sequences they have the same function; usually arise due to gene duplication events orthologs- consist of similar sequences and are found in different organisms; likely to have the same function because they have the same sequences

PCR components

primers, themostable DNA polymerase, deoxyribonucleotide triphosphates

directed evolution

process that mimics the processes of natural selection to evolve proteins toward a user-defined goal (tools for metabolic engineering directed evolution and whole-genome shuffling

most common hosts for gene cloning

prokaryotic: E. coli eukaryotic: Saccharomyces cerevisiae (type of yeast) they hosts are engineered to lack RecA proteins and restriction enzymes, which might otherwise destroy the inserted sequence or attempt to correct the recombinant DNA

sticky ends

restriction enzymes cut the target DNA in such a way that they leave _______ of short, single stranded sequences on both ides of the resulting restriction fragments (blunt cuts do not produce sticky ends)

Creation of recombinant DNA

scientists can insert the DNA of one organism into another organism's genome

production of desirable proteins

scientists have engineered bacterial produce massive amounts of insulin, which can be used to treat diabetes also have used genetic engineering to create bacteria that specialize in producing alpha, beta, and gamma interferons, which are the body's "antiviral molecule" and can be used to treat viral infections

protoplast fusion

scientists use enzymes (like cellulase) or other means to remove the cell walls of multiple cells, creating protoplasts; put protoplasts together and apply an electric shock, causing them to fuse, forming a somatic hybrid; causes a large scale change to the genome; most widely seen using fungi

DNA ligase

seals strands together, catalyzing phosphodiester bonds between the single-strand breaks

bioprospecting

search for species that have medical and commercial uses for humans

plasmids

self-replicating piece of extra chromosomal DNA found in prokaryotes; can be easily transferred form one organism to another via conjugation or transformation; carry inserted sequences of the mid-size range (20,000 base pairs )

meticillin

semisynthetic form of penicillin tht kills staph

gel electrophoresis

separates nucleic acids adn proteins on the basis of their size and electrical charge, using a gel that is a polymer (like agarose or polyacrylamide)

recombination DNA technology

set of procedures used to combine genetic material from multiple sources and create DNA sequences that are otherwise not found in biological organisms; often from two or more sources

motifs

short patterns of amino acids known to have a specific function; several are involved in gene expression; examples: helix-turn-helix, zinc finger, and leucine zipper; functional genomics can analyze these; well preserved in multiple taxa

structural proteomics

study of 3D structure of proteins

metagenomics

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

functional proteomics

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

proteomics

study of proteomes, or entire collections of proteins produced by an organism; can provide info about genomic function that mRNA studies cannot

comparative genomic

study of the differences among the genomes of different organisms; provides info on what makes a pathogen virulent, and it has given researchers insights into what kinds of vaccines or therapies may be useful to fight against virulent pathogens

functional genomics

study of the function of genomes; computers can give scientists clues about what proteins a genome codes for

lipidomics

study of the lipidome- entire lipid profile of a cell; allows research to assess the impact of the environment on a cell's membrane

metabolomics

study of the metabolome, or the entire set of small-molecule metabolites present within a cell at a given time; allows researchers to asses the physiological status of a cell at any given point in time

Genomics

study of the organization of genomes, the information they store, and the gene products that they code for

structural genomics

study of the physical nature of genomes Human genome project has sequenced the entire human genome often involved in bioinformatics- study of the genome using computers; combines computer science, biology, stats, and math scientist use computers in a process called annotation to determine where genes start and end, which allows them to locate genes on the genome; computers search for open reading frames (ORFs) by attempting to identify relatively long sequences of codons that are not interrupted by a stop codon

artificial chromosomes

synthetic chromosomes that contain fragments of DNA integrated into a host chromosome can carry larger fragments, yeast of up to 1M base pairs; these vectors were used in the Human Genome porgect

high-throughput sequencing

technology that allows researchers to rapidly and cheaply sequence and analyze a large amount of DNA

what happens when DNA containing genes of interest are cut with the same restriction enzyme

the complementary bases on the sticky ends of the restriction fragments form hydrogen-bonded base pairs; allows DNA from different sources to be combined in a lab; can be sealed by DNA ligase;l results in recombinant DNA

cloning vector categories

- plasmids, bacteriophages, cosmids, artifical chromosomes

biofuels

microbial fermentation of crop residues consisting of cellulose and hemicellulose produces ehtanol, a biofuel that is widely used to power cars adn other motor vehicles; involved in the production of methane gas and hydrogen

introducing mutations

microbiologists can use a number of techniques to introduce mutations, one of them is site directed mutagenesis

basic process process for constructing cDNA

1) beginning with a single-stranded RNA molecule, apply a short poly-T primer (reverse transcriptase can only add nucleotide to an existing 3' hydroxyl group, thus it requires a primer) 2) add reverse transcriptase and the four nucleotides (ACGT); enzyme will begin to synthesize a complementary strand of DNA from the RNA template, producing a DNA-RNA hybrid 3) add an enzyme called RNaseH to cut up the RNA and regenerate the RNA primers; if you want a double stranded DNA molecule, you need to get rid of the existing RNA molecule 4) add DNA polymerase and DNA ligase to synthesize a new strand; DNA polymerase will fix gaps b/w primers which will eventually be replaced with DNA; DNA ligase will seal fragments together makes a double stranded DNA molecule from a single stranded RNA template; double stranded DNA molecule can then be inserted into vectors and used in traditional cloning tecniques or aomplied using PCR

method to determine whether a DNA molecule contains a specific DNA sequence or gene

1) cute the DNA with restriction enzymes (produces a larger number of DNA fragments) 2) separate the DNA fragments by size on an agarose gel (crates a smear rather than distinct bands because of the large number of fragments produced) 3) place a sheet of nitrocellulose paper on top of the gel and apply pressure from the top to ensure contact between the gel and the paper (will transfer DNA from gel to paper) 4)place the paper in a heat-sealed bag with a radioactive probe (probe will hybridize with (bind to) complementary sequences, if they are present) 5) wash the filter and place it on x-ray film to produce an image of the DNA bands Xray image is called an autoradiograph. if the DNA contained the gene of interest, the DNA bands will be difficult

process to create a genomic library

1) extract DNA from cell of interest 2) use restriction enzymes to cut the DNA into fragments of a specific size 3) insert fragments into a vector 4) insert the vector into a host organism, such as e. coli 5) once the vector is inside the host cell, it can be easily amplified or retrieved for analysis

site-directed mutagenesis

1) identify the sequence of nucleotides in the gene of interest 2) create a complementary DNA sequence in the lab, an ensure that the complementary sequence has the mutation that you ultimately want to introduce to the target organism 3) insert the short, complementary sequence into the target cell 4) the target cell may then "swap" the target sequence for the complementary sequence that was introduced, creating recombinant DNA with the desired mutation

technique of RFLP

1) isolate DNA of interest 2) amplify the DNA using PCR techniques 3) Cut up the DNA with restriction enzymes, producing restriction fragments 4) use gel electrophoresis to separate the restriction fragments according to their lengths on an agarose gel 5) use computers to analyze the result

steps of gene cloning

1) isolate plasmid DNA from a bacterial cell 2) isolate the DNA of interest 3) insert the gene of interest into the plasmid DNA, forming recombinant DNA 4) insert the recombinant DNA into a bacterium, such as E. coli 5) culture the bacteria, growing many identical copies

STR analysis

Compares genomes based on the number of short-tandem repeats (repeating microsatellite sequences on a DNA strand)

artifical chromosomes categories`

P1 artifical chromosomes (PACs), bacterial artifical chromsomes (BACs), and yeast artificial chromosomes (YACs);

thermostable DNA polymerase

PCR involves increasing the temperature of the mixture, so it requires a heat resistant polymerase that will not denature at high temperatures these polymerases were isolated from thermophilic prokaryotes; these prokaryotes produce a heat-resistant DNA polymerase called Taq polymerase that is commonly used in PCR

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; typically done when the gene of interest resides on a chromosome that has not been sequenced

DNA microarray analysis

a tool that allows scientists to observe the pattern of DNA expression for thousands of genes at a time; tool related to functional genomics; they are chips with a collection of tiny DNA spots attached to a solid surface; basic process on page 16

restriction enzymes

a type of endonuclease that is an important tool that allows scientists to cut up DNA at specific sites; standard cloning techqiues use this; naturally produced by bacterial as a defense against viral infection

restriction sites

also called restriction sequences; recognized by restriction enzymes; short nucleotide sequences, usually between 4-8 nucleotide that are cut by restriction enzymes at specific points within the restriction sites. typically palindromes

in silico analysis

analysis carried out by a computer; scientists can figuring out the function of a gene exclusively through this; do not have to see translation occurring

penicilin

antibiotic; microbiologists are able to promote the production of them by growing bacteria in a medium with a high level of lactose, and then gradually bringing ammonia levels down over time scientists can then take natural penicillin into the lab and modify it to create semisynthetic penicillin, which is even more effect than natural penicillin

antibiotics

antimicrobial agents that kill or neutralize bacteria; produced by species of fungi and bacterial as a means of "chemical welfare"; do this in the presence of aother species of bacteria that is a strong competitor for resources

fermentation

mass culture of microorganisms

chemostat

bioreactor in which the volume is kept constant by continually adding fresh medium while removing the culture liquid; controls growth rate of bioorganisms

metagenomic library

can be used in genomic sequence analysis (to determine the sequence of genes) or in function-driven analysis (to analuze the proteins that genes produce)

coding sequences

compared in functional genomes with known genes in existing databases to reach conclusions about what the products of translation might be; open reading frames presumed to encode proteins

ribotyping

compares ribosomal RNA; allows researchers to find similarities and differences amount various domains. identify highly conserved regions among individuals from the various domains of life

procedures for genetic manipulation

creation of recombinant DNA, introducing mutations, protoplast fusion, metabolic engineering

deoxyribonucleotide triphosphates

dATP, dcTP, dGTP, dTTP; these will be added to the growing DNA strand during replICATION

genetic engineering

deliberate modification of an organism's genome sequence

ethidium bromide

dye used to stain the DNA

biosurfactants

emulsifies and solubilizes another substance, like oil useful in oil recovery efforts, dispersing oil spils, and in bioremediation (using organisms to remove pollutants from a contaminated site)

primary metabolite

essential for proper growth, development, and reproduction, and immediate removal of a primary metabolic will significantly impact the microbe's ability to survive and reproduce

what premise does this technique rely on

every organism's restriction sites will be located at slightly different locations on the genome, so the specific pattern of restriction fragments on the electrophoretic gel will be different from person to person; this means every person has a different DNA fingerprint that cna used as forensic evidence or evidence of paternity

biopolymers

examples: dextran and polyhydrobutyrate (PHB) gell agents and modifying the flow of liquids; adding them to a fluid slows its movement through a system, which can be useful in many industrial applications

Metabolic engineering

genetic manipulation of genetic processes in order to increase the production of certain substances directed evolution and whole-genome shuffling

cosmids

hybrid between a plasmid and a phage; larger fragments of 30000 to 47000 base pairs

SDS polyamid

if researchers wanted to neutrlize the charge and simply separate proteins based on their molecular weight

primers

in PCR you need the correct primers that bind upstream from the target sequence

thermocylcer

instrument that cyclically increases adn decreases the temperature of a solution in PCR the solution goes from 90 to separte the DNA strands, and then is cooled to about 50 degrees to allow the primers to hydrogen-bond to the complementary sequence. then heated to 70 degrees to allow the heat-stable DNA polymerase to add to nucleotides ot teh 3' ends of the primers,

reverse vacinology

involves the use of bioinformatics to screen the entire genome of a pathogen in order to identify genese that are good vaccine targets