Microbiology Exam 5

genotype

the nucleotide sequence of the genome

phenotype

the physical properties of the organism

mutation

a heritable change in the base sequence of the genome

wild-type strain

the strain isolated from nature

mutant

a strain carrying a change in its nucleotide sequence

mutant phenotype

an altered phenotype relative to the parental strain

selectable mutations

mutant has an advantage under certain environmental conditions and will outcompete and replace the parent (wild type)

non-selectable mutations

mutation does not provide an advantage so it will remain rare in the population

selectable marker gene

intentionally add certain gene to acquire certain trait for easy screening of mutants

point mutations

chemical changes in just one base pair of a gene (silent, nonsense, or missense)

silent mutation

does not affect amino acid sequence

nonsense mutation

a codon becomes a stop codon; short and incomplete polypeptide

missense mutation

an amino acid changed; altered polypeptide

frameshift mutations

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide
-DISASTROUS effect

back mutation (reversion)

when a mutated gene reverses to its original base composition (original sequence is restored)
-often occur in point mutations

mutagenesis

the production of mutations; usually very low rate in spontaneous mutations so process can be sped up

ames test

simple test to screen chemicals for potential mutagenicity
-screen for an increase in the rate of back mutations in auxotrophic bacteria in the presence of the chemical disc

transversions

a purine and a pyrimidine are interchanged

transitions

a purine or pyrimidine is swapped for the other (A to G, G to A, C to T, or T to C)

transferring genetic material

two ways to do it:
-vertical gene transfer (VGT)
-horizontal (lateral) gene transfer (HGT or LGT)

genetic exchange in prokaryotes

3 mechanisms of genetic exchange:
1. transformation
2. transduction
3. conjugation

transformation

free DNA is incorporated into a recipient cell and brings about genetic change

transduction

a bacterial virus (bacteriophage) transfers DNA from one cell to another

conjugation

genetic transfer from a donor cell to a recipient cell requiring cell-to-cell contact (mating)

rolling circle replication

transfer plasmid DNA from donor to recipient cell (happens during conjugation); some viruses also use the same mechanism

F (fertility) plasmid

circular DNA that contains genes that regulate DNA replication and contains several transposable elements

-contains tra genes that encode transfer functions

-is an episome

F-

cell without F plasmid

F+

cell with non-integrated F plasmid

HFr (high frequency og recombination)

cell with integrated F plasmid
-high rates of genetic recombination occurred between genes on the donor chromosome and those of the recipient

transposable elements

DNA fragment that move (or copy) as a unit from one location to another
-found in all 3 domains of life
-move by a process called transposition
-two main types in Bacteria: insertion sequence and transposons

transposition

movement of DNA segments within and between DNA molecules

insertion sequence

2 inverted repeats (IR) + transposase gene

transposons

2 IR + transposase gene + other genes
(e.g. antibiotic resistant gene)

transposase gene

codes for enzyme that catalyzes transposition

restriction enzymes

recognize specific base sequences within DNA (recognition sequences) and cut the DNA at specific sites; common in prokaryotes
-type of defense mechanism against hostile foreign DNA

palindromes

inverted repeat sequences; used in restriction enzymes

type II restriction enzymes

cleavage site located within the recognition sequence; form three different ends

sticky (cohesive) ends

5'-overhang and 3'-overhang

blunt ends

cuts are straight through both DNA strands at the line of symmetry

CRISPR (clustered regularly interspaced short palindromic repeats)

segments of prokaryotic DNA containing short repetitions of base sequences; each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid

CRISPR-Cas9

binds to a target viral dsDNA and cuts both strands; essentially like magic scissors

gel electrophoresis

separate DNA fragments by their size and shape; the phosphodiester bond in DNA is - charged and it migrates towards + pole; fragments are stained for observation
-larger fragments move slowly
-compact shape move fast

nucleic acid hybridization

single stranded DNA or RNA can be used to screen for homologous sequences by utilizing complementary base pairing (hybridization)

procedure of hybridization

label known nucleic acid (DNA or RNA) with radioactivity or a fluorescent dye, hybridize probes with unknown samples, if probe sticks then this indicates a match in the sequence

southern blotting

hybridization technique where DNA in the gel is labeled with DNA or RNA probe

northern blotting

hybridization technique where RNA in the gel is labeled with DNA or RNA probe

western blotting

hybridization technique where protein in the gel is labeled with antibody

splicing

introns are removed and exons are joined by spliceosome
-spliceosome contains small nuclear ribonucleoproteins (snRNPs)

introns

junk DNA sequences

exons

expressed sequence of DNA; codes for a protein

reverse transcriptase

makes a DNA copy of RNA (cDNA); cDNA can be made from mRNA, tRNA, or rRna; provides a means of synthesizing eukaryotic genes from mRNA transcripts (synthesized gene is free of introns)

PCR (polymerase chain reaction)

method to amplify DNA; rapidly increases the amount of DNA in a sample

process of PCR

primers are known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides; repetitively cycled through denaturation, priming, and extension; each subsequence cycle DOUBLES the # of copies for analysis

denaturation, priming, and extension

otherwise known as melting, annealing, and synthesis; 3 basic steps of PCR

what is PCR used for?

gene mapping, studying genetic defects and cancer, forensics, taxonomy, and evolutionary studies
**amplifies certain strands of genes

shotgun sequencing

sequences are assembled by pairing their overlaps to generate the long, continuous sequence

pyrosequencing

DNA sequencing technology that is used for whole-genome shotgun sequencing; fast and cost-effective analysis

procedure of pyrosequencing

1. DNA fragmentation
2. Separate and sequence small DNA fragments
3. Assemble pieces of DNA sequences together
4. Annotation; finding ORF, putative regulator sequences, etc.

microarray

track the expression of thousands of genes; used to identify and devise treatments for diseases based on the genetic profile of the disease

plasmids as cloning vectors

requirements to use:
-small size; easy to isolate DNA
-independent origin of replication
-presence of selectable markers
-polylinker (multiple cloning site)
-vector transfer carried out normally by chemical transformation or electroporation

desirable features in cloning host:

1. fast growth rate
2. can be grown in large quantities using ordinary culture methods
3. nonpathogenic
4. genome that is well delineated
5. capable of accepting plasmid or bacteriophage vectors
6. maintains foreign genes through multiple generations
7. will secrete a high yield of proteins from expressed foreign genes

competence

capable of accepting plasmid or bacteriophage vectors

selectable markers

vector genes that make it possible to pick out cells harboring a recombinant DNA molecule constructed with that vector

shuttle vectors

vectors that are stably maintained in two or more unrelated host organisms; able to easily move genes between multiple hosts
-usually requires multiple selection markers for the different hosts
-usually requires multiple origin of replications for the different hosts

expression vectors

allow experimenter to control the expression of cloned genes; multiple cloning site is located immediately downstream of a promoter region
-allow high levels of protein expression
-promoter is repressed unless activated by an inducer
-strong transcription terminator sites prevent read-through

Ti plasmid vector

used to introduce foreign DNA into plants; contains genes that mobilize DNA for transfer to the plant; segment of the Ti plasmid that is transferred to the plant is called the T-DNA

Agrobacterium tumefacines

plant pathogen with the plasmid used in Ti plasmid vectors

crown gall

caused by Ti plasmid vectors

3 steps in gene cloning

1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism

isolation & fragmentation of source DNA

-isolation of source DNA (e.g. genomic DNA, cDNA from RNA, or PCR products)
-fragmentation of source DNA by restriction enzymes
***most vectors are derived from plasmids or viruses

inserting DNA fragment into cloning vector

-DNA is generally inserted in vitro
-DNA ligase works with sticky or blunt ends

DNA ligase

enzyme that joins two DNA molecules (either with sticky or blunt ends)

introduction of cloned DNA into host organism

-transformation is often used to get recombinant DNA into host
-some cells will contain desired cloned gene, while other cells will have other cloned genes

electroporation

a technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing the cells

lac Z gene in cloning

polylinker (multiple cloning site) inside gene that allows for screening
-no foreign DNA inserted = blue colonies
-foreign DNA inserted = white colonies (lacZ gene is inactivated)

inactivated lacZ gene

cannot process X-gal (galactose homolog) so no color develops during the screening process

barriers of expression in mammalian genes

-differences in codon usage
-presence of introns in eukaryotes
-degradation by host intracellular proteases
-toxicity to prokaryotic host
-formation of inclusion bodies
-improper protein folding
-lack of post-translational modification

advantages of using bacterial system

-quick replication (mass production)
-cheap production cost

codon usage

differences in use between prokaryotes and eukaryotes for codons

inclusion bodies

granules of storage material such as sulfur that accumulate within some bacterial cells

post-translational modification

changes made to polypeptides following translation (disulfide bridge or glycosylation)

industrial microbes

usually are fungi (yeast) or streptomyces (bacteria)

properties of useful industrial microbes

-high yield of desired products
-grow rapidly
-easy to inoculate
-large scale liquid culture
-inexpensive medium
-non-pathogenic
-amenable to genetic manipulation (genetic engineering)

primary metabolites

produced during the exponential growth phase
-alcohol is an example; formed as part of energy metabolism
-alcohol production is parallel with growth curve; CANNOT be significantly overproduced (usually the waste product)

secondary metabolites

produced near the end of growth phase or in the stationary phase; not essential for growth and reproduction
-formation highly depends on growth conditions
-often produced as a group of related compounds
-often significantly overproduced
-often produced by spore-forming microbes during sporulation

fermenter

vessel where the microbiology process takes place; most are aerobic processes

impeller

helps supply fermenter with O2 by mixing the culture

sparger

helps supply fermenter with O2 by pumping in high pressure air for aeration

antibiotics

compounds that kill or inhibit the growth of other microbes
-typically secondary metabolites

antibiotic producers

filamentous fungi or bacteria of the Actinomycetes group

biotransformation

the process in which enzymes convert a drug into a metabolite that is itself active, possibly in ways that are substantially different from the actions of the original substance

extremozymes

enzymes that function at some extreme environment (i.e. pH or temperature)
-produced by extremophiles

carrier enzyme

bonding of enzyme to a carrier, such as bead, fiber, film, or membrane

cross-linking enzyme

cross-linking of enzyme molecules

commodity ethanol

glucose obtained from cornstarch and fermented mostly by yeast (Saccharomyces cerevisiae)
-used as an industrial solvent, a gasoline supplement, etc.

malolactic fermentation

secondary fermentation by lactic acid bacteria (primarily fermentation by yeast)
-high quality dry red wines and a few white wines use this process

vinegar

results from conversion of ethanol to acetic acid
-performed by the acetic acid bacteria (Acetobacter and Gluconobacter)

citric acid

a widely used food industry additive and in the leavening of bread; produced by the mold Aspergillus niger

-citrate acts as a iron chelator to scavenge iron for uptake into the cell

-purified by addition of lime (CaO)