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MICR 3050 Exam 4 Study Guide

by: Stephanie Erickson

MICR 3050 Exam 4 Study Guide MICR

Marketplace > Clemson University > MICR > MICR 3050 Exam 4 Study Guide
Stephanie Erickson
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This 36 page Study Guide was uploaded by Stephanie Erickson on Monday April 25, 2016. The Study Guide belongs to MICR at Clemson University taught by in Spring 2016. Since its upload, it has received 59 views.


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Date Created: 04/25/16
UNIT 4 STUDY GUIDE Chapter  41.1  –  41.3,  41.5  –  41.6   1.Understand,  in  general,  the  production  of  beer,  ale,  and  distilled  beverages.  Be  able  to   compare  and  contrast  the  processes.   • Mashing-­‐  process  used  to  release  fermentable  sugars  from  grains     o Activates  enzymes  to  yield  malt  (mixed  with  water  to  form  mash)   o Mash  heated  with  hops  in  brew  kettle     § Hopes  provides  flavor  and  assists  in  clarification  (antimicrobial)     • Later  removed   § Wort-­‐  clear  liquid  containing  fermentable  carbohydrates     o Wort  is  inoculated  with  desired  yeast   o LAGER  BEER  uses  bottom  yeast  (sink  to  the  bottom  b/c  not  enough   CO2)   § Takes  7-­‐12  days  to  ferment     o ALES  use  top  yeast    (float  b/c  a  lot  of  CO2)     § Takes  5-­‐7  days  to  ferment     • Storage-­‐  lagering     • Beer  can  be  pasteurized  or  sterilized  by  filtration     • Distilled  spirits   o Whiskey  and  bourbon  begin  with  sour  mash  (mash  inoculated  with   homolactic  bacterium)     o Ferments  then  distilled  to  concentrate  alcohol     § Begins  colorless  but  color  develops  during  aging  (10+  years)     Chapter 43.1 – 43.2 1. Know the potential waterborne diseases discussed and how drinking water is purified. • Typhoid  fever-­‐  salmonella  typhi,  major  public  health  threat  before   drinking  water  treatment     • Cholera-­‐  often  occurs  in  areas  with  inadequate  or  no  sewage  treatment   • Protozoan  infections**-­‐  Giardia,  Cryptosporidium,  Etamoeba,   cycloformums   a. May  be  present  in  surface  waters  and  survives  water  treatment   facilities  as  cysts   b. Resistant  to  chlorination*     Water is purified by filtration and chlorination! Coagulation (form and remove floc)àLayers of sand filter (removes organic and inorganic compounds)à chlorination (kills remaining microorganism and prevents growth)à storage Chlorine is a disinfectant but causes problems with oxidizing organic matter Could use UV light, ozone, or peroxide instead but chlorine is most effective because it leaves a residual*** Non point sources of pollution is worse because it’s harder to treat Coming from parking lots, agricultural fields, golf courses etc. 2. Describe the goals of wastewater treatment. • To get rid of pathogens, organic matter (feces), nitrogen and phosphorous, organic chemicals (such as makeup), and inorganic chemicals (copper, mercury) • Nitrogen and phosphorous create algae-blooms which lead to dead zones 3. Know the terms associated with wastewater treatment (ex. BOD, clarifiers, sludge, floc, etc.). • BOD- organic matter • Fluc- formed by bacteria during secondary treatment? • Sludge- muddy looking residue • THM- carcinogenic chlorinated organics 4. Distinguish between primary, secondary, and tertiary wastewater treatment. Primary: most basic form Basically a settling tank, no bacteria is used Grates and screens Separates solids Still has substantial organic matter Secondary: uses bacteria to eat up organic matter Tertiary: nitrogen removal USES BACTERIA Nitrogen is present in sewage as ammonia (NH3) Ammonia à nitrate Nitrification in aerobic tank Bacteria will use nitrite if there’s no oxygen present 70% of the power is blowing air (oxygen) to the bacteria in the tank Nitrateà nitrogen gas Anoxic tank to denitrificate (nitrogen gas bubbles out of wastewater) Beneficial because it reduces energy required for wastewater treatment Wastewater disinfection is heading towards UV light Damages DNA and prevents bacteria from reproducing However, it doesn’t penetrate very well surface level 5. Describe indicator organisms, their use in the analysis of water quality, and the methods used to detect them. Indicator organisms- Suitable for analysis of all types of water, Present wherever enteric pathogens are present Survives longer than hardiest enteric pathogen Does not reproduce in contaminated water Detected by highly specific test Harmless to humans Its level in water reflects degree of fecal pollution • Typical  to  use  coliforms  because  they’re  easy  to  isolate  and  identify     • Detected  with  MPN  method  &  membrane  filter  (used  for  large  bodies  of  water)   Chapter 6.1 – 6.5 11.  Describe  viruses  (structure,  genomes,  morphological  types).     Viruses-­‐  genetic  elements  that  replicate  independently  of  a  cell’s  chromosome,  but   requires  a  living  host  in  order  to  reproduce       -­‐Obligate  intracellular  parasites       -­‐Infect  all  types  of  cells  (including  bacteria  and  fungi)       -­‐  Acellular  infectious  agents  (not  living  things!)       -­‐Contain  DNA  or  RNA       -­‐  Virion=  the  complete  virus  particle  (nucleic  acid  surrounded  by  a  protein  coat)         -­‐  Can  be  segmented;  meaning  it  can  have  more  than  one  genome       -­‐  Contains  a  nucleocapsid  (nucleic  acid  +  protein  coat)     *Mega  virus  is  1000  nm       -­‐  Viral  envelopes-­‐  outer  flexible,  membranous  layer  (usually  host  derived)       12.  Compare  and  contrast  bacterial  and  animal  viruses.   Bacterial  viruses:     Most  are  naked  (no  envelope)       Bacteriophage     Animal  viruses:     Can  be  all  types,  mainly  dsDNA  and  ssRNA       Most  are  enveloped       The  entire  virus  is  taken  into  the  cell  via  endocytosis         Or  by  the  virus  fusing  its  cell  membrane  with  the  cell’s  membrane     Describe bacteriophages. Bacteriophages – viruses that infect bacterial cells Bacteriophage-­‐  virus  that  infects  and  replicates  within  a  bacterium     • Phages  replicate  within  the  bacterium  following  the  injection  of  their  genome  into   the  cytoplasm     • Mainly  double  stranded  DNA   • Most  inject  the  nucleic  acid       • 1/3  of  bacterial  mortality  is  caused  by  viruses     • Even  if  many  bacteriophages  attach  to  a  cell  and  inject  their  nucleic  acid,  it  is  only   the  first  bacteriophage  that  attached  whose  genome  is  replicated     Distinguish between virulent (lytic) and temperate (lysogenic) bacteriophages and their life cycles using T4 phage and lambda phage as examples, respectively. Bacteriophages two options: (1) Reproduce as virulent phages - Begin multiplying immediately after entering the host and lyses the bacterial host cell for release - Ex: T4 phage (2) Remain within the host cell without destroying it - Many integrate their genome into the host genome into host genome in a relationship called lysogeny - Ex: Lambda LYTIC  (virulent)  BACTERIOPHAGES     o Begins  multiplying  immediately  after  entering  host     o Lyses  bacterial  host  cell  for  release     LYSOGENIC  (temperate)  BACTERIOPHAGES     o Reproduce  as  virulent  phages  (lytic  pathway)  OR  remain  within  host  cell  without   destroying  it     o Integrate  genome  into  host  genome  in  a  relationship  called  lysogency     o Useful  if  there  aren’t  many  host  cells  available     o Example:  Lambda*   Viruses  incorporate  into  the  chromosome  during  prophase     Induction  may  occur  and  genome  will  pop  out     Explain lysogeny and how the lytic cycle may be induced in lysogens. Lysogeny – nonlytic relationship between a phage and its host A. Prophage – integrated bacteriophage genome a. Uses integrase enzyme to integrate into the host chromosome b. Always integrates between galactose and biotin operons = attachment site c. Replicates with the host genome B. Lysogen – the infected bacterial host a. Appears normal b. Prophage may change the phenotype of its host = lysogenic conversion Phage can switch from lysogenic to lytic cycle upon induction… Induction of Lytic Cycle - Process by which temperate phage reproduction is initiated - Results in a switch to the lytic cycle - Triggered by a drop in the level of lambda repressor o Usually cause by exposure to UV light or chemical mutagens that cause DNA damage - Excisionase – binds integrase enzyme and enables integrase to reverse the integration process Lysogency-­‐  nonlytic  relationship  between  a  phage  and  its  hose     • Bacteriophage  integrates  into  genome  during  prophase     • Uses  integrase  enzyme  to  integrate  into  host  chromosome     • Always  integrates  between  galactose  and  biotin  operons  (attachment  site)     • Replicates  with  host  genome     Lysogen-­‐  infected  bacterial  host     • Appears  normal     • Phage  may  switch  from  lysogenic  to  lytic  cycle  upon  induction     Induced  when  there’s  a  drop  in  the  lambda  repressor   INDUCTION:  process  by  which  temperate  phage  reproduction  is  initiated         Results  in  switch  to  lytic  cycle       Usually  caused  by  exposure  to  UV  light  or  chemical  mutagens       Cells  inadvertently  break  down  the  lambda  repressor;  not  intentional  just  happens     Excisionase-­‐  binds  integrase  enzymes  and  enables  it  to  reverse  integration  process       Describe the effects of animal viruses on their hosts. 1. Acute à rapid replication of the virus within the host cell, the cell then dies and the viruses are released a. Ex: cold 2. Latent à viral genome hangs out in the cytoplasm until it is activated through certain events that can trigger it to go into a lysogenic life cycle a. Virus stops reproducing and remains dormant for some time b. During latency, symptoms, antivirus-antibodies, and viruses are not detectable i. Ex: (1) cold sores caused by the Herpes virus (2) varicella-zoster: chicken-pox, then shingles 3. Chronic à genome of the virus incorporates into the chromosome and starts making viruses all the time a. Virus almost always detectable b. Clinical symptoms mild or absent for long periods i. Ex: Hepatitis B and HIV 4. Cancer à virus inserts an oncogene that it carries or sometimes it activates a proto-oncogene a. Proto-oncogene: the oncogene that you already possess/normally have – usually fine, doesn’t cause cancer until it malfunctions (viruses inserting can cause it to malfunction) b. If the plasma membrane changes that means that it's a tumor Latent  viral  infections:   • Virus  stops  reproducing  and  remains  dormant  for  some  time     • Symptoms,  antivirus-­‐antibodies,  and  viruses  are  not  detectable     • Example:  HSV  (chicken  pox-­‐shingles)     Chronic  viral  infection:   • “Budding  off”     • Virus  almost  always  detectable     • Clinical  symptoms  mild  or  absent  for  long  periods  of  time     • Example:  Hepatitis  B,  HIV     Distinguish between acute, latent, and chronic viral infections (know examples of each). Acute:     What  you  typically  think  of  when  you  get  a  virus-­‐  flu  like  symptoms       Immune  system  rids  body  of  virus     Latent:       Persistent  (lasts  for  years)     Doesn’t  incorporate  into  the  chromosome  until  the  cell  gets  stressed  and  then  it   goes  into  activation  and  makes  viruses  that  cause  acute  symptoms     Chronic:       Persistent-­‐  can  reoccur  whenever  stressed  (as  mentioned  above)       Slow  release  of  viruses  constantly,  becomes  part  of  the  genome       Symptoms  take  a  long  time  to  show  up       Budding  is  hard  on  cells  and  will  eventually  kill  them     Cancer:       Can  be  caused  by  a  virus,  only  Cervical  cancer/  HIV  is  directly  associated  with  it       Virus  inserts  itself  into  a  proto-­‐oncogene  (normal  gene  in  your  body  that  is   susceptible)  and  turns  it  into  an  oncogene  (cancer  gene)       Then  the  oncogene  is  mistakenly  inserted  and  transferred  to  other  cells       Understand the replication process of HIV (a retrovirus). Outline of Basic Steps: (1) RNA à (2) Reverse transcriptase, used to transform RNA to DNA à (3) Provirus stage à (4) mRNA à (5) Released by budding Retro-­‐  backwards     • Uses  reverse  transcriptase  to  go  from  RNAà  ddDNA     • Retroviruses  are  enveloped,  segmented,  single  stranded,  +  sense   • Attachment  is  specific     • Released  by  budding     • Uncoats  @  cell  membrane,  sheds  envelope  and  capsid     • Travels  to  the  nucleus  and  gets  DNA  inside  with  virus  and  integrates     • Called  a  provirus  b/c  it  stays  in  the  genome  and  regulates  mRNA  for  the  virus  to   build  more     Explain the role of viruses in causing cancer (oncoviruses). - Cancer à virus inserts an oncogene that it carries or sometimes it activates a proto-oncogene o Proto-oncogene: the oncogene that you already possess/normally have – usually fine, doesn’t cause cancer until it malfunctions (viruses inserting can cause it to malfunction) o If the plasma membrane changes that means that it's a tumor Epstein-Barr virus Burkitt’s lymphoma nasopharyngeal carcinoma Hepatitis B virus Hepatocellular carcinoma Hepatitis C virus Hepatocellular carcinoma Human herpesvirus 8 Kaposi’s sarcoma Human papillomavirus Cervical cancer HTLV-1 Leukemia Chapter 16.1 – 16.2, 16.4 – 16.8, 17.2 Know the basic structure of DNA. - Composed of 4 building nucleotides or bases o A = T o C = G - Watson & Crick (1953) discovered DNA structure - Nucleoid: location of chromosome and associated proteins o Irregularly shaped region, usually not membrane bound o Usually one, closed circular, double-stranded DNA molecule - Plasmids: extra-chromosomal DNA o Usually small, closed circular DNA molecules o Exist and replicated independently of chromosome o Contain few genes (non-essential) - Composed  of  4  building  nucleotides/bases     o A=  T  (double  bond,  2  H  bonds)   o C=  G  (triple  bond,  3  H  bonds  [stronger])     - 5’à  3’  antiparallel     - T  and  C  are  pyrimidine     - A  and  G  are  purine     - Sugar  phosphate  backbone     Understand the terms associated with bacterial genetics. - Gene: DNA segment that codes for a polypeptide, rRNA, or tRNA o Ex: hisC à HisC protein (when talking about the actual gene = italicized, talking about the protein = not italicized) - Genotype: specific set of genes an organism possesses (the nucleotide sequence) - Phenotype: set of observable characteristics o Ex: His+ or His- - Wild-type Strain: strain isolated from nature o The most common form of any gene is going to be carried by the wild- type strain - Mutation: stable, heritable change in nucleotide sequence o Genotype is altered – may or may not have an effect on the phenotype of an organism § Ex: hisC1, hisC2, etc. - Gene-­‐  DNA  segment  that  codes  for  a  polypeptide,  rRNA,  or  tRNA     - Genotype-­‐  specific  get  of  genes  an  organism  possesses  (the  nucleotide  sequence)     - Phenotype-­‐  set  of  observable  characteristics     - Wild  type  strain-­‐  strain  isolated  from  nature,  most  prevalent  strain     - Mutation-­‐  stable,  heritable  change  in  nucleotide  sequence  resultant  from  an   altered  genotype   -   (May  or  may  not  have  effect  on  the  phenotype  of  an  organism)   Explain how mutations arise. - Spontaneously à arise without exposure to external agents o May result from errors in DNA replication - Induced à develop after exposure to a mutagen o Caused by agents that directly damage DNA (mutagens) § Base analogs – can substitute for a normal nucleotide base § DNA modifying agents § Intercalating agents – inserting into the DNA, can get bigger or smaller when a loop is formed - Site-directed mutatgenesis à technique whereby a gene with a specific mutation can be constructed in vitro - Spontaneously     o May  result  from  errors  in  DNA  replication     - Induced     o Develop  after  exposure  to  a  mutagen     o Directly  damage  DNA     - Site-­‐directed  mutagenesis     o Technique  whereby  a  gene  w/  a  specific  mutation  can  be  constructed   in  vitro     Compare and contrast point and frameshift mutations and their effects. A. Point Mutations - Alteration of a single pair of nucleotides o Transition à purine substitutes with another purine or with pyrimidine o Transversion à pyrimidine and purine switch - The addition or deletion of a nucleotide pair which causes a framshift B. Frameshift Mutations - Caused by the deletion or insertion of base pair(s) - Result of: o Errors in DNA replication o Damage to DNA o The action of mobile genetic elements such as transposons - It alters the reading frame which can lead to nonfunctional protein or death C. Larger Mutations - Insertions - Deletions - Inversions - Duplications - Translocations Point  mutations:   • Alteration  of  a  single  pair  of  nucleotides     • Transition-­‐  purine  à  purine     • Transversion-­‐  purine  à  pyrimidine     • Can  have  as  little  as  no  effect     Frameshift  mutations:   • Caused  by  deletion  or  insertion  of  base  pair(s)   • Results  in  errors  in  DNA  replication,  damage  to  DNA,  transposons     • Can  alter  reading  frame  and  cause  nonfunctional  proteins  or  death  of  cell     Distinguish between the various point mutations. **Only way to know if there is a silent mutation is to look at the genotype/genome because it will code for the same protein and produce the same phenotype** • Missense-­‐  results  in  a  faulty  protein     o UACà  AAC     • Nonsense-­‐  incomplete  protein,  creates  stop  codon     o UACà  UAG  (stop)     • Silent-­‐  normal  protein;  happens  to  code  for  the  same  thing     o UAC  (tyrosine  codon)  à  UAU  (tyrosine  codon)   Understand the expression of mutations (forward vs. reverse). - Wild-type: most prevalent form of the gene - Forward mutations: o Wild type à mutant form - Reverse mutations: o Mutant phenotype à wild-type phenotype - Revertants: gone from wild-type to mutation to wild-type o Same-site Revertants or Reversions – the mutation that changes a base, the base just changes back to the original o Second-site Revertants or Suppressor Mutants – something downstream or upstream mutates to fix the problem caused by the original mutation and compensates for the problem (pretty rare) Forward=  wild  type  à  mutant  form     Reverse=  mutant  phenotypeà  wild  type  phenotype     • Typically  only  happen  with  point  mutations     Describe the phenotypic effects of mutations in bacteria. - Morphological: change in colonial or cellular morphology o Default shape for a bacteria = coccus - Lethal: kill the organism - Conditional: expressed only under certain environmental conditions - Resistance: resistance to pathogen, chemical, or antibiotic - Biochemical: change in metabolic capabilities o Auxotrophic mutant § Unable to make an essential macromolecule such as an amino acid or nucleotide § Has a conditional phenotype § Wild-type strain from which it arose is called a prototroph - Morphological-­‐  change  in  colonial  or  cellular  morphology     - Lethal-­‐  kills  the  organism   - Conditional-­‐  expressed  only  under  certain  environmental  conditions   - Resistance-­‐  resistance  to  pathogen,  chemical,  or  antibiotic     - Biochemical-­‐  change  in  metabolic  capabilities     -   Ex:  Prototroph  à  auxotroph  (forward  mutation)     -   Unable  to  make  an  essential  macromolecule  such  as  an  amino  acid     Distinguish between prototrophs and auxotrophs. - Auxotrophs: mutants that are unable to make an essential macromolecule such as an amino acid or nucleotide - Prototroph: the wild-type strain from which auxotrophic mutants arise - Prototrophs-­‐an  organism  that  is  capable  of  synthesizing  all  its  metabolites  from   inorganic  material,  requiring  no  organic  nutrients     - Auxotrophs-­‐  a  mutant  organism  that  requires  a  particular  additional  nutrient  that   the  normal  strain  does  not     - Describe how to detect and isolate mutants (including the replica plating technique). - Mutant Detection à observable changes in phenotype (screening) - Selectable mutations à confer some type of advantage to the organisms that possess them - Selection à placing organisms under conditions where the growth of those with a particular genotype will be favored - Replica Plating o Can be used to detect auxotrophic mutants • Detect:  observe  changes  in  phenotype  (via  screening)     • Selectable  mutations-­‐  confer  some  type  of  advantage  to  the  organism  that  posses   them  (ex:  drug  resistance)     • Selection:  place  organisms  under  conditions  where  the  growth  of  those  with  a   particular  genotype  will  be  favored     • Replica  plating-­‐  can  be  used  to  detect  auxotrophic  mutant     o Can  be  used  for  positive  or  negative  selection     o Auxotrophs  cant  grow  on  minimal  media     Know the difference between screening and selection. Screening à observation of changes in phenotype Selection à placing organisms under conditions where the growth of those with a particular genotype will be favored Describe the possible fates of donor DNA during horizontal gene transfer. • DNA  is  most  stable  on  a  chromosome     • “Plasmids,  if  they  don’t  use  them,  they  lose  them”     Describe homologous recombination in bacteria. Genetic Recombination: one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence - Homologous Recombination o Usually involves a reciprocal exchange between a pair of DNA molecules with the same nucleotide sequence § Bacterial DNA has to have a lot of similarity in sequence, sequence homology = same sequence o DNA strand breakage and reunion leads to crossover o Process of Homologous Recombination: § DNA strand is nicked § Single-stranded binding (SSB) protein and RecA protein complex formed § Recipient DNA invaded § Crossover leads to exchange which is then ligated to form two recombinant DNA molecules § **Can’t occur without special enzymes – can’t incorporate on its own • Nucleases = “scissors” • Ligase = “glue” Homologous  recombination-­‐  usually  involves  a  reciprocal  exchange  between  a  pair  of   DNA  molecules  with  the  same  nucleotide  sequence     • DNA  strand  breakage  and  reunion  leads  to  crossover     • Crossing  over  is  similar  to  that  in  eukaryotes     • Homologous=  the  same     • REQUIREMENTS  FOR  THIS  TO  WORK:   o Homology-­‐  foreign  DNA  must  have  the  majority  of  the  same  sequence   as  the  host  DNA  (165  base  pairs  to  match  up  would  result  in  high  level   recombination)     o The  cell  has  to  be  RecA  positive!     § RecA  can  invade  the  chromosome  and  get  into  the  hydrogen   bonding  and  breaks  them  when  enough  base  pairs  match  up   § RecA  then  stops  and  enzymes  come  in  to  crossover  DNA     § Ligase  glues  the  DNA  back  together     Compare and contrast insertion sequences and transposons. Transposable Elements – segments of DNA that move about the genome in a process called transposition - Can be integrated at target sites (specific site or sequence) in the chromosome - Have to contain the transposase gene which recognizes the target sequence, cuts the DNA, and glues in the sequences - Bounded by inverted terminal repeats - A. Insertion Sequences o Only carry the gene for transposase o Short segments (~1000 base pairs) - B. Transposons (Tn) o Contain genes other than those used for transposition o Larger than IS Transposable  elements-­‐  segments  of  DNA  that  moves  about  the  genome  in  a  process   called  transposition  (another  method  of  allowing  foreign  DNA  in)     1/3  of  the  human  DNA   Insertion  sequence-­‐     • Only  carry  the  gene  for  transposase  (gives  gene  ability  to  jump  around)     • Short  segments     Transposons-­‐     • Contain  genes  other  than  those  used  for  transposition     • Larger  than  IS     • Made  when  two  insertions  sequences  hop  in  and  travel  together  with  whatever  is   in-­‐between  them     Understand the mechanisms of transposition (simple and replicative). A. Simple Transposition: It cuts the DNA like rungs of the ladder – ABCD on top and bottom slide apart and the transposable element is inserted à there are holes left in the DNA when it slides apart but DNA polymerase comes and fills in the holes (A’B’C’D’ on the left and ABCD on the right) forms direct repeats B. Conservative transposition: transposon hops from plasmid to plasmid C. Replicative transposition: transposon does not hop from one location to another; it puts copies itself in other locations - For this to occur, DNA molecules have to come together - It  is  site  specific  and  looks  for  a  certain  site  of  nucleotides     - Duplicates  the  target  sequence  on  either  side     GATCCA     transposable  element     GATCCA   CTAGGT   transposable  element       CTAGGT   Describe bacterial plasmids, their significance in a bacterial host, and their role in horizontal gene transfer. Be able to read a plasmid map. Bacterial Plasmids - Small, autonomously replicating DNA molecules that can exist independently or as episomes (can integrate onto the chromosome of the bacteria) o Must have an origin or replication (ori) o Copy number – can exist as a single copy or as multiple copies § Multicopy number plasmid > 40 copies • When the cell divides they can be inherited but may not be divided evenly § Single copy number plasmid o Carry genes that may confer a selective advantage o Cell can somehow tell if they need to keep around the plasmid, “if they don’t need it, they lose it” ‘ - Resistance (R) Plasmids o Also called “R factors” o Have genes for resistance to antimicrobials o Some are conjugative § Indicated by the presence of the “tra” gene, which encodes for all functions of conjugation such as the ability to make sex pili, send the plasmid over it, etc. § Plasmids will also only contain “oriT” is they are conjugative – the origin or transfer: the first part that goes into the second cell because plasmids enter linear and then roll themselves to reform the circular plasmid o Usually do not integrate into the chromosome – which is good because genes on chromosomes are much more stable and the R plasmids encode for antibiotic resistance - Fertility (F) Plasmids o Also called F factors o Conjugative plasmids § Tra region § Can transfer copies of themselves to other bacteria during conjugation o Many are also episomes – a plasmid that can incorporate onto the bacteria chromosome Tn  –  transposon     Ori-­‐  origin  of  replication  and  of  transfer       Never  put  a  gene  of  interest  in  the  origin     Tra-­‐  gene  for  conjugation       Transfer  resistance  plasmids  to  other  bacteria     You  can  have  a  combination  of  R  and  F  plasmids       Know  why  plasmids  are  useful  as  cloning  vectors.     Cloning  vectors-­‐  small  piece  of  DNA,  taken  from  a  virus  or  plasmid  that  can  be  stably   maintained  in  an  organism  and  into  which  a  foreign  DNA  fragment  can  be  inserted  for   cloning  purposes.   *plasmids  have  multiple  copy  numbers       Describe the process of bacterial conjugation. *“Rolling Circle Replication” – the way it goes in linear and rolls back up into a plasmid *At the end it’s supposed to be 2 F+ cells • Involves  a  pilus  to  connect  an  F+  cell  (donor)  and  a  F-­‐  cell     • One  strand  of  the  F+  plasmid  is  transferred  to  the  F-­‐  cell     • Both  cells  end  up  being  F+  cells     Understand the process of transformation (natural and artificial) and the concept of cell competence. Transformation - Uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell’s genome o Competent cells can take in naked DNA from their environment – can make cells artificially competent - Plays an important role in horizontal gene transfer in nature - A stable transformation is when the DNA can be actually incorporated into the chromosome - Artificial Transformation o Transformation done in the laboratory with species that are not normally competent o Variety of techniques used to make cells temporarily competent § Calcium chloride treatment à makes cells more permeable to DNA o Methods of Transformation § Heat Shock Method: competent cells + DNA solution à hold at 0°C for 15 minutes à heat at 42°C for 90 seconds à chill suspension rapidly to 0°C à Add recovery medium, incubate at room temperature à plate and incubate appropriately à transformants § Electroporation: give cells an electric shock that makes them more permeable Transformation-­‐  cell  takes  in  DNA  from  the  environment  (naked  DNA  probably  from   dead  cells)     • Cells  have  to  be  competent,  meaning  willing  to  pick  up  DNA     • Not  all  cells  are,  so  you  can  do  this  artificially  in  the  lab  with  heat  shock     • Resultant  cells  are  called  transformants  or  recombinants     • Plays  an  important  role  in  horizontal  gene  transfer  in  nature     Know the significance of the following abbreviations: RecA, IS, Tn, tnp, F , F , tra, + − ori. RecA = Protein Complex needed for Homologous Recombination IS = Insertion Sequences Tn = Transposons tnp = Gene designation for the Transposase gene F+ = F plasmid donor F- = F plasmid recipient tra = Conjugation Gene – encodes for all functions of conjugation ori = Origin of Replication Compare and contrast generalized and specialized transduction. A. Generalized Transduction - Occurs during the lytic cycle or lytic part of lysogenic - Almost any part of bacterial genome can be transferred - During viral assembly, fragments of host DNA are mistakenly packaged into phage head o Transducing particle (defective) = virus that has the bacteria DNA in it B. Specialized Transduction - Carried out only by temperate phages that have established lysogeny - Only specific portion of bacterial genome is transferred - Occurs when prophage is incorrectly excised Transduction-­‐  virus  makes  a  mistake  and  injects  bacterial  DNA  into  the  host  and  the  cell   then  becomes  transduced  or  recombinant     • Another  form  of  horizontal  gene  transfer     • Virulent  bacteriophages  reproduce  using  lytic  life  cycle   • Temperature  bacteriophages  reproduce  using  lysogenic  life  cycle     Generalized-­‐  almost  any  part  of  the  bacterial  genome  can  be  transferred   • Occurs  during  lytic  cycle     • During  viral  assembly,  fragments  of  host  DNA  are  mistakenly  packaged  into  phage   head     • But  by  size  not  by  sequence  so  anything  can  get  into  the  viral  head     • Viruses  make  this  mistake  and  create  defective  viruses     Specialized-­‐  carried  out  by  temperate  phages  that  have  established  lysogeny     • Only  specific  portion  of  bacterial  gene  is  transferred     Occurs  when  prophage  is  incorrectly  excised Understand how to detect recombinants. Look  for  selectable  markers  and  set  up  media  to  select  for  what  you  want     Antibiotic  resistance  is  a  great  selectable  marker  on  plasmids  (pGLO  with  amp   resistance)     Chapter 15.1 – 15.4 42.  Describe  PCR  and  its  uses.  Know  the  three  steps  of  PCR  (in  order)  and  what  is  needed   for  each  step.   Polymerase  Chain  Reaction-­‐  synthesizes  large  quantities  of  a  DNA  fragment     • Step1:  Denature  template  DNA  with  heat   • Step  2:  Annealing-­‐  primers  bind  to  target  DNA     • Step  3:  Extension-­‐  copies  of  target  DNA  are  synthesized     • Number  of  copies  goes  up  exponentially     Describe the following “tools” and techniques and their uses: restriction enzymes, DNA ligase, synthetic oligonucleotides, PCR, primers, thermocycler, and Taq polymerase. A. Restriction Enzymes – “the scissors” - Recognize and bind to specific sequences in the DNA called recognition sites - Cleave DNA at this site or a defined distance from it o “Sticky” ends – cut half of the DNA down, across the strands and back down across the other = over hang of single-stranded DNA § “Sticky” ends are easier to use when making recombinant DNA o “Blunt” ends – cut straight across - Type II restriction enzymes always cut at the recognition site - Type I and III have a recognition site then cut a defined distance away B. DNA Ligase – “the glue” - Enzyme that seals new base-pairings during DNA replication C. Synthetic Oligonucleotides - Synthetically made DNA primers: starting point for DNA polymerase to start adding bases in replication - Work just like normal DNA – you can modify your DNA by adding an oligo that contains the restriction site/sequence you want - Can be only up to 100 bp in length D. PCR - Synthesizes large quantities of a DNA fragment – can make billions of copies, theoretically you would just need one DNA - Reaction Mix: o Target DNA – template you start with o Primers – determine what part of the DNA is going to be amplified (will flank the region of DNA to be amplified) § Since G and C are bound by 3 and A and T are bonded by 2 – need to make sure you can work at very high temperatures o Thermostable DNA polymerase (Taq polymerase) – can work at very high temperatures o Deoxyribonucleotide triphosphates (dNTPs) – are the bases: A, T, C, & G - Three Repeated Steps of PCR: o Denaturing à target DNA denatured with heat (95°C for 1 minute) o Annealing à primers bind to target DNA § 30 cycles of: • 95°C for 30 sec • 55°C for 1 min • 72°C for 1 min o Extension à copies of target DNA are synthesized (hold at 4°C) E. Primers - Little pieces of synthetically made DNA determine what part of the DNA is going to be amplified (will flank the region of DNA to be amplified) o Since G and C are bound by 3 and A and T are bonded by 2 – need to make sure you can work at very high temperatures F. Thermocycler - Machine used for PCR - Cycles through the different temperatures – copies of the target gene go up exponentially because making you are making copies that can also be used as templates to make more and more - Tiny tubes with very thin walls are filled with eth reaction mix and the next day the product of the PCR will be in the tubes G. Taq Polymerase - DNA polymerase adds the bases to the form the new strand – thermostable can work at very high temperatures Know why plasmids are useful as cloning vectors. Replicates independently of microbial chromosome so may be maintained in a single cell - Easy to purify - Small size - Independent origin of replication - Multiple copy number - Presence of selectable markers - Unique cloning sites (polylinker) – lots of different recognition sites **Understand how to manipulate DNA (think like a “gene jockey”). **If given a hypothetical plasmid map and gene of interest, be able to describe how you would design a recombinant plasmid based on the characteristics of the plasmid and the gene. Describe how you would introduce your recombinant plasmid into a bacterial host, and how you would select for transformants. Would be rather simple Chapter 32.2-32.3 Know the locations of the normal microbial flora in/on the human body and its role in maintaining good health. Know where bacteria should not be found in the human body. Normal microbial flora is found on the surfaces of the body but should not be found in the internal tissues: brain, muscles, organs (except the intestines), lymph nodes, blood, or in a fetus Locations of Normal Microbial Flora: - Skin - Nose - Conjunctiva (eyes) - Outer Ear - Mouth and Oropharynx - Small Intestine - Large Intestine - Urethra - Vagina Normal microbial flora is associated with healthy body tissue and is not pathogenic Describe the characteristics of the bacteria found in and on the skin, nose, nasopharynx, oropharynx, respiratory tract, eye, external ear, mouth, stomach, small intestine, large intestine, and genitourinary tract. A. Skin Microbiota - Has both resident and transient microbiota o Resident: can actually survive and grow and colonize your skin § Skin has an acidic pH, high salt concentration and is very dry – bacteria have to be able to survive in these conditions § Includes Gram-positive bacteria because they like dry environments especially Strep and Staph o Transient: might get on your skin but cannot survive there and spend a little bit of time there before they are washed off or die B. Nose Microbiota - Gram-positive bacteria C. Nasopharynx Microbiota - May contain low numbers of potentially pathogenic microbes D. Oropharynx Microbiota - Alpha-hemolytic streptococci: don’t break down blood completely – yellow or green zone of inhibition on blood agar - Diphtheroids: polymorphic shapes, nonpathogenic - Gram-negative cocci - Anaerobes in tonsillar crypts E. Respiratory Tract Microbiota - There should be no normal microbiota - Only particles smaller than ~10 um in diameter reach in the lungs F. Eye Microbiota - Small numbers of bacteria are found on the conjunctiva (thin layer of skin that convers the whole eye) of the eye – predominant bacterium is Staphylococcus epidermidis G. External Ear Microbiota - Similar to skin flora - Coagulase-negative staphylococci and Corynebacterium o Coagulase = the enzyme used to clump red blood cells o Coagulase-negative are usually nonpathogenic § S. aureus is the only coagulase-positive Staph strain H. Mouth Microbiota - Contains organisms that survive mechanical removal by adhering to gums and teeth à contribute to formation of dental plaque, dental caries, gingivitis, and periodontal disease o Can form a biofilm if ideal conditions exist - Anaerobes in the spaces between the teeth and gums I. Stomach Microbiota - Most microbes killed by acidic conditions (pH = ~2) o Some survive if they pass through the stomach very quickly or if ingested in food particles o Some bacteria can make urease which help them survive the pH of the stomach by catalyzing the urea in the stomach to produce CO2 and ammonia – ammonia cloud around itself to help it survive J. Small Intestine Microbiota - The pH and the umber of bacteria increase as you move down the small intestines K. Large Intestine Microbiota - Largest microbial population of the body à most of the microbes present are anaerobes - Colonic bacteria produce essential vitamins like B12, K, thiamine, riboflavin - Some can colonize exfoliated host cells, food particles, and sloughed mucus - Replaced rapidly because of their high growth rate (2 doublings per day) L. Genitourinary Tract Microbiota - Kidneys, ureter, and bladder are usually free of microbes - Distal portions of urethra: o Few microbes found – can cause Urinary Tract Infections - Female genital tract o Complex microbiota in a state of flux due to the menstrual cycle – some help to maintain acidic conditions Know the discussed examples of bacteria found in each of the mentioned locations. Skin Propionibacterium acnes Nose Staphylococcus aureus and S. epidermidis Nasopharynx Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae Oropharynx Streptococci, alpha-hemolytic Respiratory Tract Should be none but microbes smaller than 10 um can enter – viruses, some bacteria, and very few fungi Eye Staphylococcus epidermidis External Ear Coagulase-negative Staphylococci and Corynebocterium Mouth Streptococcus sp. Stomach Helicobacter pylori Small Intestine Entrerococcus faecalis and lactobacilli Large Intestine Bacteriodes thetaiontamicron Genitourinary E. coli and Lactobacillus acidophilus Tract (female tract) Describe the conditions of each location that support (or do not support) microbial growth (ex. pH of intestines). Skin – inhospitable environment - Slightly acidic pH - High concentration of NaCl - Many areas low in moisture - Inhibitory substances o Lysozyme and cathelicidins Respiratory Tract – inhospitable environment - Continuous stream of mucus generated by globlet cells - Ciliated epithelial cells - Phagocytic action of alveolar macrophages - Lysozyme in mucus Stomach – inhospitable environment - Very acidic conditions (pH 2) that kill more microbes Large Intestines: - Bacteria eliminated from body by peristalsis (wave-like muscle contractions), desquamation (shedding of the outermost membrane or layer of a tissue), and movement of mucus Understand how normal microbial flora can cause infection and sometimes disease. Relationship between Normal Microbiota and the Host - Usually mutually beneficial o Prevent colonization by pathogens o Produce vitamins o Stimulate immune response - Opportunistic pathogens o Members of normal microbiota that produce disease under certain circumstances - Compromised host o Debilitated host with lowered resistance to infection Chapter 32.1 – 32.2, 32.4 – 32.6, 33.1 – 33.8 (Selected Topics) Define immunity and immunology and describe the human immune system. - Immunity: ability of host to resist a particular disease or infection - Immunology: the study of immune system - Immune system: composed of widely distributed cells, tissues, and organs; recognizes foreign substances or microbes and acts to neutralize or destroy them o Made up of 2 different types of immune responses § Nonspecific à innate, natural – just react to anything that is foreign, everyone is born with nonspecific responses § Specific à acquired, adaptive – have to have an exposure to the antigen/disease to react to it • Two types of specific: (1) cell-mediated (2) antibody mediated, also called humoral Compare and contrast nonspecific (innate) and specific (adaptive) immunity. A. Nonspecific (Innate) Immunity - Act as the body’s first line of defense - The body’s built in ability to recognize and destroy pathogens or their products o Offers resistance to any microbe or foreign material - Does not rely on previous exposure to the disease - Lacks immunological memory - Going to react to the same extent every time it is exposed to the foreign body - Includes physical barriers (skin, etc.), chemical mediators (lysozymes, urea in urine, etc.), and phagocytes (little PacMan that engulf foreign bodies) B. Specific (Adaptive) Immunind - Act as the body’s 2 line of defense - Recognizes specific antigens on pathogens - Have a “memory” à effectiveness increases on repeated exposure to the antigen o Takes exposure, which helps your body get ready for repeat exposure - Can discriminate between self and non-self o If people’s immune system can’t do this, it means that they have an autoimmune disorder - Usually takes a little longer because it the body needs to be activated to act against a particular agent - Includes T cells, B cells, and antibodies Know the roles and characteristics of the following cells of the immune system: macrophages, dendritic cells, neutrophils, T cells CCTLs), T 1 cells, H 2 cells, H plasma B cells, memory B cells. Monocytes – precursors that start circulating in the blood (usually for about 8 hours) and then differentiate into two types of phagocytes, either: - Macrophages – largest phagocytes that ingest and kill foreign cells; strategic participants in certain specific immune reactions through the signaling of other specific responses - Dendritic Cells – relatives of macrophages that reside throughout the tissues (usually bonded to tissues and skin); responsible for processing foreign matter and presenting to lymphocytes Neutrophils – are another important type of phagocytes in the blood (like macrophages and dendritic cells); they are the first inflammatory cells to arrive at the scene - Can be at the site of injury within 30 minutes à attracted by chemotoxins secreted by the damaged cells - Engulf invaders and then they become activated to secrete more chemical signals T Cell Types: - Make up 70-85% of lymphocytes in the blood - Require antigen binding to T cell receptors (TCRs) for activation and continued replication - Differentiate into effector cells (helper T cells and cytotoxic T cells) and memory T cells - Play a major role in B cell activation - Cytotoxic T Cells – T Cells (CTLs) c o Kills cells infected with viruses, other intracellular pathogens, and cancer cells – directly kills the cell when it binds to it § Contains granules with porphyrin that makes holes into which the granzymes are inserted – kills the cell by apoptosis not lysis so that the contents of the cell are not released when it dies o Recognizes antigens embedded in MHC I proteins o Present the antigen on the surface of the cell o CD8 protein coreceptor helps to stabilize the interaction between MHC I proteins – holds everything together and makes the binding stable - Two Types of Helper T Cells (T ) H o Both regulate other lymphocytes and secrete cytokines o Recognize antigens embedded in MHC II proteins o CD4 protein coreceptor that helps to stabilize the interaction between MHC II proteins by holding everything together at the binding site o T 1 Hells à stimulate inflammation and other immune cells § Releases a lot of different types of cytokines – stimulates other types of immune cells, even the macrophage that it is directly attached to, which increases its phagocytic activity and increases inflammation throughout the body o T 2 Hells à assist B cells B Cell Types: - Lymphocytes activated by binding of specific antigens, but usually require TH2 triggering as well - Not mobile – have to be very sick to get to a B cell in the lymph nodes or spleen - B cell has bound antigen on its surface in MHC II molecules – T helper cell recognizes this presentation and binds to the B cell (that is already partly activated) it starts to release interleukins and cytokines that stimulate that B cell to differentiate into memory B cells and plasma cells - Plasma B Cells à produce about 2000 antibodies per second, only lives in the body for a few weeks - Memory B Cells à cells that have already been partially activated and only needs to bind to the appropriate antigen to start working; can last for years o Takes a lot longer for the primary (first time being exposed) it can take several days or weeks until the antigen is even identified o Secondary antibody response happens a lot faster – heightened response, going to make a 1000x more antibody production, doesn’t take several days or weeks § The concept of secondary immune response is how vaccines work 50.  Know  the  role  of  phagocytes  in  nonspecific  immunity  and  how  some  can  initiate  a   specific  immune  response.     Phagocytes-­‐  one  of  several  leukocytes  (white  blood  cells)  with  the  capacity  to  engulf   and  destroy  foreign  substances     • A  nonspecific  response     • Results  in  inflammation     • Includes  neutrophils,  macrophages,  and  dendritic  cells     • Phagosome  +  lysosome  à  phagolysosome     • Some  act  as  antigen  presenting  cells     o Links  a  nonspecific  response  to  a  specific  immune  response     • In  most  cases,  specific  immunity  is  initiated  by  antigen  presentation  ^       Compare and contrast cell-mediated and humoral (antibody-mediated) immunity. Describe the activation and differentiation of T cells and B cells. A. Cell-Mediated Immunity – T cells (T lymphocytes) • 70 to 85% of lymphocytes in blood • Mature in thymus • Mobile • Require antigen binding to T cell receptors (TCRs) for activation and continued replication • Differentiate into o Effector cells o Memory T cells • Major role in B cell activation B. Humoral Immunity (Antibody-Mediated) • B cells (B lymphocytes) o Mature in bone marrow o Not very mobile o Activated by binding of specific antigen, but usually require T 2 H triggering as well o Differentiate into § Plasma cells (which produce antibodies) § Memory cells • Antibodies (immunoglobulins, Ig) o Glycoprotein molecules that are able to combine with antigenic determinants o Produced by activated B cells o Very mobile 52.  Describe  T  cells  and  B  cells  and  their  roles  in  specific  immunity.     T  Cells-­‐  helper  T  cells  and  cytotoxic  T  cells     • Helper  T  cells  activate  B  cells  and  cytotoxic  cells     • Cytotoxic  cells  can  kill  directly  and  give  rise  to  memory  T  cells     B  cells-­‐  plasma  cells  (produce  antibodies)  and  memory  B  cells     o B  cells  give  rise  to  memory  B  cells  and  plasma  cells     o Plasma  cells  secrete  antibodies     Distinguish between a primary and secondary immune response. - Primary Response: takes a lot longer for the primary (first time being exposed) it can take several days or weeks until the antigen is even identified - Secondary Response: happens a lot faster – heightened response, going to make a 1000x more antibody production, doesn’t take several days or weeks Explain how antibodies eliminate pathogens. Distinguish between the types of acquired immunity (active vs. passive, natural vs. artificial). Natural vs. Artificial Immunity: - Natural Immunity – acquired through the normal life experiences of a human and is not induced through medical means - Artificial Immunity – that produced purposefully through medical procedures (also called immunization) Active vs. Passive Immunity: - Active Immunity – the consequence of a person developing his or her own immune response to a microbe - Passive Immunity – the consequence of one person receiving performed immunity


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