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LAB 2 Flashcards

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1
Q

UV light

A
  • used to control microbial growth of objects (nonliving things)
  • adjacent thymine molecules in DNA cross to form thymine dimer
  • leads to mutations during DNA replication
  • kills microbes by causing mutations
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2
Q

UV light experiment

A

-4 nutrient agar plates are inoculated for s. marcescens -inoculated for confluent growth
-swabbed on with cotton swab
-exposed to UV light
-plate 1 was exposed to UV light half covered for 1 min
-plate 2 was exposed to UV light hald covered with paper (5 mins)
-plate 3 is under UV light but covered with plastic lid (5 mins)
-plate 4 is control
-incubated
results:
-expect to see growth in 4, 3
-half growth in 2 under the paper
-complete growth under the paper and colonies on the other side bc of the short UV time for plate 1
-length of time is a factor

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3
Q

slide smear

A
  • place a drop of water on center of slide
  • use aseptic procedure to pick up bacteria culture with inoculating loop
  • mix bacteria in drop of water to make a suspension of cells
  • spread suspension on the slide -> smear
  • sterilize the loop
  • air dry smear
  • then heat fix smear by passing the slide through a flame of a bunsen burner quickly 4 times (so cells are attached to the slide and dont wash off)
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4
Q

staining the slide

A
  • pour methylene blue on the smear and cover the smear with methylene blue
  • wait 1 minute for the stain to react with the smear
  • rinse methylene blue off of the slide using water
  • blot dry the smear by placing the slide between 2 sheets of paper towels
  • once the slide is dry -> observe under microscope
  • determine shape and arrangement of cells under 100x objective lens
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5
Q

bacillus subtilis

A
  • streptobacili arrangement
  • rod shape
  • rod shape shape chain
  • also see some single bacili
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6
Q

escherichia coli

A
  • rod shape cell
  • bacili
  • no specific arrangement
  • single cells
  • no chains or clusters
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7
Q

anitobiotics

A
  • made by microbes
  • antibiotics are found in nature
  • purified by the pharmaceutical companies
  • penicillin
  • mold makes penicillium notatum -> purified into penicillin
  • penicillin inhibits the formation of peptidoglycan
  • penicillin is selectively toxic to bacteria (not toxic to host -> humans) bc humans have no peptidoglyan
  • gram neg- resistant to penicillin
  • gram +- sensitive to penicillin
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8
Q

chloramphenicol

A
  • produced by streptomyces
  • inhibits protein synthesis on the 70S ribosomes
  • eukaryotic cells have 80S ribosomes
  • antibiotic selectively inhibits protein synthesis on the 70S ribosome
  • high concentration and prolongs use can cause aplastic anemia
  • tetracycline, streptomycin, erythromycin are made by streptomyces
  • gram + and neg are sensitive to tetracycline and streptomycin and chloramphenicol
  • gram neg is resistant to erythromycin and gram pos it sensitive -> bc erythromycin is large and cannot penetrate the outer membrane and interfere with ribosomes
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9
Q

gentamicin

A
  • antibiotic
  • produced by micromonospora
  • inhibits protein synthesis on the 70S ribosomes
  • gram + and neg are sensitive to gentamicin
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10
Q

antibiotic sensitivity test

A
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11
Q

antibiotic sensitivity test

A
  • done to see if a bacterium is sensitive or resistant to various antibiotics
  • isolate the pathogen from the sample
  • based on results the physician will find treatment
  • done every day in clinical labs
  • plates are inoculated with bacteria for confluent growth
  • antibiotic discs are placed on the medium and the plates are incubated
  • after incubation the plates are observed for the zone of inhibition (absence of growth) around the disc
  • absence of growth -> sensitive to antibiotic
  • antibiotic diffuses into the medium and comes into contact with bacteria (stops or kills bacteria if they are sensitive)
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12
Q

influence of O2 on the growth of bacteria

A
  • micrococcus luteus
  • escherichia coli
  • clostridium sporogenese
  • inoculate each of the organisms on 2 plates of nutrient agar by using streaking for isolation procedure
  • incubate one plate of each organism in the presence of O2
  • incubate the other plate of each organism in the absence of O2
  • look for growth after
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13
Q

microccocus luteus

A
  • expect to see growth on the plate incubated with O2
  • do not expect growth on plate incubated with no O2
  • obligate aerobe- needs O2 for growth
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14
Q

Escherichia coli

A
  • facultative anaerobe- growth with or without O2 but grows better with O2
  • expect to see growth on both plates
  • expect to see larger colonies on the plate incubated with O2
  • expect to see smaller colonies on the plate incubated without O2
  • why does it grow better in O2 -> O2 helps the bacteria make more ATP (38)
  • without O2 it only makes 2 ATP
  • ATP helps cell make macromolecules to grow faster
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15
Q

clostridium sporogenes

A
  • obligate anaerobe- grows only without O2
  • O2 is toxic bc the bacteria doesnt have the enzymes to neutralize the toxin
  • expect to see growth on the plate incubated without O2
  • do not expect growth on plate incubated with O2
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16
Q

growing obligate anaerobes

A
  • anaerobic jar to growth obligate anaerobes like costridium sporogenes
  • place plates in anaerobic jar
  • place envelope with chemicals
  • methylene blue- O2 detector
  • blue in presence of O2
  • clear in absence of O2
  • chemicals react -> release Hydrogen
  • hydrogen combines with O2 -> water (removes O2)
  • check oxygen indicator after 5-10 min
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17
Q

endospore staining bacillus subtilis culture: prep

A
  • smear has to be prepared:
  • drop of water on slide -> using aseptic procedure pick up culture and put it in the water
  • mix the bacteria in the drop of water
  • spread the suspension onto the slide
  • air dry
  • heat fix- pass through flame 4 times quickly (so cells get attached to slide and dont wash off)
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18
Q

endospore staining bacillus subtilis culture

A
  • cover the smear with a piece of paper towel
  • pour malachite green on to the smear that is covered with the paper towel
  • wait 30 mins
  • rinse the malachite green off of the slide with water
  • cover the smear with safranin
  • wait 1 min
  • rinse safranin off of the slide
  • wait for the slide to dry
  • observe under 100x objective lens
  • pink -> vegetative cells
  • green -> endospores
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19
Q

biochemical tests

A
  • indirectly checking for enzyme by directly looking for the presence of the product or the disappearance of the substrate
  • enzyme profile/activity of bacteria
  • each species has a unique set of enzymes (bc it has unique set of genes)
  • therefore, the enzyme profile helps us to identify bacteria
  • give the bacteria the substrate
  • if the bacteria makes the enzyme -> the enzyme will convert substrate to products
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20
Q

substrate

A

-is a substance with which the enzyme reacts

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21
Q

exoenzymes: starch hydrolysis test

A
  • exoenzyme- enzymes produced in the cell and then released into the environment
  • once the enzyme is outside it breaks down macromolecules into smaller molecules in the environment
  • smaller molecules cross the plasma membrane and they are used by the cell for energy or to make structure of the cell
  • exoenzyme: amylase
  • substrate- starch
  • product- maltose
  • amylase breaks down starch and produces maltose
  • use 2 plates of starch plates
  • one plate gets inoculated with B. subtilis
  • the other plate is inoculated with E. coli
  • we make a single line inoculation
  • after incubation we expect a single line of growth across the medium
  • we look for the disappearance of the substrate (starch)
  • add iodine- indicator for starch
  • if medium turns purple -> starch is present and bacteria did not use it
  • bacteria is negative for starch hydrolysis -> negative for making amylase
  • if there is clear area around the growth -> no starch in the medium
  • bacteria broke down the starch -> bacteria is positive for starch hydrolysis -> amylase is present
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22
Q

results of starch hydrolysis test

A
  • positive for starch hydrolysis -> b. subtilis
  • negative for starch hydrolysis -> e. coli
  • e. coli- amylase is not present and starch is present in the medium -> purple
  • b. subtilis- amylase is present -> clear around the starch strip
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23
Q

nitrate reduction test

A
  • we want to see if the bacteria can use nitrate
  • 3 possibilities:
  • some bacteria make the enzyme nitrate reductase and convert nitrate to nitrite
  • some bacteria make 2 enzymes: nitrate reductase which converts nitrate to nitrite -> NITRITE reductase then converts nitrite to nitrogen gas
  • some bacteria dont make either enzymes -> bacteria cant use nitrate
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24
Q

nitrate reduction test procedure

A
  • 3 tubes of nitrate broth
  • one tube for each bacteria:
  • e. coli
  • m. luteus
  • p. aeruginosa
  • inoculate and incubate
  • we add 2 chemicals (nitrite indicators) to see if nitrite is present
  • if nitrite is present the chemicals will react and turn red (nitrate reductase must be present)
  • add a pinch of zinc to the tubes that didnt turn red
  • if the medium becomes red after adding zinc -> zinc converted nitrate to nitrite (reduction) -> this means nitrate wasnt used and there are no enzymes present
  • if the medium doesnt become red after adding zinc -> we know the bacteria made 2 enzymes (nitrate and nitrite reductase) -> no nitrate or nitrite in the tube bc nitrogen gas is present in this tube
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25
Q

red after adding nitrite indicators

A
  • nitrite is present
  • nitrate reductase is present
  • e. coli
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26
Q

no red after adding nitrite indicators but turns red after adding zinc

A
  • neither of the enzymes are present
  • nitrate was present
  • zinc converts the nitrate to nitrite
  • reduction
  • m. luteus
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27
Q

no red after adding nitrite indicators and zinc

A
  • 2 enzymes are present
  • nitrate and nitrite are not present
  • nitrogen gas is present
  • p. aeruginosa
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28
Q

differential medium

A

-helps us to differentiate one group of bacteria from another

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29
Q

blood agar: beta hemolysis

A
  • common differential medium
  • has 5% sheeps RBCs
  • blood agar is red
  • differentiates hemolytic bacteria from nonhemolytic bacteria
  • some bacteria make the enzyme beta hemolysin which breaks down RBCs -> there will be clearing of RBCs around the colonies on the blood agar
  • beta hemolysin- complete hemolysis
  • when beta hemolytic bacteria grow on blood agar complete clearing of RBCs round their colonies
30
Q

alpha hemolysis

A
  • some bacteria make the enzyme alpha hemolysis
  • this enzyme converts hemoglobin to methemoglobin (green)
  • when this bacteria grows we expect to see green colonies
  • partial hemolysis
31
Q

gamma hemolysis

A
  • no hemolysis
  • no color change in medium
  • some bacteria dont make any enzymes that cause damage to the RBCs
  • stays red
32
Q

selective medium

A
  • allows the growth of some bacteria
  • this medium prevents the growth of certain other bacteria
  • NaCl agar- facultative halophiles (grow with or without salt) such as S. aureus will grow while other bacteria such as e. coli cannot grow
  • 6.5% NaCl in the NaCl agar
  • looking for growth and colonies
  • NaCl agar is selective for S. aureus
33
Q

MacConkey agar

A
  • selective and differential medium
  • selective for G- bacteria (only gram neg will grow)
  • it has crystal violet and bile salts that prevent the growth of G+
  • differential medium bc it has lactose in it
  • lactose differentiates lactose fermenters from non-lactose fermenters
  • fermenters make pink colonies
  • non-fermenters make colorless or white colonies
  • uninoculated macconkey agar is pinkish purple on its own
34
Q

lactose fermenter

A
  • e. coli
  • gram negative
  • pink and present on MacConkey agar plate -> therefore it is G- and lactose fermenter
35
Q

lactose nonfermenter

A
  • p. vulgaris
  • present but colorless/white on the macconkey agar
  • this means the bacteria is G- and lactose nonfermenter
36
Q

triple sugar iron agar composition

A
  • differential medium
  • TSI medium
  • three sugars
  • .1% glucose
  • 1% lactose
  • 1% sucrose
  • peptone
  • iron
  • phenol red- pH indicator
37
Q

triple sugar iron agar

A
  • TSI medium
  • slant
  • inoculate needle is used
  • if the entire medium is yellow -> bacteria fermented one or both of the disaccharides (sucrose and lactose) in addition to glucose -> E. coli
  • if there is space at the bottom is also tells us the bacteria has reduced a lot of gas (makes breaks in the agar as well)
  • if entire medium is red -> none of the sugars were fermented -> P. aeruginosa (negative for fermentation)
  • red slant and rest of the medium is yellow -> only glucose is fermented (not disaccharides) -> s. marcescens
  • yellow slant and most of the medium black -> bacteria fermented a disaccharide in addition to glucose and also produced hydrogen sulfide (black) -> p. vulgaris
38
Q

hydrogen sulfide rxn

A
  • amino acid cystine- substrate
  • cystine desulfhydrase- enzyme
  • hydrogen sulfide- product
  • medium has iron- iron is the indicator for hydrogen sulfide
  • hydrogen sulfide combine with iron to make a black precipitate
39
Q

IMViC test

A
  • I- indole
  • M- methyl red
  • V- voges (proskeauer)
  • C- citrate
  • differentiate E. coli from E. cloacae (enterobacter cloacae) and K. pneumoniae (kelbsiella pneumoniae)
  • these 3 bacteria have similar enzyme bacteria -> when it comes to IMViC test they are different
  • these three bacteria will be used in all of these tests
40
Q

indole test

A
  • we are looking to see if the bacteria can make tryptophanase enzyme
  • we give the bacteria the substrate -> tryptophan (amino acid) with tryptone broth
  • 3 tubes of tryptone broth are each inoculated with the three bacteria
  • indole is the product
  • we add dropperful of kovacs reagent and react with indole -> top layer becomes red or bright pink
  • top layer is somewhat orange for negative test
  • if the top becomes red or bright pink -> indole is present -> tryptophanase is present -> positive
  • e. coli is positive
  • e. cloacae is negative
  • k. pneumoniae is negative
41
Q

methyl red test

A
  • some bacteria ferment glucose and release acids
  • 3 tubes of MRVP broth -> inoculate with the 3 bacteria
  • incubate
  • add 10 drops of methyl red
  • if medium is red -> positive for acid (fermentation took place)
  • various enzymes depending on what kind of acid is produced
  • yellow color for negative
  • example of an enzyme -> lactate dehydrogenase
  • e. coli is positive -> acids and fermentation -> red
  • e. cloacae is negative -> glucose is still present -> no fermentation -> yellow color
  • k. pneumoniae is positive
42
Q

voges-proskauer test

A
  • 3 tube of MRVP medium (glucose in it) -> inoculate with 3 bacteria
  • incubation
  • we want to see if the medium has acetyl methyl carbinol (neutral)
  • add 10 drops of barritts reagent A and B
  • shake the tube every few minutes for 20 minutes
  • if medium is red -> positive for making acetyl methyl carbinol -> enzyme is present (acetolactate dehydrogenase)
  • enzyme is acetolactate dehydrogenase, substrate is glucose, and product is acetyl methyl carbinol
  • e. coli is negative -> yellow, glucose is present, no enzyme present
  • e. cloacae is positive -> positive for enzyme and acetyl methyl carbinol
  • k. pneumoniae is negative
43
Q

citrate test

A
  • want to see if the bacteria use citrate as the only source of carbon
  • citrate medium has the pH indicator -> bromthymol blue
  • green -> neutral
  • blue -> basic
  • yellow -> acidic
  • when bacteria use citrate ammonium ions are released
  • environment becomes basic -> blue
  • if blue -> ammonium ions are present bc citrate was used -> citrase is present
  • enzyme is citrase
  • e. coli is negative -> green -> no ammonium ions -> negative for using citrate and the enzyme citrase
  • e. cloacae is positive -> basic -> citrate was used and released ammonium ions -> citrase is present
  • k. pneumoniae is positive
44
Q

urea hydrolysis test

A
  • some bacteria make urease and break down urea
  • ammonium ions are leased medium becomes basic
  • pH indicator becomes fuchsia when it is basic
  • urea broth has substrate (urea)
  • if substrate has the urease enzyme it will use urea and release ammonium ions
  • p. vulgaris (proteus vulgaris) -> strong positive -> urease and a lot of ammonium ions is present -> basic
  • k. pneumoniae- weak positive -> has enzyme urease but not a lot -> broke down some urea and release some ammonium ions
  • e. coli is negative -> no urease -> urea is present
  • e. cloacae is negative
45
Q

motility

A
  • allows us to see is bacteria can swim in the environment
  • motility medium is semisolid
  • stab inoculation
  • use an inoculating needle
  • distinct red line of growth in the medium:
  • growth is concentrated along the stab line
  • these bacteria have no choice but to stay along the line bc they have no flagella to move away
  • growth is red because of tetrazolium chloride in the medium. it reacts with the cells and color the cells red (bacteria is not red)
  • if the test is positive there is diffused growth
  • growth is not concentration along stab line -> entire medium is red if bacterium is strong positive for motility
  • bacteria have the flagella and help them to move into the medium and grow throughout the medium
  • e. coli- weak positive
  • e. cloacae- strong positive
  • k. pneumoniae- negative
46
Q

samples used in restriction enzyme analysis of lambda DNA

A
  • intact lambda phage DNA
  • Pstl digest of Lambda phage DNA
  • EcoRI digest of Lambda phage DNA
  • Hindlll digest of Lambda phage DNA
  • Pstl, EcoRI, Hindlll -> restriction enzymes
  • lambda phage infects E. coli -> its DNA is about 49,000 base pairs long (e. coli has 4 mil)
47
Q

gel electrophoresis

A
  • slab of gel made up of a substance known as agarose
  • on one end of the gel there are well -> where we place the sample that is in a dye
  • gel will be immersed in a buffer in chamber
  • connect the chamber to a power supply and turn on the power
  • DNA fragments are separated based on the size
  • 40 minutes -> take out gel
  • stain gel blue
  • this is done to stain the DNA bands such that the bands are visible
  • gel is porous
  • DNA fragments migrate through the gel
  • electric current push the DNA
  • DNA is negatively charged bc of the phosphates
  • migrate from negative to postiive
  • smaller fragments migrate faster
  • each band has many copies of a fragment
48
Q

purpose of gel electrophoresis

A
  • see the DNA
  • become familiar with restriction enzymes
  • electrophoresis
  • analysis of DNA bands
49
Q

applications of gel electrophoresis

A
  • used to identify bacteria and viruses based on the DNA finger printing of these organisms
  • genetic screening- electrophoresis is the first step
50
Q

restriction enzymes

A
  • molecular scissors
  • recognize specific sequence cut DNA
  • make cuts in the DNA
  • each enzyme recognizes different sequence
51
Q

restriction enzyme analysis of lambda DNA

A
  • Lambda phage DNA is treated with three restriction enzymes: EcoRI, Hindlll, Pstl
  • each restriction enzyme recognizes a different sequence -> makes different cuts
  • lanes:
  • 1,5- uncut lambda DNA
  • 2,6- Pstl cut lambda DNA
  • 3,7- EcoRI cut lambda DNA
  • 4,8- HindIII cut lambda DNA
  • lane 1 and 5 only have one band bc there is one piece of DNA -> no fragments -> also large band (higher up)
  • lane 2 and 6- have many bands (10) -> there are many recognition sites for PstI
  • lane 3 and 7- 4 DNA bands
  • lane 4 and 8- 5 DNA bands
  • DNA finger print of lambda phage
  • can identify species and bacteria bc they all have unique print
52
Q

staphylococcus: blood agar

A
  • s. aureus and s, epidermidis look the same under the microscope-> both cocci, gram +, and exist in grape like clusters -> cant tell apart
  • bc of this we use various selective differential media
  • blood agar: differential medium
  • tells us if bacteria is hemolytic or non
  • staphylococcus aureus- beta hemolytic -> clearing of RBC colonies -> golden brown color
  • makes the enzyme beta hemolysin -> breaks down
  • staphylococcus epidermidis- gamma hemolytic -> no damage to RBC -> stays red
  • no enzyme is present to destroy RBC
53
Q

staphylococcus: mannitol salt agar

A
  • mannitol salt agar:
  • mannitol- carbohydrate
  • salt- 7.5%
  • pH indicator- phenol red
  • differentiates and is selective for s. aureus and s. epidermidis
  • selective for staphylococcus bc they are facultative halophils -> only they will grow -> salt aspect makes medium selective
  • mannitol and the pH indicator makes the medium differential
  • mannitol is fermented and acids are released -> pH indicator becomes yellow
  • mannitol is fermented and no acids are released -> neutral -> red color
54
Q

streptococcus

A
  • 3 species:
  • streptococcus pyogenes
  • streptococcus lactis
  • streptococcus faecalis (enterococcus faecalis)
  • all look the same under microscope: gram +, exist in chains, cocci
  • use differential and selective medium to differentiate them
  • we use: blood agar 37oC, NaCl agar 37oC, and brain heart infusion agar (BHIA) 45oC
  • bacitracin disc (antibiotic disc) is placed in section 1 of the plate for blood agar
55
Q

streptococcus: blood agar

A
  • bacitracin disc is placed in section 1 of streaking for isolation
  • incubate at 37oC
  • s. pyogenes- beta hemolytic -> clearing of RBCs -> sensitive to bacitracin disc (clearing)
  • s. lactis and s. faecalis -> gamma hemolytic (no enzyme or clearing) -> resistant to bacitracin (no clearing around disc)
56
Q

streptococcus: NaCl agar

A
  • 6% NaCl
  • 37oC incubation
  • only s. faecalis will grow
  • s. lactis and s. pyogenes cannot grow
57
Q

streptococcus: BHIA

A
  • most bacteria will grow bc its a nutrient rich medium
  • only s. faecalis will grow
  • selective factor is the temperature -> 47oC
  • s. pyogenes and s. lactis will die at this temperature
58
Q

streptococcus and staphylococcus: catalase test

A
  • used to differentiate staphylococcus and streptococcus
  • hydrogen peroxide (H2O2) is converted to oxygen and water via catalase
  • place a drop of hydrogen peroxide on a slide
  • add bacteria to the drop using aseptic procedure
  • if the bacteria is positive for catalase -> bubbles will be seen in the sample
  • if bacteria is negative for catalase -> no rxn
  • staphylococcus will be positive
  • streptococcus will be negative
59
Q

indirect ELISA

A
  • serological test
  • done to see if the patients serum has antibodies against a specific antigen
  • that tells us if the patient has been exposed to the pathogen
  • plastic plates with well in them are used
60
Q

serological test

A
  • antigen antibody rxns

- based on the fact antibodies are specific for the antigen

61
Q

indirect ELISA test ex.

A
  • we want to find out if pt has antibodies for HIV (positive for HIV)
  • get sample from pt (blood)
  • attach HIV protein to the wall of the well
  • the well is a tiny cup that holds all the reagents
  • pts serum is then added
  • if the pts serum has pts antibodies for HIV it will attach to HIV protein
  • antigen antibody complex is formed
  • well is then rinsed to removed any free floating antibodies
  • secondary antibodies are then added -> antibodies against human antibodies
  • they get the secondary antibodies by injecting human antibodies into animals -> animal immune system makes antibodies against human antibodies
  • attach enzyme molecules to the secondary antibodies -> enzyme linked antibodies
  • secondary antibodies are antibodies against human antibodies AND enzyme molecules are attached
  • secondary antibodies attach to the antibody in the antigen antibody complex
  • pt antibody is sandwiched between antibody and secondary
  • well is rinsed for any free floating secondary antibodies
  • colorless substrate is then added
  • if there is enzyme -> converts colorless substrate to blue product -> pt is positive for HIV -> pt serum has antibodies specific for HIV
  • secondary antibody was able to attach to antibody antigen complex
  • if pt is not blue -> negative for HIV -> no antibodies for HIV antigen
  • quick
62
Q

molds

A
  • belong in the kingdom fungi
  • eukaryotic
  • multicellular
  • made up of filamentous structure
  • produce asexual spores
63
Q

Rhizopus nirgicans

A
  • common black bread mold
  • asexual spores -> sporangiospores
  • enclosed in a sac called sporangium
  • spores are black
64
Q

penicillium notatum

A
  • produces antibiotic penicillin
  • some species are used in cheese production
  • asexual spores -> conidiospores
  • formed in chains
  • not enclosed in sac
  • looks like fingers
65
Q

yeast

A
  • kingdom fungi
  • unicellular
  • reproduce by budding (axesual)
  • circular or oval
  • ex. saccharomyces cerevisiae
  • beneficial yeast -> bread, wine
  • ferments sugar to release alcohol and CO2 in absence of O2
66
Q

candida albicans

A
  • yeast
  • normal flora
  • opportunist
  • if one undergoes prolonged antibacterial therapy -> overgrows and causes thrush, vaginitis
  • pseudohyphae- cell is elongated (tubular)
67
Q

yogurt

A
  • streptococcus lactis
  • lactobacillus bulgaricus
  • lactobacillus acidophilus
  • live gram positive bacteria
  • casein milk protein
  • warm the milk and add a spoon of yogurt -> room temp
  • inoculate the milk
  • ferments lactose and release acids
  • acids coagulate -> milk becomes solid
  • we call the solid substance yogurt
68
Q

genetic transformation of bacteria and gene regulation

A
  • transform E. coli cells with plasmid
  • plasmid has ampicillin resistant gene
  • green fluorescent protein (GFP) gene
  • natural source of the gene is jelly fish -> aequorea victoria
  • GFP gene is attached to the promoter of the gene that codes for enzyme for arabinose (carbohydrate) fermentation
  • promoter comes from bacteria
  • GFP doesnt have its own promoter
  • DNA from different sources are inserted into the plasmid
  • genetically engineered plasmid
69
Q

pGLO pasmid

A
  • genetically engineered plasmid
  • ampicillin resistant gene- selection marker
  • allows us to select the cells that have picked up the plasmid
  • has GFP gene -> attached to the arabinose promoter that comes from bacteria cell
70
Q

resistance and regulation of gene expression experiment

A
  • e. coli cells that are sensitive to ampicillin are placed in tube
  • pGLO plasmids are added to the cells
  • tube is incubated at room temp for 10 mins
  • some e. coli will come in contact with plasmid and pick it up and some wont get the chance
  • suspension is plated onto different medium
  • suspension is plated onto:
  • nutrient agar with ampicillin and no arabinose -> GFP gene is not expressed
  • nutrient agar with ampicillin and arabinose -> GFP is expressed
  • only the cells that picked up the plasmid would grow on both these plates
  • ampicillin is selective for plasmid -> resistant cells (cells with pGLO plasmid) grow -> transformation is successful
  • colonies with arabinose have GFP -> arabinose activates GFP gene -> RNA polymerase attached to promoter of GFP gene -> mRNA -> translation
  • just bc the cell has the gene it doesnt mean it is expressed -> environment plays a major role

Microbiology (8 decks)

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