LAB 2 Flashcards

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

bacillus subtilis

A
  • streptobacili arrangement
  • rod shape
  • rod shape shape chain
  • also see some single bacili
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

escherichia coli

A
  • rod shape cell
  • bacili
  • no specific arrangement
  • single cells
  • no chains or clusters
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

gentamicin

A
  • antibiotic
  • produced by micromonospora
  • inhibits protein synthesis on the 70S ribosomes
  • gram + and neg are sensitive to gentamicin
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

antibiotic sensitivity test

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

substrate

A

-is a substance with which the enzyme reacts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
red after adding nitrite indicators
- nitrite is present - nitrate reductase is present - e. coli
26
no red after adding nitrite indicators but turns red after adding zinc
- neither of the enzymes are present - nitrate was present - zinc converts the nitrate to nitrite - reduction - m. luteus
27
no red after adding nitrite indicators and zinc
- 2 enzymes are present - nitrate and nitrite are not present - nitrogen gas is present - p. aeruginosa
28
differential medium
-helps us to differentiate one group of bacteria from another
29
blood agar: beta hemolysis
- 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
alpha hemolysis
- 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
gamma hemolysis
- no hemolysis - no color change in medium - some bacteria dont make any enzymes that cause damage to the RBCs - stays red
32
selective medium
- 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
MacConkey agar
- 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
lactose fermenter
- e. coli - gram negative - pink and present on MacConkey agar plate -> therefore it is G- and lactose fermenter
35
lactose nonfermenter
- p. vulgaris - present but colorless/white on the macconkey agar - this means the bacteria is G- and lactose nonfermenter
36
triple sugar iron agar composition
- differential medium - TSI medium - three sugars - .1% glucose - 1% lactose - 1% sucrose - peptone - iron - phenol red- pH indicator
37
triple sugar iron agar
- 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
hydrogen sulfide rxn
- 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
IMViC test
- 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
indole test
- 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
methyl red test
- 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
voges-proskauer test
- 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
citrate test
- 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
urea hydrolysis test
- 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
motility
- 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
samples used in restriction enzyme analysis of lambda DNA
- 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
gel electrophoresis
- 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
purpose of gel electrophoresis
- see the DNA - become familiar with restriction enzymes - electrophoresis - analysis of DNA bands
49
applications of gel electrophoresis
- used to identify bacteria and viruses based on the DNA finger printing of these organisms - genetic screening- electrophoresis is the first step
50
restriction enzymes
- molecular scissors - recognize specific sequence cut DNA - make cuts in the DNA - each enzyme recognizes different sequence
51
restriction enzyme analysis of lambda DNA
- 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
staphylococcus: blood agar
- 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
staphylococcus: mannitol salt agar
- 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
streptococcus
- 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
streptococcus: blood agar
- 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
streptococcus: NaCl agar
- 6% NaCl - 37oC incubation - only s. faecalis will grow - s. lactis and s. pyogenes cannot grow
57
streptococcus: BHIA
- 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
streptococcus and staphylococcus: catalase test
- 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
indirect ELISA
- 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
serological test
- antigen antibody rxns | - based on the fact antibodies are specific for the antigen
61
indirect ELISA test ex.
- 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
molds
- belong in the kingdom fungi - eukaryotic - multicellular - made up of filamentous structure - produce asexual spores
63
Rhizopus nirgicans
- common black bread mold - asexual spores -> sporangiospores - enclosed in a sac called sporangium - spores are black
64
penicillium notatum
- produces antibiotic penicillin - some species are used in cheese production - asexual spores -> conidiospores - formed in chains - not enclosed in sac - looks like fingers
65
yeast
- 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
candida albicans
- yeast - normal flora - opportunist - if one undergoes prolonged antibacterial therapy -> overgrows and causes thrush, vaginitis - pseudohyphae- cell is elongated (tubular)
67
yogurt
- 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
genetic transformation of bacteria and gene regulation
- 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
pGLO pasmid
- 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
resistance and regulation of gene expression experiment
- 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