Lab Quiz 3 Flashcards
Molecular cloning
Recombinant DNA molecules are assembled and expressed in a host organism
Enable the alteration of genetic information for research and industrial purposes
Cutting, pasting, and rearranging of pieces of DNA
Often involves the insertion of a gene-of-interest into a plasmid
restriction enzymes
Perform the cutting
Bind and cleave (digest) a restriction site
Different restriction enzymes have different
restriction sites
Cleave both strands of DNA
Restriction site
A specific DNA sequence where the restriction enzyme(s) cleave
Are palindromic – the 5’ –> 3’ sequence is identical on
both strands
Plasmid
A circular DNA molecule that replicates independent of the chromosome
Used as vectors to facilitate the transfer of a gene into an organism
During cloning, the plasmid is digested by a restriction enzyme to allow the insertion of the gene-of-interest
Plasmid cutting
A plasmid that has been cut by a restriction
enzyme in one location will become a single linear piece of DNA
A plasmid cut in two locations will yield two linear pieces of DNA, whose combined size is equivalent to the full-size plasmid
Gel electrophoresis
Used to visualize the size and quantity of DNA in a sample
An electrical current is passed through a gel. Since DNA has a intrinsic negative charge (due to its phosphate backbone), DNA moves away from the negative
electrode and toward the positive electrode
The gel consists of an agarose matrix that enables
DNA to be separated by size: smaller DNA fragments weave through the pores of the agarose
matrix easier than larger DNA fragments.
A DNA marker (or ladder) is included on a gel as a
reference to indicate fragment size
Do larger or smaller pieces go to the negative end (up top) on gel electrophoresis?
Larger
Within a bacterial cell, a plasmid naturally exists in a _________ conformation
supercoiled
What converts a plasmid to a relaxed, circular conformation?
When a plasmid is isolated from bacterial cells, the isolation process can damage the DNA backbone (nick in one strand)
When isolating plasmid from a bacterial cultures, many
copies of the plasmid are harvested, some in a supercoiled confirmation and some in the relaxed,
circular conformation - which move faster?
Supercoiled plasmids move slightly faster through the gel matrix than expected (compared to linear DNA) while circular plasmids migrate a little slower
All microbial species are unique, often due to the ______ they produce.
enzymes
Microbial enzymes catalyze the _________ of biological molecules, thereby enabling microbes to grow on particular substrates
metabolism
Microbiologists take advantage of differences in biochemical activities to distinguish between
microbial ______.
species
Enteric bacteria
Gram negative rods that ferment glucose
Can be identified using a group the IMViC tests
IMViC: Indole (I), Methyl Red (M), Voges-Proskauer (V), Citrate (C)
Indole Test
Determines if the bacteria can metabolize the amino acid tryptophan to produce indole using the
enzyme tryptophanase.
Media containing tryptophan (ex.SIM stab) is inoculated and incubated.
After growth occurs, Kovac’s reagent is added and will react with indole if present.
+ : red color in the layer atop the media.
- : yellow color in the layer atop the media
Methyl Red Test
Determines if mixed acids are produced during fermentation of glucose
Mixed acid fermentation produces three acids (acetic, lactic, succinic).
Glucose-containing MR-VP broth is inoculated. Then methyl red (pH indicator) is added after growth in MR-VP broth.
+ : carbohydrate utilization test: red color in the medium
- : carbohydrate utilization test: yellow color in the medium
Voges Proskauer (VP) Test
Determines if bacteria produce acetoin during fermentation of glucose. Acetoin is an organic molecule used by some bacteria to store energy.
Glucose-containing MR-VP broth is inoculated. After growth, Barritt’s reagent A (alpha-naphthol) then Barritt’s reagent B (potassium hydroxide) are added.
+ : development of a red color 15min after the addition of Barritt’s reagents indicating the presence of diacetyl, the oxidation product of acetoin.
- : absence of a red color 15min or more after the addition of Barritt’s reagents.
Citrate Test
Determines if the bacteria can utilize citrate as a sole
carbon source; if a microbe can use citrate as its sole source of carbon and energy for growth
Media contains the pH indicator bromothymol blue to detect the production of alkaline products that increase the pH of the media.
+ : media turning blue or the appearance of bacterial growth on the slant. Sometime the blue is faint.
- : media remaining green.
SIM Stab
A single medium that tests for three characteristics: sulfur reduction, indole production, and motility.
SIM medium is inoculated by stabbing through the agar with an inoculation needle.
SIM stab contains 2 sources of sulfur: sodium thiosulfate and cysteine (an amino acid).
Hydrogen sulfide gas is produced if the bacteria possess enzymes that metabolize either source of sulfur (either thiosulfate reductase cysteine desulfurase).
In the media is the H2S indicator ferrous ammonium sulfate, which reacts with hydrogen sulfide gas to produce an insoluble, black precipitate.
