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BIO 219 Lab Quiz 1
bioluminescence, alcoholic precipitation of nucleic acids
44
Biology
Undergraduate 2
07/02/2011

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Term
Term Project: Make E. Coli glow using the lux operon from V. fischeri
Definition
Glowing Bacteria V. fischeri -- isolate chDNA -- repurify chDNA -- digest chDNA and plasmid DNA for shotgun cloning of lux operon -- ligate chDNA into plasmid DNA to create V. fischeri Genomic library -- Transform E. Coli with Plasmid library -- screen E. Coli for plasmids containing lux operon (glowing colonies) -- re-isolate plasmids from lux positive E. Coli for mapping
Term
Gram Negative Bacteria V. fischeri
Definition
Chromosome, Pilus, Cell Membrane, Cell Wall, Capsule (slime layer), plasmid, flagellum, inclusion, ribosomes, pilus (fimbria)
Term
Bioluminesence 
Definition

96% of all deep sea marine organisms have a symbiotic relationship with bacteria

exact benefit to fish unknown (defense, communication, attracting prey)

bacteria (nice cushy, nutrient rich environment)

Term
Quorum Sensing
Definition

Production of light requires a lot of energy and oxygen (bacteria won't glow unless their environment allows optimal growht)

Threshold cell density required (process by which a colonizing bacteria knows that "critical mass" has been achieved is called QUORUM SENSING)

Term
Auto - Induction
Definition
Bacteria produce a diffusible compound (N-Acyl homoserine lactone), the more bacteria, the more inducer.  Once the levels are high enough, bacteria knows that nutrient levels are good and energy can then be expended
Term
Luciferase
Definition
Enzyme that catalyzes the light emitting reaction (heterodimer of alpha and beta subunits), mixed function oxidase**, reaction requires the simultaneous oxidation of Flavin mononucleotide and a long chain aldehyde
Term
Three enzymes needed to generate the long chain aldehyde that Luciferase will oxidize
Definition

1. Acyl Transferase

2. Acyl - Protein Synthetase

3. Acyl- Reductase

Term
Acyl - Transferase
Definition
removes fatty acids from their normal biosynthetic pathway for use in this reaction
Term
Acyl - Protein synthetase
Definition

"activates" the fatty acid to form R-CO-AMP

ATP DEPENDENT!

Term
Acyl - Reductase
Definition

Reduces the activated fatty acid to form the necessary aldehyde

NADPH DEPENDENT 

Term
Biochemistry of Bioluminescence
Definition

FMNH2 + O2 + R-CHO --> FMN + R-COOH + H2O +    LIGHT

Term
Lux I
Definition
Encodes for the autoinducer
Term
Lux R
Definition
Encodes for the transcriptional activator that binds to the inducer
Term
Lux C
Definition
encodes for the acyl-reductase enzyme
Term
lux D
Definition
encodes for the acyl transferase enzyme
Term
Lux A
Definition
encodes for alpha subunit of luciferase - has the active site of the enzyme
Term
Lux B
Definition
encodes for the beta subunit of luciferase - absolutely necessary for the activity of the enzyme
Term
lux E
Definition
encodes for the acyl-protein synthetase enzyme
Term
Operon
Definition
genetic regulatory system in which genes coding for functionally related proteins are clusted along the DNA. This feature allwos protein synthesis to be controlled coodinately in response to the needs of the cell
Term
Chromosomal DNA
Definition
in a bacteria, it is a large circular piece of DNA. for our project, the chromosomal DNA comes from V. fischeri and we will be isolating the lux operon from this
Term
Restriction Digest
Definition
enzymatic reactions that cut DNA into smaller pieces.  Mediated by enzymes that will digest the DNA at only specific sequences
Term
Plasmid
Definition
small circular DNA, used to move DNA from one organism into a second, vector
Term
Ligation
Definition
enzymatic reaction that will "paste" two pieces of DNA together.  Pieces can be from the same organisms or two different sources
Term
Recombinant DNA
Definition
term for what is produced when DNA from two different sources (chDNA + plasmid DNA)
Term
Shotgun cloning
Definition
is the practice of randomly digesting a large piece of DNA to smaller pieces that can be ligated into plasmids for transport to other organisms
Term
Genomic Library
Definition
collection of cloned DNA fragments from a genome.  Each part of the genome is represented int he library, the library can be screened for the presence of a sequence of interest (in our project the genome is from V. fischeri and the sequence of interest is the lux operon)
Term
Clone
Definition
a bacteria housing a recombinant plasmid that contains apiece of genomic dna, it doesn't have to contain the gene you're looking for
Term
TES buffer - Tris/EDTA/NaCl
Definition
Tris/NaCl: helps keep pH stable and isotonic
Term
EDTA
Definition
chelates (sequesters) divalent cations like Mg 2+, aids the lysozyme removing Mg from LPS layer, inhibits DNases
Term
Lysozyme
Definition
enzyme that destroys the cell wall
Term
Proteinase K
Definition
digests proteins: stable, more efficient in SDS
Term
SDS
Definition

strong ionic detergent:

dissolves cell membrane and denatures protein

Term
Phenol, Phenol: chloroform
Definition
removes lipids, protein and polysaccharaides: clean up
Term
NaCl/Sodium Acetate
Definition
helps precipitate DNA by charge shielding
Term
Alcohol
Definition
Precipitates DNA
Term
Adding two volumes of cold EtOH
Definition

isopropanol can be used but results in more protein contamination

When using isopropanol, you use less of it which reduces overall product (advantage if your tube has limited capacity)

Term
Factors affecting nucleic acid precipitations
Definition

1. Cations for Charge Shielding

2. Temperature during the precipitation step

3. Concentration of the nucleic acids

4. Time and speed of Centrifugation

Term
Charge Shielding
Definition

There must be at least .2M of a monovalent cation present to shield the high negative charge of the phosphate in nucleic acids 

 

Without shielding, intrastrand repulsion is too great, and there will be no aggregation 

Term
Most common salt for routine DNA isolations
Definition
Sodium Acetate, Sodium chloride may be used if the sample contains/will contain SDS (allows detergent to remain soluble in ethanol solution)
Term
Temperature During Precipitation
Definition
it was commonly thought that precipitation needed to be at lower temperatures, but the recovery is actually better at 0 c (on ice) or at room temperature
Term
Concentration of nucleic acids
Definition

precipitations occur effectively within 10 minutes when the nucleic acid concentration is greater that .5 microgram/mL

if the concentration is less than .5 microgram/mL, incubation time must be increased (from 30 minutes to overnight)

Term
Centrifugation
Definition

nucleic acids are usually collected at 12000 - 14000 x g between 4 C and room temperature

ten minutes is long enough if the concentration is high (> microgram/mL)

if the concentration is low, centrifuge for 30 -60 min OR increase the g force

Term
RNase
Definition
digests RNA
Term
Isoamyl alcohol
Definition
defoaming agent
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