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Bio 219 final drexel (1)
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Bio 219 final drexel (1)
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Bio 219 final drexeloperon - ANS-group of genes operating together which allows for a controlled cooperation of
protein synthesis
chromosomal DNA (bacteria) - ANS-negatively supercoiled in a circle
restriction digestion - ANS-Use of restriction enzymes to cleave DNA into fragments
quorum sensing - ANS-The ability of bacteria to sense the presence of other bacteria via
secreted chemical signals.
Auto-induction - ANS-Bacteria release N-Acyl Homoserine lactone. As density of bacteria
increase so does the inducer. When inducer's density is high enough it diffuses back in to the
cell
lux operon - ANS-encodes genes for self-regulation and for the production of luminescent
proteins
Luciferase - ANS-enzymes that catalyzes light reaction. Heterodimer of alpha and beta
subunits.
structure of the lux operon - ANS-LuxR, luxI, luxC, luxD, luxA, luxB, luxE (RICDABE)
LuxI - ANS-Encodes to catalyze the AHL synthesis (auto inducer)
LuxR - ANS-Encodes for a DNA binding transcriptional activator that binds the high
concentrations of autoinducer
LuxC - ANS-encodes for the Acyl-reductase enzyme
luxD - ANS-encodes for the acyl-transferase enzyme
LuxA - ANS-Encodes for the alpha subunit of luciferase (activation site)
luxB - ANS-encodes for the beta subunit of luciferase - absolutely necessary for the activity of
enzyme
Luxe - ANS-Encodes for acyl-protein synthetase enzyme
acyl transferase - ANS-Removes fatty acids from bio synthetic pathway for use (luxD)
, acyl synthetase - ANS-Activates the fatty acid to form R-CO-AMP (luxE)
ATP dependent
Acyl-reductase - ANS-Reduces the activated fatty acid to form the necessary aldehyde
NADPH dependent
Resuspension Buffer (Buffer ATL) - ANS-contains sodium dodecyl sulphate (SDS) that can
solubilize the membrane proteins. The detergent disrupts the lipid bilayer and brings the
proteins into solution. SDS helps release the DNA binding proteins by denaturing them and
binding both membrane and non-membrane proteins as monomers
Proteinase K (ProK) - ANS-Aids the release of nuclei acids and deactivates nucleases
lysis buffer - ANS-solution used to break the cell membrane and release cell contents
shotgun cloning - ANS-creation of a genomic library by cloning random fragments of a genome
What makes a good plasmid vector? - ANS-Size
Large enough to hold workable quantities of foreign DNA
Small enough to be retained by the host
Small enough to be able to distinguish from host chDNA
High copy number
50 to 100 per cell
Origin of replication (ORI)
Must be recognizable by the host's replication machinery
Must be able to copy often and independently of host DNA
Multiple cloning site (MCS)
Region of DNA containing recognition sequences for many restriction enzymes
Engineered so that these sites are unique on the vector
Cutting with enzymes in MCS = linearization, not fragmentation
Selectable markers
For uptake of vector
For uptake of foreign DNA
Often use resistance to antibiotics, b-gal (blue/white colonies), Green Fluorescent proteins
RNA polymerase promoter sequences
So mRNA can be made off of the inserted DNA
Usually near the multiple cloning site
Often one on each side so directionality of insert not an issue
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