Lecture 21 + 22 Flashcards
conjugative plasmids
transmitted during conjugation, carry a variety of info
resistance plasmids (R)
protect against the environmental factors, MDR (multiple drug resistance) plasmids
Colicinogenic plasmids (Col)
codes for proteins that kill other microbes
Degradative plasmids
contain genes for novel catabolic enzymes
Virulence plasmids (vir)
increases the pathogenicity of a bacteria (e.g. toxins)
Why are plasmids useful for us?
Putting a gene into a plasmid may allow that gene product (protein) to be expressed at high levels
vector
a plasmid that has been streamlined and modified to make it amenable to carrying a payload of DNA
components of vectors
- origin of replication
- positive selection gene (to select for vector-carrying cells)
- insert differentiation gene (to detect empty vs full vectors)
- cloning sites (loading area)
Foreign DNA is degraded by….
cutting at specific sequences (endogenous/original DNA is not)
R/M system
Host DNA is modified by methylation at a specific sequence, unmethylated DNA is restricted (cut) at the same sequence
EcoRI restriction endonuclease
Cuts within a palindromic sequence only if unmethylated, leaving staggered cuts (sticky ends)
Type I restriction enzyme
not useful; R+M; a specific sequence is recognized by a multi-subunit protein, a random sequence is cut hundreds of bases away
Type III restriction enzyme
not useful; R; a specific sequence is recognized by a multi-subunit protein, a random sequence is cut ~25 bp away
Type II restriction enzyme
most useful; R; a specific sequence is recognized (4-8 bp palindrome) by a single protein and is cut within that sequence
Restriction enzymes recognize…. bp sequences
even… can’t have odd number because of palindromic sequence
Improper condition (restriction enzymes) may give…
star activity (relaxed specificity)
double digest
reactions use 2 enzymes at once (if they utilize compatible buffers or buffer can be changed)
Restriction digest components
- DNA (what we want to cut)
- buffer (to ensure optimal enzyme function)
- water (to get buffer to right conc.)
- enzyme (cut the DNA)
_________________ in DNA backbone give it a negative charge.
phosphate groups
agarose gel electrophoresis
(-) charged DNA + salt solution + electricity = DNA pulled to positive anode
agarose gel electrophoresis process
- agarose poured and cooled with comb
- gel immersed in buffer (TAE, TBE, SB)
- DNA+dye+glycerol placed in wells
- current applied
Supercoiled circles travel…
faster than their apparent size
Relaxed circles travel…
slower than their apparent size
lower agarose concentration
better for larger bands
