Lecture 21 + 22

Question 1

conjugative plasmids

Answer 1

transmitted during conjugation, carry a variety of info

Question 2

resistance plasmids (R)

Answer 2

protect against the environmental factors, MDR (multiple drug resistance) plasmids

Question 3

Colicinogenic plasmids (Col)

Answer 3

codes for proteins that kill other microbes

Question 4

Degradative plasmids

Answer 4

contain genes for novel catabolic enzymes

Question 5

Virulence plasmids (vir)

Answer 5

increases the pathogenicity of a bacteria (e.g. toxins)

Question 6

Why are plasmids useful for us?

Answer 6

Putting a gene into a plasmid may allow that gene product (protein) to be expressed at high levels

Question 7

vector

Answer 7

a plasmid that has been streamlined and modified to make it amenable to carrying a payload of DNA

Question 8

components of vectors

Answer 8

• 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)

Question 9

Foreign DNA is degraded by….

Answer 9

cutting at specific sequences (endogenous/original DNA is not)

Question 10

R/M system

Answer 10

Host DNA is modified by methylation at a specific sequence, unmethylated DNA is restricted (cut) at the same sequence

Question 11

EcoRI restriction endonuclease

Answer 11

Cuts within a palindromic sequence only if unmethylated, leaving staggered cuts (sticky ends)

Question 12

Type I restriction enzyme

Answer 12

not useful; R+M; a specific sequence is recognized by a multi-subunit protein, a random sequence is cut hundreds of bases away

Question 13

Type III restriction enzyme

Answer 13

not useful; R; a specific sequence is recognized by a multi-subunit protein, a random sequence is cut ~25 bp away

Question 14

Type II restriction enzyme

Answer 14

most useful; R; a specific sequence is recognized (4-8 bp palindrome) by a single protein and is cut within that sequence

Question 15

Restriction enzymes recognize…. bp sequences

Answer 15

even… can’t have odd number because of palindromic sequence

Question 16

Improper condition (restriction enzymes) may give…

Answer 16

star activity (relaxed specificity)

Question 17

double digest

Answer 17

reactions use 2 enzymes at once (if they utilize compatible buffers or buffer can be changed)

Question 18

Restriction digest components

Answer 18

• DNA (what we want to cut)
• buffer (to ensure optimal enzyme function)
• water (to get buffer to right conc.)
• enzyme (cut the DNA)

Question 19

_________________ in DNA backbone give it a negative charge.

Answer 19

phosphate groups

Question 20

agarose gel electrophoresis

Answer 20

(-) charged DNA + salt solution + electricity = DNA pulled to positive anode

Question 21

agarose gel electrophoresis process

Answer 21

• agarose poured and cooled with comb
• gel immersed in buffer (TAE, TBE, SB)
• DNA+dye+glycerol placed in wells
• current applied

Question 22

Supercoiled circles travel…

Answer 22

faster than their apparent size

Question 23

Relaxed circles travel…

Answer 23

slower than their apparent size

Question 24

lower agarose concentration

Answer 24

better for larger bands

Question 25

higher agarose concentration

Answer 25

better for small pieces

Question 26

restriction mapping

Answer 26

used to grossly identify/verify DNA pieces of interest; using a series of single and double digests to understand locations of restriction sites relative to each other on a DNA fragment

Question 27

map

Answer 27

The positions of cut sites relative

to one another or known gene/sequence locations

Question 28

strategies for making a map

Answer 28

• determine if it’s linear or circular
• make single digest maps then combine them
• digests with fewer bands are easier to map first
• compare single and double digest sizes to determine the identity of the bands
• keep track of all sizes/distances

Question 29

reptation

Answer 29

• above ~20kb DNA fragments will not separate

- fragments up to 1Mb can be resolved

Question 30

pulsed field gel electrophoresis

Answer 30

alternates the direction of movement to counter reptation

Question 31

PFGE Application

Answer 31

• cut bacterial chromosomal DNA with rare enzyme

- compare fragments to create strain-specific patterns

Question 32

pulsenet

Answer 32

surveys and tracks food borne illnesses; illnesses studied using PFGE to track strains and look for clusters or outbreaks

Question 33

Core of molecular biology and cloning experiments

Answer 33

manipulating DNA