Genetics

Question 0

Direct selection

Answer 0

Mutants can grow
Observe after simple plating on specific medium

Antibiotic resistance
Ability to use specific nutrient (ex lactose)
Ability to synthesize nutrient (separate slide)
Morphology (LPS, pili, flagellin -> colony shape)

Question 1

Types of mutations

Answer 1

Silent (redundant code or conservative/similar amino acid)
Point -> missense or nonsense
- transition = purine->purine, etc
- transversion = purine -> pyrimidine, etc
Deletion, insertion, frameshift
Reversion - restores phenotype
- true - reverse of original mutation
- suppressor - restores phenotype but not genotype

Question 2

Nutrition selection

Answer 2

Prototroph - can synthesize necessary metabolites
- grows on minimal medium (C, N, P only)
Auxotroph - missing enzyme, requires supplementation (ex Arg auxotroph)

Can assess complementation (order of enzymes)
ex Missing enzyme #3 -> can make earlier precursors -> diffuse to bordering areas that are missing earlier enzymes -> robust growth

Can select with penicillin (kills ONLY growing cells = prototrophs)
Minimal medium + penicillin -> prototrophs killed -> grow autotrophs on enriched medium

Question 3

Selection of antibiotic resistance

Answer 3

Can prove spontaneous generation by replica plating
-> same colonies appear on non-selective and antibiotic medium
(Darwinism > Lamarkism)

Can also isolate colonies by direct plating onto antibiotic medium

Question 4

Conditional mutants

Answer 4

Only mutant under certain circumstances

Temperature sensitive
- “permissive” low temp - protein folds correctly
- non-permissive high temp -> abnormal folding -> loss of fx
Can include mutations in essential genes (no growth at high temp)

Question 5

Types of recombination

Answer 5

Homologous (normal repair mechanism, high frequency, conserved)

Site-specific - small region of homology + recombinase
- ex lysogenic lambda bacteriophage

Illegitimate - no homology, random, low frequency
- ex transposition

Question 6

Population genetic study

Answer 6

Most bacteria cannot be cultured

• require symbiosis
• fastidious conditions

Study via genetics - ex “small subunit RNA” - 16S
- generic probe PCR -> array or sequencing

Question 7

Whole genome sequencing

Answer 7

Only identification for intracellular and difficult to culture

Short genome! 4-5 kB with 4k-5k genes
- selected for speed of replication = no junk

Question 8

Bacteriophage

Answer 8

Virus that infects bacteria
Either RNA or DNA, variety of structures
Specific for organism (capsid binding)

Either lytic or lysogenic or temperate (either)

Grow on active bacterial plate -> plaques
Morphology, size help identify

Question 9

Virulent phage

Answer 9

aka lytic

Adsorption -> introduction -> transcription -> replication of DNA, translation of capsid proteins -> packaging -> lysis and release
-> clear plaques (kills cells)

Not binary fission (duh)

Question 10

Lambda phage

Answer 10

Temperate - either lytic or lysogenic
Turbid plaques - some lysis but not all

Lysogenic phase:
Circularizes -> site specific recombination -> prophage
(represses lytic genes -> replication in genome)
Host cell stress -> excises -> lytic phase

Question 11

Complementation

Answer 11

Multiple genes in same biosynthetic pathway -> same phenotype

Co-infect to test number of different genes

• > rescue phenotype = different genes/groups
• > absent phenotype = same gene/group

Question 12

Restriction enzymes

Answer 12

Bacterial protection -> cleave foreign DNA
Palindromic sequences
Modify their own DNA to resist cleavage (EcoR1 methylase)

Also used for lab recombination

Question 13

Transposons

Answer 13

Mobile genetic elements
Integrated into multiple areas
- nonhomologous (illegitimate)
- requires insertion sequence element - ex transposase enzyme
Can also carry antibiotic resistance, toxins
May replicate during transfer -> spread via plasmids
May also disrupt host genes

Question 14

Transformation

Answer 14

Uptake of naked DNA
 -> integration to homologous chromosome
 -> autonomous plasmid
Wide range of bacteria
 - can increase "competence" via electroporation, CaCl2

Question 15

Generalized transduction

Answer 15

Random uptake of host DNA into phage
-> homologous recombination with recipient
(does not rely on integration of phage DNA)

Can select for integration of antibiotic or metabolic markers
Ex - distance measured by co-transduction of genes

Question 16

Specialized transduction

Answer 16

Messy excision of integrated phage DNA ->
uptake of nearby DNA -> recipient cells -> homologous integration
(Also carry phage DNA, can become permanent phage genome)

Must be a temperate (integrated) phage (ex lambda)
Can carry toxins (ex Shigella)

Question 17

Plasmid structure

Answer 17

Circular, supercoiled, double strand
Small to very large (400 kb)
Extrachromosomal (can separate with centrifuge)

Origin of replication - autonomous
Control copy number via encoded repressor mechanisms
(same repressor mechanism -> can’t grow in same cell = “incompatibility group)

Question 18

Plasmids and resistance

Answer 18

“R” plasmid = type of F with antibiotic resistance
Rtf = resistance transfer factor
- R plasmid with IS sequences -> insert into other Rtf’s -> multiple resistance

Selection only if antibiotic exposure (otherwise slows replication)
Can duplicate -> highly resistant if shortened course
Can transfer beween species

Question 19

Conjugation

Answer 19

Sexual cell-cell transfer
F+ donor -> pilus/bridge, transfer genes (tra), origin of transfer

Hfr = high-frequency transfer
Includes insertion sequences -> integration into host genome -> can carry host genes after excision (F’ plasmid)

Mapping - constant origin of transfer and directionality
- interrupt transfer -> map sequence of phenotypes