Topic 9: Microbial Genetics Flashcards

1
Q

Reasons why bacteria are ideal genetic research tools

A
  • Single chromosome and plasmids
  • One chromosome for easy detection of mutations
  • In early studies, nutritional mutants were used
  • Allowed study of one gene based on its inability to use or produce a particular nutrient
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Wild Type

A
  • Strains of bacteria or archaea that are essentially same as when originally isolated from nature
  • It is from wild type strains that mutants are generated for further analysis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Mutant

A

-Strain derived from wild type that contains mutation
- Named after phenotype, always in comparison to phenotype of original environmental strain
- Have genetic change that disrupts or alter the function that was associated with the wild type

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Alleles

A
  • Refer to gene variant associated with the mutant strain
  • Can be gain of function, where a mutant can do a function the wild one couldn’t (ex: antibiotic resistant mutant vs antibiotic-sensitive wild type)
  • Or, can be loss of function alleles (ex: antibiotic resistant wild type, antibiotic sensitive mutant)
  • Change of function alleles (ex: yellow colonies for wild type and red colonies for mutants)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Auxotrophs

A
  • Specific type of strain (either wild or mutant) that can’t make an organic compound that is required for growth
  • Most common example of auxotrophic mutants are those that can’t make specific amino acids
  • Also include strains that can’t make individual nucleotides of vitamins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Prototroph

A
  • Related strain that is capable of producing the organic compound that auxotroph cant
  • Usually parental strain that gave rise to the auxotrophic mutant
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Genotype

A
  • description of alleles within an organism, generally reflects differences from wild-type
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Phenotype

A
  • observable properties of a strain, ignores what genes are involved
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Selection and Screening

A

Screening is more tedious than selection.

Phenotypic Selection:
- Used for selectable mutations (i.e., mutants grow under conditions that would kill wild-type cells)
- Useful in genetic research (e.g., to find antibiotic-resistant strains)

Screening:
- Used for nonselectable mutations (i.e., mutants that do not have a growth advantage over wild-type cells)
- Large numbers of colonies must be screened (tedious)

Screening by comparing plates:
- Duplicate plates are created and one lacks a particular nutrient
- a mutation has occurred where a colony grows on the full support plate but doesn’t on the partial support plate

Screening on a single plate:
- Use differential media to look for a difference in colour

Replica Plating and Patching
- Increase the throughput of colonies screened for mutants of interest by “copying” cells from a master plate onto differential media with either a wooden block (replica plating) or by robotic or manual colony transfers (patching, colonies on gridded plate, compare two)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Restriction and Modification Enzymes

A
  • Restriction enzymes cut DNA at a specific recognition site.
  • Recognition sites are usually palindromic
  • Similar ends of cut DNA can be paired together and ligated
  • Restriction enzymes are always paired with modification enzymes
  • Often in a single operon
  • Recognize the same site as the paired restriction enzyme
  • Methyltransferase activity protects DNA from restriction enzyme activity.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Explain how plasmids can be used to clone DNA for screening or selection

A
  • Restriction enzymes allow researchers to stitch together fragments of useful DNA into recombinant molecules
    – Recombinant molecules can be used to clone a bacterial gene of interest
  • Vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell:
    – Plasmid vectors
    – Phage vectors
    – Cosmids
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Plasmid Cloning Vectors

A
  • Plasmid Cloning Vectors were first used in the 1970s by Cohen at Stanford.
  • He cut fragments from two plasmids carrying antibiotic resistance genes with the same restriction enzyme
  • The transformed strain exhibited traits from both plasmids
  • Today plasmid cloning vectors are engineered with desirable plasmid traits for easier gene cloning:
    — Origin of replication
    — Selectable marker gene
    — Multiple cloning site
    — Small size
    — High copy number

Alternate hosts
- Certain host cell types may be restricted by the origin of replication
- Shuttle-vector plasmids have multiple types of origins, which expands the range of host cell types the plasmids can be inserted into.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Phage Vectors

A
  • Mix viral DNA with fragment of interest
  • Lysogenic lambda phage can carry ~20-kb fragments
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Cosmids

A
  • Phage genomes that omit nearly all the phage DNA, leaving more room for the fragment
  • critical phage cos packaging recognition sites remain
  • Other elements include a multiple cloning site and an antibiotic selection marker
  • Cosmids can typically carry 35‒45 kb fragments
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Transformation

A
  • Introduction of extracellular DNA directly into an organism
  • Doesn’t require cell-to-cell contact
  • Some bacteria are naturally competent for transformation, take directly from environment
  • Other bacteria can be artificially induced to become competent by:
    — Treatment with calcium cations
    — Electroporation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Conjugation

A

Conjugation: the transfer of DNA from cell to cell via direct contact/sex pilus formation.

Mechanism of Conjugation
– F plasmid carries the gene to form sex pilus “bridge” between two cells
– F plasmid can be copied and sent across the bridge (origin of transfer first) into a recipient cell
– Turns an F– cell into an F+ cell

Hfr Strains
– The F plasmid integrates into the host chromosome by homologous recombination, thus it is an episome (DNA that can integrate into the chromosome or exist autonomously).
— Creates new opportunities to pass genetic information
– Incorporated F plasmid sends the host cell DNA next to its incorporation site across the sex pilus.
— Can be used to “map” the location of genes in the host chromosome
— Generation of F′ plasmids

Triparental Conjugation
– Conjugation can still occur using recombinant plasmid lacking the required tra gene and a helper plasmid with the gene.

17
Q

Transduction

A
  • Virus accidently packages a fragment of host cell DNA
  • Virus delivers random host genome fragment instead of viral DNA to the next cell
  • Virus is usually unable to replicate because it lacks the viral genome
  • Few transducing phage when they occur
  • Homologous recombination must occur to integrate into a recipient genome
18
Q

Specialized Transduction

A
  • Lysogenic bacteriophage integrates at specific site
  • Virus excises incorrectly, including some host genome
  • All virus particles are transducing phage following lysis
  • Homologous recombination must occur to integrate into a recipient genome
19
Q

explain the following conjugation terminology: F+, F−, F′, Hfr, tra, oriT

A

F+: F-plasmid found autonomously within the cell

F-: cell that does not contain a F-plasmid

F′: an incorporated F plasmid excises itself by homologous recombination

Hfr: high frequency of recombination
- F-plasmid is found incorporated into the chromosome
- can then transfer the entire chromosome over the mating bridge during conjugation (from a donor to a recipient)
- Can have multiple insertion sites dependant on homology
- Can produce F’ bacteria, which can conjugate
- Can be used to map the location of genes in the host chromosome

Tra: encode all of the proteins needed for conjugation

oriT: origin of transfer, directional

20
Q

explain how mobile genetic elements can mobilize DNA through replicative and non-replicative transposition

A

Replicative transposition:
- Copies the element and moves the copy to another location
- Has res site and resolvase gene

Non-replicative transposition:
- Cuts and pastes the element into a new location

21
Q

Application of Transduction

A
  • Historically, co-transduction frequency was used to map bacterial genomes; genes that were closer to a known “marker” gene would be transduced with that marker more frequently than ones farther away
  • Can also be used to modify bacteria