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BIOL215 > Week 2 L1 - Plasmids > Flashcards

Week 2 L1 - Plasmids Flashcards

1
Q

Structure of genomes - Prokaryotic vs Eucaryotic

A 
Prokaryotic cell:
* 1-5 μm
* usually circular, ds DNA (10^6-10^7bp)
* usually one chromosome
* 1-many plasmids
Eukaryotic cell:
* 50-100 μm
* linear, ds DNA (10^7-10^9 bp)
* many chromosomes
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2
Q

What is a plasmid?

A
  • Small molecules of DNA that replicate independently
  • Found naturally in bacteria and some yeasts
  • Double-stranded, circular DNA molecule
  • 1-200 kilobases (kb) in size
  • Capable of autonomous (self) replication
  • Can be infectious (self-transmissible via conjugation)
  • Can integrate into the main chromosome - episome
  • Have been modified for use in genetic engineering
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3
Q

What is the origin of replication (ori)?

A

Sequence needed for plasmid to replicate in the host.

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4
Q

What is the origin of transfer (oriT)?

A

Sequence needed for the transfer of the plasmid from one bacterium to another. Optional.

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5
Q

Two examples of various genes with promoters?

A

– Virulence genes

– Antibiotic resistance genes

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6
Q

Types pf plasmids?

A
  • Fertility (F) plasmids
  • Resistance (R) plasmids
  • Bacteriocin plasmids
  • Virulence plasmids
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7
Q

What’s a fertility plasmid?

A

Carry instructions for conjugation

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8
Q

What are resistance plasmids?

A

Carry genes for resistance to one or more antimicrobials

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9
Q

What are bacteriocin plasmids?

A
  • Carry genes for proteinaceous toxins called bacteriocins

* Bacteriocins can kill bacterial cells of the same or similar species that lack the plasmid

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10
Q

What are virulence plasmids?

A

Carry instructions for structures, enzymes or toxins that can enable a bacterium to become a pathogen (e.g. E. coli strains)

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11
Q

What are the three methods of transferring DNA between cells?

A
  • Transformation (produces transformants)
  • Conjugation (produces transconjugants)
  • Transduction (produces transductants)
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12
Q

What is transformation in regards to DNA transfer?

A

• Unidirectional transfer of extracellular DNA into cells – recipients are called Transformants
• Bacterial transformation was 1st observed by Frederick Griffith in 1928
• Bacterial species vary in their ability to take up DNA
• Cells that have the ability to take up DNA from their environment – COMPETENT
– Results from alterations in cell wall and cytoplasmic membrane that allow DNA to enter cell

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13
Q

What are the two types of transformation in regards to DNA transfer?

A

Two types:
1. Natural Transformation
• Bacteria are naturally able to take up DNA
• Example: pathogens in the genera of
– Bacillus
– Streptococcus
– Staphylococcus
2. Engineered Transformation
• Bacteria are altered to enable them to take up DNA (chemical treatment or electric shock)
– Competency and transformation are important tools in recombinant DNA technology/cloning.

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14
Q

What is conjugation in regards to DNA transfer?

A
  • A process in which there is unidirectional transfer of genetic information through DIRECT cellular contact between donor and recipient bacterial cell
  • Conjugation needs physical contact between donor and recipient bacterial cell
  • Recipients that have received a piece of donor DNA – Transconjugants
  • Conjugation is mediated by – conjugation pili
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15
Q

How and when was conjugation discovered? (Experiment by Joshua Lederberg and Edward Tatum).

A

Conjugation was discovered in 1946 (Joshua Lederberg and Edward Tatum)
• They studied two E. coli strains that differed in their nutritional requirements
– Strain A: met, bio, thr+, leu+, thi+
– Strain B: met+, bio+, thr, leu, thi
• Plated each strain individually, and had no colony growth (auxotrophic cells). Mixed the two together, and had growth of colonies (prototrophic colonies).
• NB. Minimal medium – contain the minimum nutrients possible for colony growth, generally without the presence of amino acids

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16
Q

What is auxotrophic?

A

An organism / cell that is unable to synthesise a particular organic compound required for its growth - they require to be on medium with the amino acid that they cannot produce.

17
Q

What is prototrophic?

A

Cells that are self-sufficient producers of the required amino acids for their growth. Can grow on any medium without the addition of these required amino acids.

18
Q

Which experiment proved that conjucation requires direct cell-cell contact?

A
  • U–tube experiment – Bernard Davis
  • Placed strains A and B in a liquid medium on either sides of the U– tube – separated by a filter with pores too small to allow bacteria to move through
  • Cells plated out on minimal medium
  • No protrotrophic colonies observed
  • Conclusion – cell to cell contact is a must for conjugation
19
Q

What did William Hayes show in regards to conjugation in 1953?

A
  • That genetic exchange in E. coli occurs only on one direction between donor and recipient
  • F factor (sex factor) – plasmid (donor cell - F+)
  • F- cells – lack the plasmid – recipient
  • F plasmid contains the gene coding for conjugation pili – hair like host cell surface components
20
Q

What 4 steps do cells undergo in regards to transfer of DNA through conjugation?

A
  • Donor cell attaches to a recipient cell with its pilus.
  • Pilus may draw cells together.
  • One strand of F plasmid DNA transfers to the recipient.
  • The recipient synthesises a complimentary strand to become an F+ cell with a pilus; the donor synthesises a complimentary strand, restoring its complete plasmid.
21
Q

What are Hfr strains in conjugation?

A

High frequency recombination strains.
• Some strains are able to transfer chromosomal genes, not just plasmid genes.
• Only occurs when the F plasmid integrates into the chromosome.
• Such strains known as Hfr strains (high frequency recombination).
• Integration is rare; occurs in only 1/10,000 cells. Reversions are similarly rare.
• Rare that all genes from donor enter the recipient - requires conjugal bridge to remain for ~100 min.

22
Q

What is an episome?

A

A portion of genetic material that can exist independent of the main body of genetic material (the chromosome) at some times, while at other times is able to integrate into the chromosome. Examples of episomes include insertion sequences and transposons. Viruses are another example of an episome.

23
Q

Plasmids and experiments

A

Plasmids that are used experimentally are called vectors. Researchers can insert DNA fragments or genes into a plasmid vector, creating a so-called recombinant plasmid. This plasmid can be introduced into a bacterium by way of the process called transformation.

24
Q

Recombinant plasmid uses?

A 
One of the most valuable tools available to the molecular biologist
• Used for:
– Gene cloning
– Genetic engineering
– Recombinant protein expression
• Can be used in:
– Bacteria
– Eukaryotic cells
– Plants
25
Q

Selectable marker

A

– Cells with the plasmid can be easily distinguishable from cells that lack the plasmid
– Typically it is a gene for resistance to an antibiotic

26
Q

Multiple cloning site (poly linker)

A

– Region of DNA containing several unique restriction sites

– For insertion of foreign DNA fragments (cloning)

27
Q

Restriction sites

A

Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes.

28
Q

Restriction enzymes

A

A restriction enzyme, or restriction endonuclease, is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites.

29
Q

What is recombinant DNA technology? Four main goals?

A

Intentionally modifying the genetic content of organisms for practical purposes.
Main goals:
• Eliminate undesirable phenotypic traits
• Combine beneficial traits of two or more organisms
• Create organisms that synthesize products that humans need
• Recombinant protein production.

30
Q

How to clone step-by-step

A
  1. Obtain bacterial cells and isolate the plasmids.
  2. Obtain a strand of DNA containing your gene of interest from a separate source, enzymatically cleave it into fragments, and isolate the fragments containing the gene of interest.
  3. Insert gene into plasmid.
  4. Insert gene-containing plasmid into bacterium.
  5. Culture bacteria.
  6. Can now either:
    - harvest copies of gene to insert into plants or animals, to eliminate undesirable phenotypic traits or create beneficial combination of traits;
    - harvest proteins coded by the gene to produce vaccines, antibiotics, hormones or enzymes.
31
Q

How to extract plasmids from cells.

A
  1. Alkaline lysis method
    • Rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA.
  2. Solution 2: NaOH and SDS (pH ~12)
    • Detergent and high pH, lyses the cells, denatures and precipitates chromosomal DNA
    • Plasmid DNA remains relatively soluble
  3. Solution 3: Na acetate (pH ~4.5)
    • Neutralizes the alkaline pH
    • Precipitates proteins and forms SDS-protein complex
    • Chromosomal DNA renatures and aggregates with proteins
  4. Centrifugation
    • Pellets the protein-chromosomal aggregates
    • Plasmids will be present in the supernatant
    • These plasmids can be precipitated with ethanol
  5. Analysis of DNA (plasmid) extracts is usually done by agarose gel electrophoresis.
    To read more, see https://askabiologist.asu.edu/alkaline-lysis
32
Q

How to extract gene of interest from DNA

A

Restriction Enzymes.
• Bacterial enzymes that cut DNA molecules only at restriction sites
– Categorized into two groups based on type of cut
• Cuts with ‘sticky’ ends (cut with BamHI)
• Cuts with ‘blunt’ ends (cut with smaI)

33
Q

Cloning with a plasmid that has the lacZ gene (example steps).

A
  1. Plasmid confers resistance to amplicillin and can make functional beta-galactosidase (the enzyme which cuts lactose into galactose and glucose). The plasmid contains a multiple cloning site in the lacZ gene.
  2. Restriction enzymes cut the multiple cloning site (polylinker).
  3. DNA fragments can be inserted into the MCS, disrupting the lacZ gene. Plasmid confers ampicillin resistance, but cannot make functional beta-galactosidase. Blue-white colony screening can be used here.
    • Bacterial cells are plated onto media containing ampicillin and X-gal
    • Only cells with the plasmid with ampR will grow
    Blue colony:
    – no DNA insertion
    – Functional β-galactosidase
    – Cleaves X-gal, generates blue/indigo precipitation in cells
    White colony:
    – DNA insertion
    – Non- functional β-galactosidase
    – Unable to cleave X-gal, generates white colonies.
34
Q

Describe the three essential features of a laboratory plasmid. What purpose does each of these features provide?

A

Plasmids have:

  • ori R (origin of replication) - a particular sequence in a genome at which replication is initiated.
  • selectable markers (antibiotic resistance) - a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection, eg. antibiotic resistance
  • unique restriction enzyme sites - sites on the plasmid that contain unique sequences that are only recognised by specific restriction enzymes.

BIOL215 (13 decks)

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