Week 2 L1 - Plasmids Flashcards

Question

Selectable marker

Answer 1

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

Answer 2

– Region of DNA containing several unique restriction sites | – For insertion of foreign DNA fragments (cloning)

Answer 3

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.

Answer 4

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.

Answer 5

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.

Answer 6

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.

Answer 7

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

Answer 8

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)

Answer 9

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.

Answer 10

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.

Week 2 L1 - Plasmids Flashcards

(34 cards)