Lecture 27

Question 1

Plasmids

Answer 1

Orivan bacteria only. Naturally occurring extra chromosome pieces of DNA in bacteria to allow for horizontal gene transfer and easy gene removal

Question 2

Feature of plasmids

Answer 2

Lab ones are small, engineered. Has Oriv, antibiotic resistance gene, promoter site and multiple-cloning site. Success=selection under selection pressure, OriV for self-replication and transferable.

Question 3

OriV

Answer 3

Origin of vegetative replication i.e normal cell growth. Without it, bacteria won’t replicate plasmids.

Question 4

Overview

Answer 4

Isolate plasmid of interest, use restriction enzyme to cut plasmid, take target DNA and ligate with plasmid, insert back into bacterial cell.

Question 5

Plasmid DNA extraction

Answer 5

Alkali solution + detergent lyses bacterial cell membrane spilling contents out. Increase pH separates DNA strands. When solution is neutralised, chrom. DNA tangles as it is large with proteins becoming insoluble while plasmid DNA is able to rejoin to complementary strands. Plasmid DNA is purified.

Question 6

Cutting plasmid DNA

Answer 6

Use restriction enzymes to hydrolyse phosphodiester bonds between sugar-phosphate backbone. R.E o.g form defence in bacteria. Enzymes cut within palindromic sequence (exceptions obs). Restriction enzymes are produced by bacteria and purified using recombinant DNA technology. Different R.E cut different sequences and may cut different. Aim is to create over hang of 5’ called sticky ends or making a blunt cut. Sticky is better as it is more specific to target gene.

Question 7

Cloning site

Answer 7

Multiple cloning site is a sequence of bp which contains recognition site for restriction enzymes specific therefore, won’t appear anywhere else in plasmid.

Question 8

Ligation

Answer 8

Once cut in enzyme is made and DNA of interest is isolated, it must be inserted into plasmid. DNA ligase does this where once DNA has formed complementary bonds, it forms phosphodiester bonds.

Question 9

PCR tails

Answer 9

PCR tails are short, extra sequences added to the 5’ ends of primers during PCR. They often include restriction enzyme sites, which aren’t part of the original gene. After PCR, these tails allow restriction enzymes to cut the PCR product at known positions, creating sticky ends that match the plasmid.

Question 10

Competent cell

Answer 10

Cell that can take up new plasmids. Can make competent cells by adding CaCl2 and heat shock. Ca2+ decreases electrostatic repulsion and cold treatment primes membranes for destabilisation (becomes rigid) increasing effect of heat shock (creates large temp. differential). Rapid transition to fluid state temporarily creates disordered regions and transient pores.

Question 11

Separating transformed and non transformed cells

Answer 11

Plasmid with target gene have antibiotic resistant gene while those without target gene don’t therefore, grown on agar dish with antibiotics to kill non-transformed cells.

Question 12

Issue

Answer 12

Plasmid doesn’t take up correct sequence therefore.

Question 13

Transformed cell

Answer 13

Once that has taken-up proteins.