4D - Recombination

Question 1

why are plasmids good cloning vectors

Answer 1

they can self-replicate. small. can be taken up by bacteria.
easy to include antibiotic resistance genes, recognition sites, and expression signals.

Question 2

vector

Answer 2

 a means of introducing
foreign DNA into an organism.
Plasmids are a popular vector in bacterial transformation

Question 3

purpose of genetic modifications of bacteria

Answer 3

allows bacteria to produce useful human proteins like insulin, interferons etc

Question 4

why are plasmids used for making recombinanation

Answer 4

they are small,
self replicating
can be taken up by bacteria.
easy to introduce antibiotic resistance genes, recognition sites, expression signals.

Question 5

key components recombination of bacteria

Answer 5

gene of interest, plasmid vector, restriction endonuclease, and ligase

Question 6

gene of interest

Answer 6

dna sequence encoding desired bacteria - its isolated and amplified using PCR
inserted into vector
mustnt have introns
-> use synthetic dna or

Question 7

how is cdna made

Answer 7

copy dna made from reverse transcriptase enzyme which functions to transcribe mRNA backwards into the cDNA.

cDNA does not contain introns due to the absence of introns in the mRNA being reverse transcribed.

Question 8

plasmid vectors 4 imp dna sequences

Answer 8

restriction endonuclease sites
antibiotic resistance gene (ampR for ampicillin tetR for tetracycline)
origin of replication - signals the start site for dna replication in bacteria

reporter gene - easily identifiable phenotype - used to see if the GOI was taken up by the plasmid

Question 9

what 2 traits must a plasmid have to be used as a vector

Answer 9

2 genes that code for observable traits (antibiotic resistance or reporter genes like gfp (fluorescent protein))

one of which must contain the restriction site

Question 10

purpose of restriction endonuclease and dna ligase in recombination

Answer 10

goi and plasmid cut w same restriction enzyme
forms complementary sticky ends which can easily form H bonds.
ligase seals the SP backbone creating a recombinant plasmid

Question 11

heat shock

Answer 11

method of bacterial transformation
- bacteria and plasmids, placed in a calcium
ion solution on ice.

• positive calcium ions -> plasma membrane more
  permeable to the negatively charged plasmid DNA.
• solution heated to around
  37–42 °C for 25–45 seconds
• > returned to the ice.
  sudden change in
  temp -> plasma membrane even more permeable
  -> allows plasmid
  vectors to cross the phospholipid bilayer and enter the bacteria’s cytoplasm

Question 12

electroporation

Answer 12

electrical current is passed through a solution containing bacteria and plasmid vectors.
-> causes the plasma membrane to become more permeable,
allowing plasmid vectors to cross the phospholipid bilayer and enter the bacteria’s
cytoplasm

Question 13

explain antibiotic selection

Answer 13

transformed bacteria (with plasmids) are grown on antibiotic rich agar plates -> only transformed bacteria survive due t AB resistance genes in plasmids.

Question 14

what happens once bacteria is transformed

Answer 14

its cultured and induced to produce target proteins which are then purified for use in medicine andresearch

Question 15

explain what is meant by universal

Answer 15

human gene can be expressed by bacteria and its downstream protein can be produced, despite the gene coming from a different species.