Genetic Engineering

Question 1

What are the 3 steps to genetic engineering?

Answer 1

1. isolate desired gene
2. insert into vector
3. transformation

Question 2

How do retroviruses uses RNA?

Answer 2

-as genetic material
-they convert RNA backwards to make DNA
-then inserts itself into the host cell nuclear DNA

Question 3

What is reverse transcriptase?

Answer 3

-to achieve DNA from RNA
-only makes a single strand
-once any DNA polymerase in the nucleus will complete the other strand

Question 4

What do you do if the mRNA for the desired protein cannot be isolated?

Answer 4

-cut DNA with restriction enzymes
-either staggered or straight

Question 5

What are staggered cut restriction enzymes?

Answer 5

-produce fragments with sticky ends
-easier to insert

Question 6

What is the easiest thing to add DNA to?

Answer 6

-prokaryotes
-as the DNA being added is not inside a nuclear membrane
-plasmids are simple molecules

Question 7

How can you open the bacterial membrane?

Answer 7

-partially denature the membrane with high temp + Ca ions
-electroporation

Question 8

What is electroporation?

Answer 8

-passing a small electric current through bacteria
-creating pores

Question 9

How would be able to tell if a prokaryote has been successfully modified?

Answer 9

-a gene given antibiotic resistance is often included
-successful transfers can be found by adding antibiotic
-bacteria accepted the desirable DNA will also be resistant and survive
= sample of successful prokaryote

Question 10

How can you insert genes into plants?

Answer 10

-electrofusion after removing the cell wall
-careful about osmosis
-using plasmid of a bacterium that infects plants
-tumefaciens is used as it causes a tumour of modified cells to form
-each can be cloned

Question 11

How can you insert genes into animals?

Answer 11

-very complex
-commercially sensitive
-insert mRNA that complements desired DNA sequence into retrovirus
-when it infects - it will deliver the mRNA and reverse transcriptase
-producing DNA that is inserted into nucleus

Question 12

Why is labelling transgenic organisms important?

Answer 12

-not every individual will successfully take up the modified DNA

Question 13

How is labelling done?

Answer 13

-adding the gene for GFP along with the gene being transferred
-successful organisms will make the target protein and the GFP = easily identified

Question 14

What happens during isolating a desired gene?

Answer 14

-either restriction endonucleases
-or reverse transcriptase

Question 15

What are restriction endonucleases?

Answer 15

-recognise a specific restriction site on the genome
-can produce sticky = exposed bases

Question 16

What happens during inserting into a vector?

Answer 16

-extract plasmid from bacteria/ use virus
-cut plasmid using the same RE at marker gene = produces same sticky ends

Question 17

What does recombinant DNA consist of?

Answer 17

-2 marker genes + desired gene
-DNA ligase form phosphodiester bonds between desired + plasmid
-blue marker + ampicillin

Question 18

What does a blue marker do?

Answer 18

-produces blue pigment
=blue colonies

Question 19

What does an ampicillin marker do?

Answer 19

-resistant gene
-can survive in a plate of ampicillin

Question 20

What happens during transformation?

Answer 20

-make bacterial membrane more permeable
-either Ca + heat shock, electroporation or electrofusion

Question 21

What is electrofusion?

Answer 21

-plasmid in a vesicle
-electric current
-creates a pore and joins them together = transgenic bacteria

Question 22

Why is it hard to mass produce transgenic bacteria?

Answer 22

-hard to control the success of plasmid taken up by desired gene

Question 23

What are the 3 possible outcomes of transgenic bacteria?

Answer 23

1. not successful in step 3
2. successful in step 3, but OG plasmid in uptake
3. successful in recombinant

Question 24

What happens if the bacteria has no plasmid?

Answer 24

-no ampicillin = dies
-doesn’t appear on plate

Question 25

What happens to bacteria with OG plasmid?

Answer 25

-blue pigment
=blue colonies

Question 26

What happens to a successful bacteria?

Answer 26

=white colonies
-cannot produce blue pigment

Question 27

What is genetic engineering?

Answer 27

-insertion of a DNA fragment into the DNA of another organism using a vector + host cell