Biotech - Recombinant DNA

Question 1

Recombinant DNA

Answer 1

This is the producto f insertion of a DNA fragment into a vector capable of independent replication in a host cell.

Question 2

Why is recombinant DNA useful?

Answer 2

Large quantities of the inserted DNA can be obtained by replication of the recombinant in a host.

Question 3

Lambda Phage

Answer 3

This is a tailed bacteriophage, with E.Coli its natural host

Question 4

What is the step after DNA replication in a host?

Answer 4

Isolation of the recombinant then isolation of the fragment itself by restriction endonuclease digestion, then GE.

Question 5

Why is Lambda Phage useful?

Answer 5

It can undergo transduction.

Question 6

Transduction

Answer 6

This is the transfer of genes from one bacteria to another, and is non-pathogenic to humans.

Question 7

Why are buffers used in RE cleavage?

Answer 7

To optimise the enzyme and ensure efficient proceeding of the reaction.

Question 8

How can cloning be used to udnerstand reaction enzyme catalyses?

Answer 8

Clone generated then excising the gene from the gene segment, atatch it to a vector and replicate the product in a microorganism

Question 9

What is Recominbant DNA generated by

Answer 9

RE and DNA ligases

Question 10

Function of RE and Ligases?

Answer 10

RE cleave DNA at specific sites
Ligases link DNA molecules together

Question 11

What is the mechanism of RE?

Answer 11

Recognise DNA sequences between 4-8 BP and cut dsDNA leaving one strand overhanging.

Question 12

PAR genes

Answer 12

(Protein Associated With RNA) are involved in a variety of cellular processes like cell polarity, mitosis and embryonic development.

Question 13

Where are PAR genese used in biotech?

Answer 13

Control localizationg and expression of recombinant proteins.

Question 14

Example of a PAR gene?

Answer 14

PAR-4 localises heterologous proteins in mammal cells so when fused with a protein of choice, protein can be targeted to specific locations.

Question 15

What can PAR genes be designed with?

Answer 15

Biocatalysts, that being biological systems speeding up chemical reactions.

Question 16

Bacteria Artificial Chromosomes

Answer 16

These are vectors accomodation DNA fragments up to 300kb

Question 17

What are the elements of BAC?

Answer 17

A selectable marker, a ORI site, a polylinker region and a BAC backbone with genes allowing maintenance in bacterial cells.

Question 18

What is the polylinker region in BAC used for?

Answer 18

Cloning of target DNA fragment containing many restriction enzyme recognition sites.

Question 19

Yeast Artifical Chromosomes

Answer 19

These can accomodate 2 MB fragments, containing same elements as BAC but with centromere and telomere.

Question 20

Transformation

Answer 20

This is DNA uptake by creating pores within the membrane by heat shock, electroporation or chemicals.

Question 21

What are the steps of transformation?

Answer 21

Insertion through membrane.
Donor DNA protected
DNA transportation to nucleus or nucleoid for integration into host genome.
DNA replication and expression.

Question 22

How does heat shock facilitate transfomraiton?

Answer 22

Increases membrane fluidity and creating temporary gaps, by disrupting lipi molecule interactions.

Question 23

How does electroporation facilitate transformation?

Answer 23

Create electrical potential difference disrupting lipid bilayer in dielectric breakdown.

Question 24

Dielectric Breakdown

Answer 24

This is a sufficiently strong electric field breaking down the insulating properties of an insulator to allow flow of charge.

Question 25

Transmembrane Potential

Answer 25

This is the difference in electric potential between interior/exterior of a membrane.

Question 26

What is the mechanism of electroporation in the membrane?

Answer 26

Hydrophilic head grooups allign to the electric field, disrupting the organisastion, by electrical field dipoles and dipole moment allignment.

Question 27

What causes the dipole in lipids?

Answer 27

Polar head group negative charge on one end and positive charge on the other., due to polar covalent bonds(OH and NH2

Question 28

How does Calcium Chloride promote membrane porosity?

Answer 28

Ca2+ binding negatively charged phosphate groups of membrane reducing repulsion between negative charged DNA.

Question 29

What is the importance of antibiotic resistance genes in Recombinant?

Answer 29

Positive selection for bacteria containing the vector.

Question 30

What does the LacZ gene do?

Answer 30

Reveal which cells contain plasmids through conversion of colourless molecules to blue products.

Question 31

Blue-White Screening

Answer 31

This is a method of identificaiton of recombinant bacteria through beta-galactosidase nezyme and lactose to glucose to galactose cleavge.

Question 32

What is the function of the LacZ gene?

Answer 32

When galactose is present, LacZ operon results in beta-galactosidase enzymes metabolising lactose

Question 33

How is blue-white screening set up?

Answer 33

Plasmids are ligated with LacZ gene, whilst hosts contain mutated version.
Gene contains RE site, which is split if vector is introduced

Question 34

How is it known that no vector insert has occured?

Answer 34

Functional LacZ gene produces functional enzymes

Question 35

How is Blue-White screening identification done?

Answer 35

Agar plate with a chromogen called X-gal

Question 36

What does B-Galactosidase do to Xgal?

Answer 36

Hydrolyses forming 5-bromo-4-chloro-indooxyl which spontaneously hybridises into an insoluble blue pigment

Question 37

When is the blue pigment produced?

Answer 37

Where failutre of vector insertion has occured