Unit 3 - Biotechnology & Gene cloning

Question 1

What two types of molecules are there for options in drug synthesis?

Answer 1

Macromolecules
- producing therapeutic proteins
Small chemical entities (SCE) drugs
- organic chemistry synthesis

Question 2

What is a protein?

Answer 2

Polymer made up of smaller units called amino acids which are linked together in a specific sequence by peptide bonds

Question 3

What are the four levels of protein structure?

Answer 3

Primary
Secondary
Tertiary
Quaternary

Question 4

What is the primary structure of a protein?

Answer 4

Sequence of amino acids in a chain

Question 5

What is the secondary structure of a protein?

Answer 5

Local folding of the polypeptide chain into helices or sheets

Question 6

What is the tertiary structure of a protein?

Answer 6

Three dimensional folding pattern of a protein due to side chain interactions

Question 7

What is the quaternary structure of a protein?

Answer 7

Protein consisting of more than one amino acid chain

Question 8

What are used to produce macromolecules?

Answer 8

Prokaryotes
- bacteria
Eukaryotes
- yeast
- cell culture
- transgenic plants
- tobacco
- transgenic animals
- goats
- sheep
- cows

Question 9

What are the advantages of using prokaryotes to produce macromolecules?

Answer 9

Easy to construct
Supports large scale production
Can express toxic proteins
No ethical issues
Low cost

Question 10

What are the advantages of using eukaryotes to produce macromolecules?

Answer 10

Can perform some forms of post-translational modification

- glucosylation

Question 11

What is a gene?

Answer 11

A sequence of DNA which encodes a protein

Question 12

How can a gene be inserted into a bacterial or eukaryotic cell so that it is expressed?

Answer 12

Genetic engineering

Question 13

Give some examples of therapeutic proteins made using genetic engineering

Answer 13

Insulin
Interferon
Monoclonal antibody

Question 14

What are chimeric proteins?

Answer 14

Proteins that do not exist in nature

Question 15

How are chimeric proteins made?

Answer 15

Created by joining two genes to make a single gene which encodes a protein with functional properties derived from each of the original proteins

Question 16

How is a therapeutic protein made?

Answer 16

Cloning
Expression
Fermentation (mass production)
Cell lysis
Soluble/insoluble protein
Refolding
Purification
Characterisation
Activity assays, interaction analysis

Question 17

How is the target gene isolated and cloned?

Answer 17

1.Harvest a copy of the gene using restriction enzymes

2. Amplify a copy using Polymerase Chain Reaction
   - PCR

3.Synthetically produce a copy

Question 18

What is PCR used for?

Answer 18

To make a copy of the gene sequence
It also allows you to incorporate restriction sites at either end of the sequence.

This means you can generate any restriction sites you want.

Question 19

What is the mechanism of DNA ligase?

Answer 19

To form two covalent phosphodiester bonds between 3’ hydroxyl ends of one nucleotide with 5’ phosphate ends of another
- ATP is required for the ligase reaction

Question 20

What different methods can be used to introduce a vector and gene into a host bacterium?

Answer 20

CaP/heat shock

Electroporation

Question 21

What different methods can be used to introduce a vector and gene into a host yeast?

Answer 21

Electroporation

DMSO

Question 22

What different methods can be used to introduce a vector and gene into a host mammalian cells?

Answer 22

CaP
Electroporation
Lipofectin

Question 23

What is electroporation?

Answer 23

Electrical shock makes cell membrane permeable to DNA

Question 24

How can clones containing the desired gene be selected for?

Answer 24

Loss of Antibiotic resistance
Nutrient utilisation-colour change
Screen for the physical presence of the gene
Screen for expression of the gene product

Question 25

Give an example of how a change of colour confirms insertion of a gene

Answer 25

LacZ gene for B-galactosidase inserted into plasmid X-gal turns blue upon digestion - modified galactose molecule Blue colonies produced - have taken up LacZ gene for B-galactosidase Destroy the ability of the gene to make the enzyme and therefore the colonies can't produce the enzyme and are white.

Question 26

How can the physical presence of a gene be screened for?

Answer 26

``` Cloned gene inserted into plasmid Plasmid cut with Sal 1 Plasmid cut with EcoR1 Plasmid cut with Sal 1 and EcoR1 Electrophoresis carried out to show bands ```

Question 27

How can the expression of the gene product be screened for?

Answer 27

Detect expression of protein on basis of size Specific antibodies which recognise the protein - Western blot Detect the presence of expression tags which have been added to the protein during cloning process Detect the biological activity - of the recombinant protein - enzymatic activity - of the expression tag

Question 28

What are the four steps to producing a therapeutic protein?

Answer 28

Transformation - double stranded recombinant plasmid DNA introduced into bacterial cell Selection - isolate bacteria containing target DNA Propagation - cell culture produces hundreds of millions of new bacteria Transfer to large scale expression host - many copies of purified plasmid isolated from lysed bacteria

Question 29

What are the stages in the production phase of production a therapeutic protein?

Answer 29

``` Transfer to large scale expression host Fermentation - large scale production Harvesting - cell lysing - soluble/insoluble proteins - refolding Purification Characterisation Activity assays, interaction analysis ```

Question 30

What are the three steps in the manufacturing process of therapeutic proteins?

Answer 30

Grow Lyse Purify

Question 31

What factors need to be considered when developing a large scale production process?

Answer 31

``` Recombinant protein expression system - seed stock - provenance of starting material Production media Downstream processing Production facilities - dedicated production facilities - bio-security Costs ```

Question 32

How can recombinant proteins be harvested from bacterial host?

Answer 32

``` Secreted proteins - easy to purify Soluble proteins trapped inside cell - release by cell lysis Proteins can form insoluble inclusion bodies - release by cell lysis - refold as soluble protein ```

Question 33

What can be used to lyse bacterial cells?

Answer 33

Enzymes Sonication French press

Question 34

What are inclusion bodies?

Answer 34

Over expression of recombinant proteins in E.coli can lead to the formation of insoluble structures

Question 35

How can therapeutic proteins be recovered from inclusion bodies in bacteria?

Answer 35

Denature the protein | - 6M urea

Question 36

How do restriction enzymes work?

Answer 36

Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences. Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs. However, some produce blunt ends.

Question 37

Describe the Polymerase chain reaction

Answer 37

Heat the DNA to separate into two strands. Add a primer which will stick to their complimentary DNA when the sample has cooled reheat the sample to a slightly lower heat than before and use DNA polymerase(taq polymerase) to add Complimentary DNA nucleotides to the strands.