Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MPharm 2 - PH2114 > Unit 3 - Biotechnology & Gene cloning > Flashcards

Unit 3 - Biotechnology & Gene cloning Flashcards

1
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is a protein?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the four levels of protein structure?

A

Primary
Secondary
Tertiary
Quaternary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the primary structure of a protein?

A

Sequence of amino acids in a chain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the secondary structure of a protein?

A

Local folding of the polypeptide chain into helices or sheets

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the tertiary structure of a protein?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is the quaternary structure of a protein?

A

Protein consisting of more than one amino acid chain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are used to produce macromolecules?

A 
Prokaryotes
- bacteria
Eukaryotes
- yeast
- cell culture
- transgenic plants
- tobacco
- transgenic animals
- goats
- sheep
- cows
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are the advantages of using prokaryotes to produce macromolecules?

A 
Easy to construct
Supports large scale production
Can express toxic proteins
No ethical issues
Low cost
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the advantages of using eukaryotes to produce macromolecules?

A

Can perform some forms of post-translational modification

- glucosylation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is a gene?

A

A sequence of DNA which encodes a protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

Genetic engineering

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Give some examples of therapeutic proteins made using genetic engineering

A

Insulin
Interferon
Monoclonal antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are chimeric proteins?

A

Proteins that do not exist in nature

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How are chimeric proteins made?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How is a therapeutic protein made?

A 
Cloning
Expression
Fermentation (mass production)
Cell lysis
Soluble/insoluble protein
Refolding
Purification
Characterisation
Activity assays, interaction analysis
17
Q

How is the target gene isolated and cloned?

A

1.Harvest a copy of the gene using restriction enzymes

  1. Amplify a copy using Polymerase Chain Reaction
    - PCR

3.Synthetically produce a copy

18
Q

What is PCR used for?

A

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.

19
Q

What is the mechanism of DNA ligase?

A

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

20
Q

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

A

CaP/heat shock

Electroporation

21
Q

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

A

Electroporation

DMSO

22
Q

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

A

CaP
Electroporation
Lipofectin

23
Q

What is electroporation?

A

Electrical shock makes cell membrane permeable to DNA

24
Q

How can clones containing the desired gene be selected for?

A

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

25
Q

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

A

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.

26
Q

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

A 
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
27
Q

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

A

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

28
Q

What are the four steps to producing a therapeutic protein?

A

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

29
Q

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

A 
Transfer to large scale expression host
Fermentation
- large scale production
Harvesting
- cell lysing
- soluble/insoluble proteins
- refolding
Purification
Characterisation
Activity assays, interaction analysis
30
Q

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

A

Grow
Lyse
Purify

31
Q

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

A 
Recombinant protein expression system
- seed stock
- provenance of starting material
Production media
Downstream processing
Production facilities
- dedicated production facilities
- bio-security
Costs
32
Q

How can recombinant proteins be harvested from bacterial host?

A 
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
33
Q

What can be used to lyse bacterial cells?

A

Enzymes
Sonication
French press

34
Q

What are inclusion bodies?

A

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

35
Q

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

A

Denature the protein

- 6M urea

36
Q

How do restriction enzymes work?

A

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.

37
Q

Describe the Polymerase chain reaction

A

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.

MPharm 2 - PH2114 (32 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions