Recombinant DNA and Cloning Vectors

Question 1

What are the most commonly used extra-chromosomal genetic elements?

Answer 1

Plasmids

Question 2

Where do extra-chromosomal genetic elements originate from?

Answer 2

A natural source but are manipulated to use in microbiology

Question 3

What are the recombinant vectors that are used?

Answer 3

• Plasmids
• Phages
• Viruses
• Artifical Chromosomes

Question 4

Where are plasmids found?

Answer 4

In many but not all bacteria

Question 5

Where are phages found?

Answer 5

Lambda - bacterial viruses

Question 6

What are phages used for?

Answer 6

They are used as therapeutic tools to treat infectious diseases

Question 7

What are examples of viruses used as recombinant vectors and what are each used for?

Answer 7

Non-primate lentiviruses - used to integrate DNA in mammalian cells
Baculoviruses - used in combination with recombinant expression in insect cells

Question 8

What artificial chromosomes are used and what for?

Answer 8

Yeast artifical chromosomes - YAC for introducing large segments DNA

Question 9

Why are plasmids essential for recombination?

Answer 9

• Discrete circular dsDNA molecules
• Means by which genetic information is maintained
• Genetic elements (replicons) that exist and replicate independently of the bacterial chromosomes so extra-chromosomal
• Normally exchanged between bacteria within a restricted host range

Question 10

What are vectors?

Answer 10

Cut down version of naturally occuring plasmids

Question 11

What are vectors used for?

Answer 11

Used as molecular tools to manipulate genes

Question 12

How can a plasmid be modified?

Answer 12

It can be used and modified to allow us to introduce a foreign DNA into the plasmid and then it will be maintained as the plasmid replicates within the cell. It can be modified to express a protein or be tagged to look at how a particular protein works within the cell.

Question 13

What are the important features of plasmid vectors?

Answer 13

1. Can be linearised at one or more sites in non-essential stretches of DNA
2. Can have DNA inserted into them
3. Can be re-circularised without loss of the ability to replicate.
4. Modified to replicate at high multiplicity (copy number) within a host cell
5. Contain selectable markers
6. Are relatively small 4-5kb in size

Question 14

Where are restriction sites found in the bacterial plasmid DNA?

Answer 14

Found at the portion that is “non-essential”

Question 15

What is formed in the plasmid and what does this do?

Answer 15

A cloning site is introduced to the plasmid and this will introduce restriction enzymes as it will contain multiple restriction sites.

Question 16

How are recombinant proteins made from recombinant DNA?

Answer 16

The transduce bacteria is where the plasmids will replicate and be maintained. This will isolate that which will express the recombinant gene. This can be used to produce recombinant proteins in bacteria.

Question 17

What are recombinant proteins needed for?

Answer 17

To investigate their properties

To develop and produce therapeutics

Question 18

Why are plasmids used as a recombinant tools?

Answer 18

• Expression of a recombinant gene in a living organism of choice
• Add or modify control elements
• Alter the properties of the gene product
• Make it useful as a therapeutic

Question 19

How are recombinant proteins used clinically?

Answer 19

Recombinant proteins or peptides constitue about 30% of all biopharmaceuticals.

• Human insulin
• Interferons
• Erythryopoietin
• Factor XIII
• Tissue plasminogen activator (TPA)

Question 20

What is an increasingly important drug class?

Answer 20

Recombinant antibodies

Question 21

What are the requirements for a plasmid in the prokaryotic system?

Answer 21

• Ability to replicate in bacteria
• Maintained at high copy number
• Selectable contains an antibiotic marker
• Easy to manipulate

Question 22

What control elements are required for expression in bacteria?

Answer 22

• The coding sequence
• Shine-Dalgarno sequence
• Promoter
• Transcriptional terminator

Question 23

What is the coding sequence?

Answer 23

The part of the gene coding for the protein not including the UTRs nor any intronic or regulatory sequences such as a promoter enhancers.

Question 24

What is the shine-dalgarno sequence?

Answer 24

The ribosomal binding site found around 8 nucleotides before the start codon in the RNA in prokaryotes.

Question 25

What is the promoter?

Answer 25

The gene element that is involved in regulation and initiation of transcription.

Question 26

What is the transcriptional terminator?

Answer 26

A sequence that terminates transcription and initiates the dissociation of transcription.

Question 27

What is a constitutive promoter?

Answer 27

Promoter that is always on.

Question 28

What is a constitutive promoter used for?

Answer 28

It allows a culture of cells to express the foreign proteins to a high level.

Question 29

When should a constitutive promoter not be used?

Answer 29

It is fine if the protein isn't toxic to E.coli. It is a bad idea if there is no cells that would be able to survive and produce a recombinant protein.

Question 30

What is a inducible promoter?

Answer 30

It is a molecular switch

Question 31

What is a inducible promoter used for?

Answer 31

It allows large cultures to be grown without expressing the foreign protein. It induces a response to a defined signal.

Question 32

What do inducible promoters use?

Answer 32

Transcriptional repressors

Question 33

What is lac Operator?

Answer 33

It is used by a inducible promoter that is de-repressed by addition of lactose mimic IPTG.

Question 34

Describe structure of lac operon

Answer 34

Comprises of genetic elements that in prokaryotes include some regulatory sequences one of which is the lac operator and a gene that lac repressor (inhibitor).

Question 35

What does the lac operon system allow?

Answer 35

It allows bacteria to be responsive to low glucose environments and switch to lactose as a carbon source.

Question 36

How is any gene regulated by the lac operon?

Answer 36

This system to regulate any gene by placing a lac operator (lacO) upstream of its transcriptional start.

Question 37

Requirements for gene regulation

Answer 37

- Copy of the coding sequence - Must contain the start codon to and including the stop codon - Easy to manipulate

Question 38

Why are some proteins best made in eukaryotes?

Answer 38

- Many pharmacologically useful proteins are heavily modified and will not be appropriately processed in bacteria. - Solution is to express them in a eukaryotic system

Question 39

How is the effect of a defective gene in a cell culture system studied?

Answer 39

- Express the protein in human embryonic fibroblasts

Question 40

What is used to study the effect of a defensive gene?

Answer 40

- Inducible Promoter - Shine-Delgarno - Insert with in frame start and stop codon - Transcriptional terminator - Origin of replication - Selectable marker - Choice of unique Restriction sites MCS

Question 41

What problem can be encountered when studying for a defective gene?

Answer 41

- Bacterial promoter doesn't work - Shine-Delgarno sequence isn't recognised - Transcriptional start is not recognised, no cap site - No polyadenylation signal - Termination of transcription not recognised by eukaryotic Polymerase II - Origin of replication doesn't work

Question 42

Compare prokaryotic and eukaryotic expression vectors

Answer 42

Prokaryotic: - Contains a promoter - Shine-dalgarno sequence - ORF (common in humans rare in E.coli - codon preference) - Terminator Eukaryotic: - Promoter - Kozak sequence - ORF - Introns can be tolerated but aren't necessary - Poly-A signal in 3' UTR - Terminator

Question 43

What happens to plasmids?

Answer 43

They transfect into the eukaryotic system

Question 44

How are plasmids grown up in bacteria?

Answer 44

- Selectable bacterial marker | - Maintained at high copy number

Question 45

What are the changes that occur in a prokaryotic vector?

Answer 45

- Substiution of promoter with a eukaryotic promoter - Introduce a 3' UTR containing polyadenylation signal - Terminator substituted with a eukaryotic transcriptional terminator

Question 46

What is a transgenic cell line (transient or stable expression)?

Answer 46

- Ability to replicate mammalian cells or integrated in the chromosomes - A selectable marker in eukaryotes is needed

Question 47

Describe the structure of the eukaryotic/prokaryotic hybrid vector

Answer 47

Contains: - Eukaryotic promoter RSV, CMV - Choice of unique Restriction sites MCS with Kozack consensus - Transcriptional terminator and polyadenylation signal BGH - 2nd Eukaryotic promoter SV40 - Eukaryotic selectable marker - Transcriptional terminator and polyadenylation signal SV40 - Bacterial origin of replication - Bacterial selectable marker ampR

Question 48

Why are viral promoters commonly used?

Answer 48

They are used in eukaryotic expression systems because they are more compact and simpler to manipulate

Question 49

What is the combined transcriptional terminator and polyadenylation signal used?

Answer 49

3'UTR of Bovine Growth Hormone (BGH)

Question 50

When are selectable markers used and give an example of one

Answer 50

G418, one of many, but must be constructed with eukaryotic promoter polyadenylation signal and terminator and SV40 virus sequences

Question 51

Why can functional analysis of a protein not occur?

Answer 51

The expressed protein was very hard to obtain in a pure enough form from the bacteria

Question 52

What are the two of the most popular 3' gene fusions protein tags?

Answer 52

6 Histidines and Glutathione S transferase (GST)

Question 53

Where are 3' gene fusions found?

Answer 53

Made at either end of the coding sequence either before the stop codon or after the start

Question 54

Explain the steps of forming recombinant proteins from recombinant DNA

Answer 54

- Insert recombinant plasmid DNA into the recombinant expression vector - Transform into E.coli - Forms colonies on agar plate containing antibiotic - Isolate colonies; confirm insert and culture - Induce and purify protein - Purify protein on affinity column (separates tagged protein) - Elute

Question 55

How is the localisation and trafficking of the protein in a cell culture system studied?

Answer 55

By expressing the protein in human embryonic fibroblasts

Question 56

What needs to be figured out when studing a protein in a human cell?

Answer 56

Where it goes in cells - cytoplasmic, nucleus or in a membrane

Question 57

How was the fate of a protein tracked?

Answer 57

- In 1971, a fluorescent protein was identified and cloned from Jelly Fish. - The green colour derives from an intrinsically green fluorescent protein, that is non-toxic and otherwise biochemically inert. - 5' gene fusions are used and a protein tag is added to track the fate of a protein.