Recombinant DNA and Cloning Vectors

Question 1

What are plasmids as recombinant vectors like?

Answer 1

• Found in many but not all bacteria
  
  • Generally have a restricted host range
  • Are transferable by various means including transformation and conjugation

Question 2

What are bacteriophages as recombinant vectors like?

Answer 2

• Include Lambda – bacterial viruses
  
  • Transfer of antimicrobial resistance through a mechanism called transduction
  • Also have a restricted host range

Question 3

What are viruses as recombinant vectors like?

Answer 3

• Non-primate Lentiviruses – vectors used to integrate DNA in mammalian cells
  
  • Baculoviruses – vectors used in combination with recombinant expression in insect cells (a eukaryotic expression system)

Question 4

What are artificial chromosomes as recombinant vectors like?

Answer 4

• Yeast artificial chromosomes YACs – used for introducing large segments DNA

Question 5

What are plasmids like functionally?

Answer 5

Discrete Circular dsDNA molecules found in many but not all bacteria
Are a means by which genetic information is maintained in bacteria
Are genetic elements (sometimes referred to as replicons) that exist and replicate independently of the bacterial chromosomes and are therefore extra-chromosomal
Can normally be exchanged between bacteria within a restricted host range (e.g. plasmid borne antibiotic resistance)

Question 6

What are vectors and what do they do?

Answer 6

Vectors are a cut down version of naturally occurring Plasmids & are used as molecular tools to manipulate genes
May also be used to mutate a gene
Could be used to insert promoters
Sometimes used in two component system to understand the interaction and association between gene products in a biological system

Question 7

What features do plasmid vectors have?

Answer 7

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

Question 8

What does a bacterial plasmid need when used as a vector?

Answer 8

The selection of a vector is important as it needs to have the correct features for inserting the gene, selecting for recombinance and may also require promotors or elements in the correct place
For example the vector must have an appropriate site in a cassette into which we will insert the gene
The arrow shows a promoter
A multiple cloning site with a variety of restriction sites e.g. Xba I where we may cut and linearise it
Also has a bacterial transcription terminator displayed as the black bar
The vector and PCR amplicon must be cut with restriction enzymes to produce compatible ends
These are then joined together by ligation using DNA ligase
We should end up with a recircularised, recombinant vector with our gene, but will only produce a protein if the vector still retains the correct in frame start and stop codons and ideally a Shine Delgarno sequence

Question 9

Why do we use plasmids as recombinant tools?

Answer 9

Plasmids add functionality over simple DNA and facilitate experimental or functional genomics:
Expression of a recombinant gene in a living organism of choice
- Prokaryote or eukaryote
Add or modify control elements
- Make it inducible or express it to high levels on demand
Alter the properties of the gene product
- For example to make it secreted extra-cellularly or into the periplasmic space,
- Or fuse it to a peptide tag or other protein
- Make it useful as a therapeutic

Question 10

What are recombinant proteins used for clinically?

Answer 10

Recombinant vectors facilitate production of recombinant drugs:
Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals:
- Human insulin - diabetes
- Interferons-α & β – viral Hepatitis or MS
- Erythropoietin – kidney disease, anaemia
- Factor XIII – haemophilia
- Tissue plasminogen activator (TPA) – treats clots as a result of embolism, stroke
Around 62 recombinant drugs approved by the FDA for clinical use between 2011 and 2016

Question 11

What control elements are required for expression in bacteria?

Answer 11

The Shine-Dalgarno sequence is the ribosomal binding site found around 8 nucleotides before the start codon in the RNA in prokaryotes
This is a benefit for translational efficiency as RNA isn’t capped
We need to have a strong bacterial promoter to initiate transcription and this needs to be added to the 5’ end of the transcription unit
Lastly we need a transcriptional terminator to allow the polymerase to end transcription and release the message

Question 12

What are the different types of promoters?

Answer 12

Constitutive or Inducible
Constitutive – always on
- Allows a culture of cells to express the foreign protein to a high level
- Fine if the protein isn’t toxic to E.coli
- Bad idea if it is
Inducible – molecular switch
- Allows large cultures to be grown without expressing the foreign protein,
- Induced in response to a defined signal

Question 13

How do inducible promoters work?

Answer 13

Inducible Promoters use transcriptional repressors
Inducible Promoters typically uses the lac Operator which is de-repressed by addition of lactose
see first year lectures
Requires the constitutive expression of lac inhibitor
The lac Operator is de-repressed experimentally by addition of lactose mimic called IPTG

Question 14

What does the DNA insert have to be like to collect and amplify DNA

Answer 14

The DNA must be easy to manipulate – to cut and re-join to with other DNA, add restriction sites using PCR or other methods
Copy of the coding sequence – generated by e.g. PCR
Must contain the start codon to & including the stop codon:
- No introns – bacteria can’t splice it – i.e. exonic sequence only
- No Cap site required
- No eukaryotic UTRs required
- No polyadenylation signal required – bacterial RNAs are not polyadenylated

Question 15

What’s the difference between prokaryotic and eukaryotic expression vectors?

Answer 15

Start codon is defined by the Kozak sequence
Polyadenylation signal is required
Introns can be tolerated but aren’t necessary
Eukaryotic DNA has a 5’ and 3’ untranslated region
The codon preference for Arginine AGG is common in humans but rare in E.coli

Question 16

So how do you switch from a prokaryotic vector to a eukaryotic vector?

Answer 16

Plasmids transfected into in a eukaryotic system :–
A vector that’s easy to manipulate – cut and re-join
Can also be grown up in bacteria:
- Selectable bacterial marker
- Maintained at high copy number
Substitution of promoter with a Eukaryotic promoter
Introduce a 3’UTR containing polyadenylation signal
Terminator must be substituted with Eukaryotic Transcriptional terminator
A choice has to be made whether to express the protein from a construct that is capable of replicating and thus being maintained in eukaryotic cells i.e. stable expression or transiently and eventually lost.

Question 17

What properties would a plasmid be required to have for stable expression?

Answer 17

Stable expression i.e. making a transgenic cell line would require a plasmid to have a number of different elements:

- Ability to replicate mammalian cells
- Or integrated in the chromosomes
- For this, we need a Selectable marker in eukaryotes

Question 18

What are 3’ gene fusions?

Answer 18

There are many tags but the most common ones are 6HIs tags where six histamines one after the other and glutathione S transferase (GST)
Gene fusions can be made at either end of the coding sequence before the stop codon or after the start codon.
The key to this is that the tag must be placed in the correct reading frame with the start or stop codon and the genes around it must remain in the same reading frame for the correct decoding of each sequence so the correct chimeric protein is translated.

Question 19

What are 5’ gene fusions?

Answer 19

The approach of using a GFP lies upon the insertion of a GFP coding sequence (minus the stop codon) either immediately before the stop or after the start codon.