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Genomics > Recombinant DNA and Cloning Vectors > Flashcards

Recombinant DNA and Cloning Vectors Flashcards

1
Q

What are the recombinant vectors used in DNA technology?

A

Plasmids
Phages
Viruses
Artificial chromosomes

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2
Q

Where are plasmids found?

A

In many but not all bacteria

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3
Q

What are phages?

A

Lambda – bacterial viruses

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4
Q

Which viruses are used as vectors?

A

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)

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5
Q

Which artificial chromosomes are used as vectors?

A

Yeast artificial chromosomes YACs – introducing large segments DNA

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6
Q

What are plasmids?

A

Discrete Circular dsDNA molecules found in many but not all bacteria
Are a means by which genetic information is maintained in bacteria

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7
Q

Why are plasmids referred to as extra-chromosomal?

A

Are genetic elements (replicons) that exist and replicate independently of the bacterial chromosomes and are therefore extra-chromosomal

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8
Q

What is the significance of plasmids being used as vectors??

A

Can normally be exchanged between bacteria within a restricted host range (eg plasmid borne antibiotic resistance)

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9
Q

What are vectors?

A

Vectors are a cut down version of naturally occurring Plasmids & are used as molecular tools to Manipulate genes

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10
Q

Outline the important features of vectors

A
  • Can be linearized at one or more sites in non-essential
    stretches of DNA
  • Can have DNA inserted into them
  • Can be re-circularised without loss of replication ability
  • Are often modified to replicate at high multiplicity (copy
    number) within a host cell
  • Contain selectable markers
    -Are relatively small 4-5 kb in size
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11
Q

When are bacterial plasmids used as vectors?

A

Using for example PCR to amplify DNA, restriction enzymes to cut it and DNA Ligases to re-join it we can manipulate DNA, make and insert recombinant genes into plasmids

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12
Q

How do we form recombinant protein from recombinant DNA?

A
  1. insert gene into vector plasmid
  2. Forms recombinant expression vector (recombinant
    DNA)
  3. Transform into E. Coli
  4. Culture on agar plate containing antibiotic
  5. Isolate colonies and culture
  6. Confirm insert by restriction mapping of clone
  7. Purify protein
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13
Q

What is the significance of using recombinant DNA to form recombinant proteins?

A

Having done this we can transduce bacteria where the plasmids will replicate and be maintained
We can isolate the which will express the recombinant gene

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14
Q

How do plasmids add functionality over DNA?

A
  • Express recombinant gene
  • Add/ modify control elements
  • alter properties
  • used as therapeutics
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15
Q

What is the advantage of being able to express recombinant genes?

A

We can choose the expression of recombinant genes in a chosen living organism either prokaryotic or eukaryotic

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16
Q

How do we modify control elements of recombinant DNA?

A

Make it inducible or express it to high levels on demand

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17
Q

In what way can we alter the properties of recombinant genes?

A

Make it secreted extracellularly or into the periplasmic space,
Fuse it to a peptide tag or other protein

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18
Q

Why is recombinant DNA significant for clinical use?

A

Recombinant vectors facilitate production of recombinant drugs
Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals

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19
Q

Give examples of recombinant proteins used in biopharmaceuticals

A

Human insulin - diabetes

Interferons-α & β – viral Hepatitis or MS

Erythropoietin – kidney disease, anaemia

Factor XIII – haemophilia

Tissue plasminogen activator (TPA) – embolism, stroke
around 62 recombinant drugs approved by the FDA for clinical use between 2011 and 2016

20
Q

When did recombinant antibodies first appear in clinics?

A

In the late 1980s

21
Q

What are the requirements for cloning a defective gene from a patient with genetic condition and expressing it in large scale bacteria to analyse ?

A 
Ability to replicate in bacteria (E. coli)
Maintained at high copy number
Modified origin of replication
Selectable contains an antibiotic marker
Ampicillin resistance gene
Easy to manipulate – cut and rejoin
Multiple cloning site (MCS)
22
Q

What is the coding region?

A

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

23
Q

What is the shine-Dalgarno sequence?

A

The shine-Delgarno sequence is the ribosomal binding site found around 8 nucleotides before the start codon in the RNA in prokaryotes.

Remember the RNA of this group of organism is not capped

24
Q

What is the promoter?

A

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

25
What is the transcriptional terminator?
The transcriptional terminator is a sequence that terminates transcription and initiates the dissociation of transcription
26
What is meant by a constitutive promoter?
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
27
What is an inducible promoter?
Inducible – molecular switch - allows large cultures to be grown without expressing the foreign protein, - induced in response to a defined signal
28
How do inducible promoters function?
> use transcriptional repressors | Inducible Promoter typically uses lac Operator which is de-repressed by addition of lactose mimic IPTG
29
Outline how the lac operon is used as a regulatory switch
The lac operon comprises of genetic elements that in prokaryotes include some regulatory sequences one of which is the lac operator and a gene the lac repressor (inhibitor) These allow bacteria to be responsive to low glucose environments and switch to lactose as a carbon source
30
How can we use a lac operon used to regulate genes?
We can use this system to regulate any gene by placing a lac operator (lacO) upstream of its transcriptional start
31
What requirements need to be met in order to insert a lac operon upstream of a gene transcriptional start?
Copy of the coding sequence – generated (eg PCR) Must contain the start codon to & including the stop codon No introns – bacteria can’t splice it – ie exonic sequence only - No Cap site required - No eukaryotic UTRs required - No polyadenylation signal required – bacterial RNAs are not polyadenylated Easy to manipulate – cut and re-join to other DNA add restriction sites using PCR or other methods
32
Why are bacterial plasmids not used as often as other vectors?
Some proteins are best made in eukaryotes Many pharmacologically useful proteins are heavily modified and won't be appropriately processed in bacteria (e.g. interferons) (usually by glycosylation) Some proteins retain biological activity, some don’t
33
How do we ensure correct use of bacterial plasmids?
Solution is to express them in a eukaryotic system
34
What elements are present when studying the effect of a defective gene in a cell culture system by expressing it in human fibroblasts?
- inducible promoter - shine-dalgarno - insert within frame start / stop codons - transcriptional terminator - origin of replication - selectable marker - choice of unique restriction sites MCS
35
What are the issues with the elements present in the defective gene expressed in human fibroblasts?
1. Bacterial promoter doesn’t work 2. Shine Delgarno sequence isn't recognised 3. Transcriptional start is not recognised, no cap site 4. No polyadenylation signal 5. Termination of transcription not recognised by eukaryotic Polymerase II 6. Origin of replication doesn’t work, but do I need it anyway?
36
What features are present in prokaryotic expression vectors?
``` prokaryotic promoter shine-dalgarno sequence AUG start codon ORF - codon preference prokaryotic terminator ```
37
What are the features of eukaryotic expression vectors?
``` enhancer eukaryotic promoter kozak sequence AUG start codon ORF - codon preference introns polyA signal in 3'UTR eukaryotic terminator ```
38
What is required for a plasmid to be transinfected into a eukaryotic system?
A vector that’s easy to manipulate – cut and rejoin 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
39
What is required for a transient / stable expression?
(ie a transgenic cell line) - Ability to replicate mammalian cells - Or integrated in the chromosomes - For this we need a Selectable marker in eukaryotes
40
Why are viral vectors used more often in eukaryotic expression systems?
Viral promoters are commonly used in eukaryotic expression systems as they are more compact and simpler to manipulate
41
What is bovine growth hormone?
Bovine growth hormone (BGH), is a peptide hormone produced by cows' pituitary glands
42
What can be identified on the BGH restriction map?
A combined transcriptional terminator and polyadenylation signal is shown from the 3’UTR of (BGH) The selectable marker, (G418) could be one of many, but must be constructed with eukaryotic promoter polyadenylation signal. Terminator and SV40 virus sequences are used in this case
43
What is the significance of the bovine growth hormone vector?
its a plasmid in a dual system | eukaryotic / prokaryotic hybrid vector
44
Which protein tags are used to make purifying expressed proteins easier?
Many different protein tags are used but two of the most popular are 6 Histidines and Glutathione S transferase (GST)
45
Where along the protein coding sequence are protein tags added?
Fusions can be made at either end of the coding sequence either before the stop codon or after the start
46
How can we assess the expressed protein in human fribroblasts' localisation in a cell culture system?
Can use green fluorescent protein tags to track the fate of a protein
47
Where is the green fluorescent protein derived from?
In 1971, a fluorescent protein was identified and cloned from JellyFish the green colour derives from an intrinsically green fluorescent protein, that is non-toxic and otherwise biochemically inert

Genomics (20 decks)

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