W2 - Recombinant DNA and Cloning Vectors

Question 1

What are the different vectors in the molecular took kit?

Answer 1

• plasmids
• bacteriophages
• viruses
• artificial chromosomes

Question 2

Why are plasmids useful?

Answer 2

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

Question 3

Why are phages useful?

Answer 3

• Lambda – a virus that grows and divides inside a bacterium
• transfer of antimicrobial resistance through a mechanism called transduction

Question 4

Why are viruses useful?

Answer 4

Non-primate Lentiviruses –vectors used to integrate DNA in mammalian cells

Baculoviruses – a member of a family of DNA viruses infecting only invertebrate animals. Some have a very specific insect host, and may be used in biological pest control. Used in combination with recombinant expression in insect cells (a eukaryotic expression system)

Question 5

Why are artificial chromosomes useful?

Answer 5

DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics.

Yeast artificial chromosomes YACs – introducing large segments DNA

Question 6

Why are plasmids an essential part of recombinant DNA cloning?

Answer 6

• 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 (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 (eg plasmid borne antibiotic resistance). Does not transfer horizontally.

Question 7

What are the features of a plasmid that enables its use?

Answer 7

1. Can be linearized (DNA helix is cut in both strands in the same place) 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. 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

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

Question 8

What is an example of using bacterial plasmids as a vector?

Answer 8

• vector must have an appropriate site in a cassette, where we insert the gene.
• There would be a bacterial promoter, multiple cloning site with a variety of restriction sites (where we can cut and linearise it) and a bacterial transcriptional terminator.

The vector and the PCR amplicon of gene must be cut with restriction enzymes to produce compatible ends. These are them joined together via DNA ligase.

Now we have a recircularised, recombinant vector containing our genes.

Question 9

Once a recombinant plasmid is made, what do we do?

Answer 9

These bacteria can now be artificially transduced where the plasmids will replicate and be maintained in the presence of a selected marker.

We can then select colonies and grow these in bulk to produce a recombinant protein.

Question 10

Why use Plasmids as recombinant tools?

Answer 10

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
- Make it secreted extra-cellularly or into the periplasmic space,
- fuse it to a peptide tag or other protein
- make it useful as a therapeutic

Question 11

What is the clinical use of recombinant proteins like?

Answer 11

• Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals.

Recombinant vectors facilitate production of recombinant drugs:
* Human insulin - diabetes
* Interferons-a & b – 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

Question 12

I want to clone the defective gene from a patient with an inherited condition and express it in bacteria in large amounts so that I can perform functional analysis on the protein?

Answer 12

Ecoli is normally the model prokaryotic organism used, so it must have the ability to replicate bacterium (prokaryotic system).

Question 13

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

Answer 13

• 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 re-join
• Multiple cloning site (MCS)

Question 14

What Control elements are required for
expression in bacteria?

Answer 14

What is necessary for the transcription and translation of recombinant gene.
- We don’t want the UTIRs nor any intronic or regulatory sequences such as a promotor or enhancer.

• Gene coding sequence insufficient AUG
• Shine-Dalgarno sequence (– 8) RBS (ribosomal binding cell site) recognition of AUG : still not enough AUG. This would be beneficial.
• Need a strong Bacterial promoter to initiate transcription and this needs to be added to the 5’ end of a transcriptional unit- no still still not enoughAUG
• To complete the transcription unit, a Transcriptional terminator is needed to end the transcription and release the message.

Question 15

How would you like the recombinant gene to be expressed? Are Promoter Constitutive or Inducible?

Answer 15

• 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 16

What is the Inducible Promoters use of transcriptional repressors?

Answer 16

Inducible Promoters typically uses
the lac Operator which is de-repressed by addition of lactose see first year lectures.
These allow the bacteria to grow in a glucose free environment and switch to lactose as a source.

We can use this system to regulate any gene by placing a Lac operator shown by LacO upstream of the transcriptional start of our gene. At the same time, express the lac inhibitor, the gene symbol of which is lacI.

Requires the constitutive expression of lac inhibitor

The lac Operator is de-repressed experimentally by addition of lactose mimic called IPTG binding to the lac inhibitor and de-repressing the lac operator.

Question 17

What are the requirements for the DNA insert?

Answer 17

• 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 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

Question 18

How do Bacterial plasmids work as a vector?

Answer 18

The vector and the PCR, one of the coding sequence of the gene, must then be cut to produce compatible ends. These are then joined together using DNA ligase.

Question 19

What are Recombinant Proteins from
recombinant DNA?

Answer 19

The plasmid can be transformed into bacteria where it will replicate, will be maintained in the presence of a selectable marker such as ampicillin. We can then pick individual clones and grow these up in bulk. The protein is then induced by the addition of the lactose mimic, such as IPTG to produce recombinant protein.

Question 20

Why are some proteins best made in eukaryotes?

Answer 20

• Many pharmacologically useful proteins are heavily modified and will not be appropriately processed in bacteria
• (e.g. interferons) (usually by glycosylation) Some proteins retain biological activity, some don’t. Solution is to express them in a eukaryotic system

Question 21

I now want to study the effect of the defective gene in a cell culture system by expressing the protein in human embryonic fibroblasts? (a human cell line)

So What’s the problem? I’ve got everything already

Answer 21

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

1. Bacterial promoter doesn’t work
2. Shine-Delgarno sequence isn’t recognised
3. Transcriptional start is not recognised, and theres no cap site
4. No polyadenylation signal
5. Termination of transcription not recognised by eukaryotic Polymaerase II
6. Origin of replication doesn’t work, but do I need it anyway?

Question 22

What is the Comparison between prokaryotic
and eukaryotic expression vectors?

Answer 22

Eukaryotics are far more complicated than corresponding points of the prokaryotes.
Ones found in prokaryotes are not found in eukatyotes.

-Codon usage has a different frequency than that of eukaryotes.
- In eukaryotes, the Shine-Delgaro is replaced by the cap on the identification of eukaryotes.
- The identification of the correct start codon is partly defined by the Kozak sequence, which resides within the 5’ UTR.
- Introns are tolerated but not necessary
- Polyadelynation signal is required in 3’ UTR.

Question 23

What are the requirements for Plasmids transfected into in a eukaryotic system?

Answer 23

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 promoterIntroduce a 3’UTR containing polyadenylation signal. Terminator must be substituted with Eukaryotic Transcriptional terminator

Transient or stable expression (ie a transgenic cell line)
*Ability to replicate mamalian cells
*Or integrated in the chromosomes
*For this we need a Selectable marker in
eukaryotes

Viral promoters are commonly used in eukaryotic expression systems because they are more compact and simpler to manipulate. Strong viral promoters like RSV, CMV are common. Combined transcriptional terminator and a polyadenylation signal comes from the BGH.

Question 24

The expressed protein was very hard to obtain in a pure enough form from the bacteria and so I can’t perform functional analysis of the protein?
Can I make it easier to purify?

Answer 24

Two of the most common used are 6His GSTransferase gene fusion can be made at either end of the coding sequence. Before stop codon, or after start codon.
They must be placed in the correct reading frame, so that the chimeric protein is correctly translated.

Before the plasma can be transformed into bacteria and we can pick clones, grow these up into bulk, lies the bacteria and purify the protein by using an affinity column. This binds the GST or 6His and separates the protein from all the others, it can then be washed.

Question 25

I now want to study the localisation and trafficking of the protein in a cell culture system by expressing the protein in human embryonic fibroblasts ?
Where did it go in my cells?
Is it cytoplasmic, in the nucleus or in a membrane?

Answer 25

In 1971, a fluorescent
protein was identified and
cloned from Jelly Fish
the green color derives from
an intrinsically green
fluorescent protein, that is
non-toxic and otherwise
biochemically inert

The molecule absorbs light at a wavelength of light wavelengths of light at 395nm and stays green until 509nm.

GFP has been widely used since around 1994 as a biological tank to intend to find a location of chimeric proteins joined to it.

Question 26

What is the 5’ Gene fusion?

Answer 26

The approach of using a GFP relies upon the insertion of GFP coding sequence minus the stop codon either immediately before the stop or after the start codon.
GFP must be placed in the correct reading frame with the start codon and the following gene must remain in the same reading frame for the correct decoding of each sequence, so that become chimeric protein is correctly translated after transaction of the cells. The location of the protein in the live cells can be tracked by florescent microscopy or they can be fixed. And additionally stained to identify cellular structures, Eg, Dappy Stain identifies nuclei.