Recombinant DNA and cloning vectors

Question 1

Define Vector

Answer 1

The term vector simply means a type of recombinant tool that can be used to transfer DNA into a biological system like a bacterium or cell.

Question 2

What are the 4 commonly used vectors?

Answer 2

1. Plasmids
2. Bacteriophages
3. Viruses
4. Or artificial chromosomes

Question 3

1. Where are plasmids found and how are they transferred?

Answer 3

Plasmids:

• They are found in many but not all bacteria
• Generally Have a restricted host range
• Are transferable by various means including transformation (but this is rare, due to the instability of large molecules of DNA in the environment.) and conjugation (this is where living bacteria form sex pili encoded by the tra genes of a conjugative plasmid).
• Experimentally, transformation is the main means by which plasmids are artificially transferred into bacteria.

Question 4

2. Give an example of a phage vector

How are they transferred

Do they have a restricted host range?

Answer 4

• Phages or more correctly bacteriophages Include Lambda, one of the best known bacteriophages (CLICK)
• They can be thought of as a type of bacterial virus.
• Phages are also naturally occurring, but can also transfer antimicrobial resistance, virulence etc through a mechanism called transduction
• Phages also have a restricted host range, their restriction and ability to lyse bacteria means they have been used as antibacterial agents

Question 5

3. Give 2 examples of virus vectors used

Answer 5

• Recombinant Viruses are used widely as vectors in eukaryotic systems including whole animals or even as recombinant vaccines
• Lentiviruses –are Non-primate viral vectors used to integrate DNA in mammalian cells
• Whilst Baculoviruses –vectors are used in combination with recombinant expression in insect cells (also a eukaryotic expression system)

Question 6

4. Describe artificial chromosomes as vectors

Answer 6

• Artificial Chromosomes most commonly Yeast artificial chromosomes or YACs
• these are very large DNA molecules used for introducing large segments of DNA for example entire genes including promotors and introns.
• YACs are similar conceptually to plasmids but are much bigger and are restricted to yeast

Question 7

Describe the structure of plasmids

Why are they referred to as replicons

Why are conjugative plasmids not normally used?

Answer 7

We will focus on plasmids, as these are the most versatile and common form of recombinant vector, and are an essential part of the molecular tool kit

• Plasmids are circular double stranded DNA molecules only found in prokaryotes, but are used as tools for introducing DNA into both prokaryotes and eukaryotes
• They are a means by which genetic information is maintained in bacteria and passed vertically to their progeny
• Plasmids are genetic elements, that are sometimes referred to as replicons. They exist and replicate in bacteria independently of the bacterial chromosome and are therefore described as extra-chromosomal
• As we know, they are normally exchanged between bacteria facilitated by conjugation, but conjugative plasmids are not normally used and recombinant vectors are therefore, not exchanged horizontally and this is an important feature

Question 8

Give some examples of how plasmids are used as molecular tools to manipulate genes

Answer 8

1. For cloning a gene, or disease causing variant of a gene and producing a recombinant protein in a biological system such as a bacterium in large quantities
2. They may also be used To mutate a gene and understanding the functional role of parts of a protein or the effects of a specific mutation on protein structure or function
3. They could be used to insert promoters in front of reporter genes allowing us to better understand the regulatory mechanisms of a genes promoter
4. They are also sometimes used in two component systems to understand the interaction and association of different gene products in a biological system for example the yeast two hybrid system

Question 9

What are the desrible features of using a plasmid as a vector?

Answer 9

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

Question 10

Lets take a simple example of how we might use a plasmid vector & insert a recombinant gene into it.

What are the important components in this?

Answer 10

• The selection of the vector is important, it needs to have the correct features for inserting the gene selecting for recombinants and may also require promotors or other elements in the correct place.
• For example the vector must have, the appropriate sites in a cassette into which we’ll insert the gene
• In the slide the vector has a bacterial promoter (blue arrow) a multiple cloning site with a variety of restriction sites (Xba I etc) where we may cut and linearize it.
• It also has a bacterial transcriptional terminator (the black bar)
• The vector and the PCR amplicon of the gene must be cut with restriction enzymes to produce compatible ends
• These are then joined together by ligation using a DNA ligase

Refer to diagram

Question 11

What do we have when the gene is inserted to the plasmid?

Answer 11

We should then have a re-circularised recombinant vector containing our gene sequence

On image

Question 12

What can we now do with the recombinant plasmid

Answer 12

We can artificially transduce bacteria where the plasmids will replicate and be maintained in the presence of a selectable marker such as ampicillin

We can then pick individual clones, grow these up in bulk to produce recombinant proteins in our bacteria, where we might choose to
• For example purify the protein produced, investigate its properties or function,
• Or alternatively develop and produce therapeutics

Question 13

Why use Plasmids as recombinant tools?

Answer 13

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 14

Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals

Give some examples of recombiant proteins

Answer 14

• 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 15

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?

What are the requirements for this?

Answer 15

My Requirements: For a Plasmid in a prokaryotic system
• 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 16

In order for transcription and translation of the recombinant gene, what is required?

Answer 16

• the coding sequence is the part of the gene coding for the protein, in a bacterium we don’t want the UTRs nor any intronic or regulatory sequences such as a promoter nor enhancers. For example introns can’t be processed in prokaryotes and eukaryotic regulatory sequences won’t work.
• The Shine-Delgarno sequence is the ribosomal binding site found around 8 nucleotides before the start codon in the RNA in prokaryotes. You should remember the RNA of this group of organism is not capped, so having a Shine –Dalgarno sequence would be a benefit for the translational efficiency
• We need to have strong bacterial promoter to initiate transcription, and this needs to be added to the 5 prime end of the transcription unit,
• However, to complete the transcription unit, a transcriptional terminator is required to allow the polymerase to end transcription and release the message

Question 17

What are the 2 types of promotor region?

Answer 17

Next we need to consider how we would like the recombinant gene to be expressed.
Here we have two options a constitutive or an inducible promotor.
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

Inducible – molecular switch
• allows large cultures to be grown without expressing the foreign protein,
• induced in response to a defined signal

Question 18

What does the lac operon comprise of?

What does it allow?

Answer 18

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 (or inhibitor).

These allow normally bacteria to be responsive to low glucose environments and switch to lactose as a carbon source.

We can use this system to regulate any gene by placing a lac operator (shown as lacO) upstream of the transcriptional start of our gene and at the same time expressing the lac repressor or inhibitor (the gene symbol of which is lacI).

Question 19

What are the requirments for the DNA insert?

Answer 19

The DNA insert
• 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 20

When the gene is inserted into the plasmid, what enzyme is it joined by?

Answer 20

DNA ligase

Question 21

Describe the fate of the plasmid?

Answer 21

The plasmid can be transformed into bacteria where the it will replicate and be maintained in the presence of a selectable marker such as ampicillin

We can then pick individual clones, grow these up in bulk

The protein is then induced by addition of a lactose mimic IPTG to produce recombinant protein

Question 22

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?

Answer 22

We have an Inducible Promoter, with a Shine-Delgarno, with an Insert that has in frame start and stop codons and the transcriptional unit has a Transcriptional terminator

The plasmid has an origin of replication, a selectable marker and a range of restriction sites available

Unfortunately in eukaryotes:

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 Polymaerase II
6. Origin of replication doesn’t work, but its questionable needs to be asked do I need it anyway?

Question 23

Comparison between prokaryotic and eukaryotic expression vectors

Answer 23

Refer to document

In eukaryotes
• the Shine-Dalgarno sequence is replaced by capping
• and the identification of the correct start codon is partly defined by the Kozak sequence in a 5’ UTR
• introns are tolerated but are not necessary
And a polyadenylation signal is required in a 3’ UTR

Question 24

What are the requirments for a plasmid that will be grown in bacteria and subsequently transfected into a eukaryotic cells?

Answer 24

• A vector that’s easy to manipulate – cut and re-join
• Can also be grown up in bacteria: so must have a Selectable bacterial marker and be Maintained at high copy number
We would have to Substitute the promoter with a Eukaryotic promoter
• Introduce a 3’UTR containing polyadenylation signal
• And a Terminator must be substituted with Eukaryotic Transcriptional terminator

Finally 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 (stable expression) or transiently and eventually be lost.

Stable expression, ie making A transgenic cell line would require our plasmid to have
• Either the Ability to replicate in mamalian cells
• Or be integrated in the chromosomes For this we also need a Selectable marker in eukaryotes

Question 25

What do viral promoters do?

Answer 25

• Viral promoters are commonly used in eukaryotic expression systems because they are more compact and simpler to manipulate

Strong viral promoters like the CMV or RSV promoters are common

Question 26

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

How do we resolve this?

Answer 26

Purify the protein

Question 27

What are the 2 main types of protein tags?

Where are gene fusions found?

Answer 27

• There are many different protein tags that are used but two of the most popular are a histidine tag (tag 6 Histidines one after the other) and Glutathione S transferase (GST)
• Gene Fusions can be made at either end of the coding sequence either before the stop codon or after the start codon. The slide shows these added before the stop codon
• But a key to this is they must be placed in the correct reading frame so that the chimeric protein is correctly translated

Question 28

How do we purify the protein?

Answer 28

On image

Question 29

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?

Describe Green Fluroscent Protein

Answer 29

Green Fluorescent Protein

In 1960s, a fluorescent protein was identified and subsequently cloned from Jelly Fish the name Green

Fluorescent protein or GFP was coined in 1971
the green color derives from its intrinsic green fluorescence, GFP is non-toxic and otherwise biochemically inert. The molecule absorbs light at a wavelength of 395nm and emits light in the green range at 509 nm

GFP has been widely used since around 1994 as biological tag to identify the location of chimeric proteins joined to it

Question 30

Describe 5’ Gene infusions

Answer 30

• We’ve already talked about gene fusions with GST and or His tagged proteins, and this approach is not really any different conceptually.
• And the approach of using GFP relies upon the insertion of the GFP coding sequence (minus the stop codon) either immediately before the stop or after the start codon. The slide shows these added after the start codon
• But again a key to this is GFP must be placed in the correct reading with the start codon and the following gene must remain in the same reading frame for the correct decoding of its sequence so that the chimeric protein is correctly translated
• After transfection of the cells the location of the protein in the live cells can be tracked by fluorescent microscopy or they can be fixed and additionally be stained to identify cellular structures for example DAPI stains nuclei