Fergus - Plasmids Flashcards
What are plasmids?
(2)
Extrachromosomal circular independently replicating DNA molecules
Found in bacteria
List the typical contents of a plasmid
(5)
Origin of replication
Antibiotic resistance gene
Multiple cloning site
Marker for recombination
Specialist regions
Give two examples of origins of replication
pBR322
ColE1
What is a multiple cloning site also called?
Polylinker
Give an example of a marker for recombination
Blue/white
Blue and white colony formation
What is an origin of replication
DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins
Why are antibiotic resistant genes often components of plasmids?
These allow for selection of plasmid-containing bacteria
What are multiple cloning sites
Short segments of DNA which contain several restriction sites allowing for the easy insertion of DNA
Where are multiple cloning sites often found in expression plasmids?
The multiple cloning site is often found downstream from a promoter
What does the ‘insert’ of a plasmid refer to?
The gene, promoter or other DNA fragment cloned into the MCS for further study
What is a promoter region?
(2)
The region that drives transcription of the target gene
It determines which cell types the gene is expressed in and the amount of recombinant protein obtained
Why do we need a selectable marker if we already have an antibiotic resistance gene?
For the use of plasmids in other cell types
What is a primer binding site?
A short single stranded DNA sequence used as an initiation point for PCR amplification or sequencing
What’s the main difference between a plasmid and a vector?
(3)
Plasmids are extra chromosomal elements of bacterial cells
Vectors are vehicles that carry foreign DNA molecules into another cells
Plasmids can be used as vectors
Give some examples of vectors
pGex
Explain how we use pGex to produce insulin
(4)
We amplify the insulin gene using PCR
The gene is amplified in such a way that restriction enzyme sites which complement pGex are incorporated
We cut the PCR product and pGex using Xho 1 and Eco R1 restriction enzymes
We mix the two products together in the presence of T4 DNA ligase - sticky ends combine to form recombinant DNA incorporated in a pGex plasmid
How do we induce protein expression by pGEX
(2)
IPTG -a form of lactose that bacteria cannot metabolise
This triggers the transcription of the lac operon and thus induces protein expression
What is bacterial transormation?
The introduction of DNA (normally plasmids) into bacterial cells
What are the two ways of inducing bacterial transformation?
Chemical treatment
Electroporation
Explain how bacterial transformation is carried out via chemical treatment
Bacterial cells are treated with ice cold CaCl2 and heat shocked in the presence of plasmid DNA
The CaCl2 creates pores in the cell walls
Transformed cells are selected using antibiotic resistance
What is electroporation
Cells are exposed to high voltage in the presence of plasmid DNA and low salt conditions
Give some uses of recombinant DNA
Production of insulin
Production of growth hormone
Why did we need an alternative for growth hormone replacement
(5)
In those with forms of dwarfism caused by lack of growth hormone -> GH can be used to treat this
GH used to be collected from cadavers
There was very poor purification of this GH
This collection is not illegal nearly every
Risk of CJD - human prion disease
List five different approaches/vectors used in the production of recombinant proteins
Bacterial expression vectors
Eukaryotic expression vectors
Baculovirus expression vectors
Vaccinia expression vectors
Transgenic animal approaches
Why do we rarely use eukaryotic cells for recombinant DNA production?
Eukaryotic mammalian cells are very expensive to use
What do we use instead of mammalian cells in recombinant DNA production?
Baculovirus expression vectors
Normally baculovirus attacks insect cells and produces lots of proteins
We modify the baculvoirus
Give an example of a virus that can be used as an expression vector for eukaryotic cells
Vaccinia virus
What is meant by transgenic animal approaches to gene expression
We design an animal e.g. goat so that the required protein is expressed in their milk
How can recombinant DNA methods be used in gene therapy?
If you are missing a protein why dont we just give you the gene to express the protein
What are the components of a bacterial expression vector (plasmid)?
(5)
Bacterial origin of replication
Selectable marker
Multiple cloning site
Strong inducible promoter with control (lac)
Method of purifying protein
Give an example of a bacterial origin of replication
pBR322
Give an example of a bacterial expression vector selectable marker
Ampicillin resistance
Give an example of a promoter for bacterial expression vectors
Lac operon
Give two methods of purifying proteins in bacterial expression vectors
GST
His fusions
GST is used as a method of purifying proteins made by bacterial expression vectors, how is this done?
(7)
GST = glutathione S transferase
GST is not normally found in bacteria
We combine the gene for GST and our cDNA (gene of protein we want to express)
A fusion protein results - part is GST and the other is our needed protein e.g. insulin
Purification on glutathione sepharose beads -> fusion protein binds to these beads
Cleave off the GST from the protein
Pure protein results
List the components of the pEt vector
(7)
T7 promoter/Lac operator
G x His residues
Multiple cloning site
Stop codons
ColE1 origin of replication
Ampicillin resistance gene
Lac repressor gene
What is the T7 promoter?
A bacteriophage (virus)
It’s RNA polymerase is 10x stronger than that of E.coli => can theoretically get 10x more protein
What is 6xHis
(3)
6 histadines in a row
This does not exist in nature (eukaryotes have a max of 3 in a row, bacteria barely have any histadines)
=> 6xHis can be used to identify our plasmids
What is the pET system
(3)
E.Coli is modified to induce a t7 RNA polymerase gene
This is regulated by the LAC operon
There is also a Lac repressor gene built into the gene
How does 6x His fusion work?
(8)
6x Histadine is tagged onto our protein
6x His Fusion protein results
6X His binds to Nc++ (nickel atom)
6x His will binds to Nickel agarose bead
Retention of 6x His (bound to protein) in the column
Wash everything else away
Add excess nickel to compete with the protein for binding
This will give us our purified proteins
List the advantages of bacterial expression of protein
Large quantities of protein rapidly produced
Cheap - need very cheap medium (LB broth)
Quick expression and purification
Often useful for antigens
Possibly active proteins
What are the disadvantages of bacterial expression of protein
(4)
Poor post-translational capabilities
Only cDNA can be used
Protein folding can be incorrect
Solubility of protein in bacterial cell can be poor
Will not splice out introns
Conditions in bacteria might be different -> different pH or salt concentrations
What are the components of a eukaryotic expression vector?
(8)
Origin of replication
Ampicillin resistant gene
Promoter
Maybe 6x His
Multiple cloning site
Poly A signal
Stop codon
Neomycin resistant gene
Ribosome binding site
Give three examples of eukaryotic promoters
CMV
SV40
RSV
What is the origin of replication in eukaryotic vectors
SV40 origin
Give an example of a eukaryotic vector
pCMV-Tag
What are the components of CMV
(10)
PCMV
Pbla
MCS
T SV40
Kozak sequence
Flag or cmyc
F1 ori
Ori SV40
Neomycin
TK poly A
What is ColE1 ori?
This allows replication in E Coli
What is Pbla
Ampicillin resistance gene
What is PCMV
Viral promoter that works in mammalian cells
High levels of expression of proteins
Cytomagalovirus promoter
What is MCS
Multiple cloning site
What is T SV40
SV40 transcription terminator
This has a poly A signal on it -> it will polyadenylate sequence (terminate)
What is a kozak sequence?
Eukaryotic translation start sequence
Ribosome binding site -> more efficient translation
What is Flag or cmyc
Epitope tags
Allow for antibody detection of expressed proteins without having to generate a specific antibody -> allows us to use commercial antibodies and carried out a Western Blot
What is F1 ori?
Filamentous phage origin of replication
This allows generation of a single stranded copy of the sequence (useful in sequencing)
What is Ori SV40?
Sv40 origin of replication - allows plasmid to replicate in eukaryotic cells
It allows plasmid to replicate in eukaryotic cells
What is neomycin?
It confers resistance to G418 in eukaryotic cells
What is TK poly A
Thymine kinase poly A signal
Poly A signal that binds to neomycin resistance
List the advantages of eukaryotic expression vectors
(6)
Genomic clones acceptable
Post-translational events generally occur
Solubility generally good
Stable cell lines can be generated -> will constantly produce proteins for us
With appropriate signals we can get secrete the protein into cell culture medium possible -> we can purify from this
6xHis purification possible
List the disadvantages of eukaryotic expression vectors
(5)
Low levels of expression
Slow growth of cultures -> eukaryotic cells divide every 24 hours
Expensive -> culture medium and equipment
Purification is more difficult
Cells can become contaminated with bacteria -> could waste a week trying to grow a culture
Name an expression vector for both bacterial and eukaryotic expression
pDual
What medium is used to grow eukaryotes?
Foetal calf medium -> very expensive
What conditions are needed to grow eukaryotes?
Need 5% Co2 and specific temperatures -> way more complex then growing bacteria
Write a note on yeast expression vectors
Benefits of bacterial expression and eukaryotic expression systems
Rapid growth, high levels of expression, protein processing, folding and post-translational modifications
