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2.1 Cell and Molecular Biology > Fergus - Plasmids > Flashcards

Fergus - Plasmids Flashcards

1
Q

What are plasmids?
(2)

A

Extrachromosomal circular independently replicating DNA molecules

Found in bacteria

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2
Q

List the typical contents of a plasmid
(5)

A

Origin of replication

Antibiotic resistance gene

Multiple cloning site

Marker for recombination

Specialist regions

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3
Q

Give two examples of origins of replication

A

pBR322

ColE1

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4
Q

What is a multiple cloning site also called?

A

Polylinker

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5
Q

Give an example of a marker for recombination

A

Blue/white

Blue and white colony formation

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6
Q

What is an origin of replication

A

DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins

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7
Q

Why are antibiotic resistant genes often components of plasmids?

A

These allow for selection of plasmid-containing bacteria

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8
Q

What are multiple cloning sites

A

Short segments of DNA which contain several restriction sites allowing for the easy insertion of DNA

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9
Q

Where are multiple cloning sites often found in expression plasmids?

A

The multiple cloning site is often found downstream from a promoter

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10
Q

What does the ‘insert’ of a plasmid refer to?

A

The gene, promoter or other DNA fragment cloned into the MCS for further study

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11
Q

What is a promoter region?
(2)

A

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

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12
Q

Why do we need a selectable marker if we already have an antibiotic resistance gene?

A

For the use of plasmids in other cell types

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13
Q

What is a primer binding site?

A

A short single stranded DNA sequence used as an initiation point for PCR amplification or sequencing

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14
Q

What’s the main difference between a plasmid and a vector?
(3)

A

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

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15
Q

Give some examples of vectors

A

pGex

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16
Q

Explain how we use pGex to produce insulin
(4)

A

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

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17
Q

How do we induce protein expression by pGEX
(2)

A

IPTG -a form of lactose that bacteria cannot metabolise

This triggers the transcription of the lac operon and thus induces protein expression

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18
Q

What is bacterial transormation?

A

The introduction of DNA (normally plasmids) into bacterial cells

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19
Q

What are the two ways of inducing bacterial transformation?

A

Chemical treatment

Electroporation

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20
Q

Explain how bacterial transformation is carried out via chemical treatment

A

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

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21
Q

What is electroporation

A

Cells are exposed to high voltage in the presence of plasmid DNA and low salt conditions

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22
Q

Give some uses of recombinant DNA

A

Production of insulin

Production of growth hormone

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23
Q

Why did we need an alternative for growth hormone replacement
(5)

A

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

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24
Q

List five different approaches/vectors used in the production of recombinant proteins

A

Bacterial expression vectors

Eukaryotic expression vectors

Baculovirus expression vectors

Vaccinia expression vectors

Transgenic animal approaches

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25
Why do we rarely use eukaryotic cells for recombinant DNA production?
Eukaryotic mammalian cells are very expensive to use
26
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
27
Give an example of a virus that can be used as an expression vector for eukaryotic cells
Vaccinia virus
28
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
29
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
30
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
31
Give an example of a bacterial origin of replication
pBR322
32
Give an example of a bacterial expression vector selectable marker
Ampicillin resistance
33
Give an example of a promoter for bacterial expression vectors
Lac operon
34
Give two methods of purifying proteins in bacterial expression vectors
GST His fusions
35
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
36
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
37
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
38
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
39
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
40
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
41
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
42
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
43
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
44
Give three examples of eukaryotic promoters
CMV SV40 RSV
45
What is the origin of replication in eukaryotic vectors
SV40 origin
46
Give an example of a eukaryotic vector
pCMV-Tag
47
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
48
What is ColE1 ori?
This allows replication in E Coli
49
What is Pbla
Ampicillin resistance gene
50
What is PCMV
Viral promoter that works in mammalian cells High levels of expression of proteins Cytomagalovirus promoter
51
What is MCS
Multiple cloning site
52
What is T SV40
SV40 transcription terminator This has a poly A signal on it -> it will polyadenylate sequence (terminate)
53
What is a kozak sequence?
Eukaryotic translation start sequence Ribosome binding site -> more efficient translation
54
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
55
What is F1 ori?
Filamentous phage origin of replication This allows generation of a single stranded copy of the sequence (useful in sequencing)
56
What is Ori SV40?
Sv40 origin of replication - allows plasmid to replicate in eukaryotic cells It allows plasmid to replicate in eukaryotic cells
57
What is neomycin?
It confers resistance to G418 in eukaryotic cells
58
What is TK poly A
Thymine kinase poly A signal Poly A signal that binds to neomycin resistance
59
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
60
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
61
Name an expression vector for both bacterial and eukaryotic expression
pDual
62
What medium is used to grow eukaryotes?
Foetal calf medium -> very expensive
63
What conditions are needed to grow eukaryotes?
Need 5% Co2 and specific temperatures -> way more complex then growing bacteria
64
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

2.1 Cell and Molecular Biology (26 decks)

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