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2.1 Cell and Molecular Biology > Fergus - Plasmids > Flashcards

Fergus - Plasmids Flashcards

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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
Q

Why do we rarely use eukaryotic cells for recombinant DNA production?

A

Eukaryotic mammalian cells are very expensive to use

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26
Q

What do we use instead of mammalian cells in recombinant DNA production?

A

Baculovirus expression vectors

Normally baculovirus attacks insect cells and produces lots of proteins

We modify the baculvoirus

27
Q

Give an example of a virus that can be used as an expression vector for eukaryotic cells

A

Vaccinia virus

28
Q

What is meant by transgenic animal approaches to gene expression

A

We design an animal e.g. goat so that the required protein is expressed in their milk

29
Q

How can recombinant DNA methods be used in gene therapy?

A

If you are missing a protein why dont we just give you the gene to express the protein

30
Q

What are the components of a bacterial expression vector (plasmid)?
(5)

A

Bacterial origin of replication

Selectable marker

Multiple cloning site

Strong inducible promoter with control (lac)

Method of purifying protein

31
Q

Give an example of a bacterial origin of replication

A

pBR322

32
Q

Give an example of a bacterial expression vector selectable marker

A

Ampicillin resistance

33
Q

Give an example of a promoter for bacterial expression vectors

A

Lac operon

34
Q

Give two methods of purifying proteins in bacterial expression vectors

A

GST

His fusions

35
Q

GST is used as a method of purifying proteins made by bacterial expression vectors, how is this done?
(7)

A

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
Q

List the components of the pEt vector
(7)

A

T7 promoter/Lac operator

G x His residues

Multiple cloning site

Stop codons

ColE1 origin of replication

Ampicillin resistance gene

Lac repressor gene

37
Q

What is the T7 promoter?

A

A bacteriophage (virus)

It’s RNA polymerase is 10x stronger than that of E.coli => can theoretically get 10x more protein

38
Q

What is 6xHis
(3)

A

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
Q

What is the pET system
(3)

A

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
Q

How does 6x His fusion work?
(8)

A

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
Q

List the advantages of bacterial expression of protein

A

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
Q

What are the disadvantages of bacterial expression of protein
(4)

A

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
Q

What are the components of a eukaryotic expression vector?
(8)

A

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
Q

Give three examples of eukaryotic promoters

A

CMV
SV40
RSV

45
Q

What is the origin of replication in eukaryotic vectors

A

SV40 origin

46
Q

Give an example of a eukaryotic vector

A

pCMV-Tag

47
Q

What are the components of CMV
(10)

A

PCMV

Pbla

MCS

T SV40

Kozak sequence

Flag or cmyc

F1 ori

Ori SV40

Neomycin

TK poly A

48
Q

What is ColE1 ori?

A

This allows replication in E Coli

49
Q

What is Pbla

A

Ampicillin resistance gene

50
Q

What is PCMV

A

Viral promoter that works in mammalian cells

High levels of expression of proteins

Cytomagalovirus promoter

51
Q

What is MCS

A

Multiple cloning site

52
Q

What is T SV40

A

SV40 transcription terminator

This has a poly A signal on it -> it will polyadenylate sequence (terminate)

53
Q

What is a kozak sequence?

A

Eukaryotic translation start sequence

Ribosome binding site -> more efficient translation

54
Q

What is Flag or cmyc

A

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
Q

What is F1 ori?

A

Filamentous phage origin of replication

This allows generation of a single stranded copy of the sequence (useful in sequencing)

56
Q

What is Ori SV40?

A

Sv40 origin of replication - allows plasmid to replicate in eukaryotic cells

It allows plasmid to replicate in eukaryotic cells

57
Q

What is neomycin?

A

It confers resistance to G418 in eukaryotic cells

58
Q

What is TK poly A

A

Thymine kinase poly A signal

Poly A signal that binds to neomycin resistance

59
Q

List the advantages of eukaryotic expression vectors
(6)

A

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
Q

List the disadvantages of eukaryotic expression vectors
(5)

A

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
Q

Name an expression vector for both bacterial and eukaryotic expression

A

pDual

62
Q

What medium is used to grow eukaryotes?

A

Foetal calf medium -> very expensive

63
Q

What conditions are needed to grow eukaryotes?

A

Need 5% Co2 and specific temperatures -> way more complex then growing bacteria

64
Q

Write a note on yeast expression vectors

A

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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