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Molecular Biology & Genetics > Vectors > Flashcards

Vectors Flashcards

1
Q

Episomal

A

Non-intergrated

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2
Q

Why is DNA transported in plasmids?

A

Separate from chromosome so can move

Free DNA is attacked by cells

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3
Q

When does ampicillin kill bacteria?

A

When they’re dividing

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4
Q

F-plasmid genes

A

Cause bacteria to make sex pili proteins
Attaches to new cell, form cytoplasmic link
Mobilisation proteins move plasmid DNA through link so plasmid present in both cells

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5
Q

Copy number (vectors)

A

How many copies can be supported

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6
Q

What does a relaxed origin of replication mean?

A

High copy number

Generally small plasmid

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7
Q

Why do plasmids have marker genes like those for antibiotic resistance?

A

To detect if the bacteria has taken up the recombinant plasmids

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8
Q

Bacteriacide

A

Antibiotic that kills bacteria immediately

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9
Q

Bacteriostatic

A

Antibiotic that kills bacteria when division occurs

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10
Q

Beta-lactomase

A

Enzyme coded for by ampicillin resistant gene

Cleaves B-lactam

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11
Q

What are important traits required by plasmids to be used in cloning?

A 
dsDNA
Relaxed origin of replication for lots of copies
Have 1 or more selectable markers
Has restriction enzyme sites
Suitable promoter
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12
Q

Why does pBluescript have 2 origins of replication?

A

1 normal

1 from filamentous bacteriophage

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13
Q

Lambda

A

Large virus
Infects E.coli
Linear dsDNA inside rigid geometric head

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14
Q

How can we use lambda as a cloning vector?

A

Delete a non-essential gene cluster and replace it with cloned sequences (up to ~20kb)
Can then be injected into cell by lambda

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15
Q

Cos sites

A

Lambda virus genome has short complimentary ssDNA ends/overhangs
Can allow the genome to circularise

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16
Q

What lambda enzyme digests cos sites?

A

Virus endonuclease

17
Q

Why would you want to digest cos sites?

A

So the circular vector can be linearised

Much superior method of gaining entry into cells

18
Q

Cosmid vector

A

Vector that has cos sites from lambda virus

19
Q

BAC vectors

A

Bacterial artificial chromosomes
Largest capacity vector
Low copy number

20
Q

Why is there no need to include a selectable marker with retorviruses?

A

They integrate their DNA straight into the cells DNA

21
Q

Baculovirus

A

Insect specific virus
Strongest gene promoter
Polyhedrin protein made to very high levels in infected cells

22
Q

How are baculoviruses used as vectors?

A

The gene of interest replaces polyhedrin coding sequence

23
Q

What factors should be considered when choosing a vector?

A

Size of fragment to be cloned
Goal of cloning
Target cells or organism
Suitability of vector

24
Q

Gene library

A

Large collection of individually cloned DNA fragments that represent all genetic sequences of an organism, cell or tissue sample

25
Q

What is the ideal gene library like?

A

As small as possible

As small fragments as possible

26
Q

Expression vectors

A

Contain necessary regulatory sequences

27
Q

Fusion peptides

A

Additional short DNA sequence in vector can fuse a novel peptide onto a recombinant protein

28
Q

What can fusion peptides do?

A 
Stabilise the protein and prevent degredation
Solubilise the protein
Direct protein targeting
Allow protein detection
Allow protein purification
29
Q

What can high levels of recombinant protein translation result in?

A

Inclussion bodies

30
Q

Inclussion bodies

A

Aggregates of insoluble protein

Caused by high levels of recombinant protein translation

31
Q

Partial restriction digest

A

Use an enzyme that cuts DNA sequence frequently
Stop halfway through
Results in a random distribution of cut sites

32
Q

What agent removes a cleaves methionine?

A

Cyanogen bromide

33
Q

How do fusion peptides allow recombinant protein purification?

A

Ligans fixed to inert surface
Pour cell lysate through column and tagged proteins bind to ligand
Release bound fusion protein

Molecular Biology & Genetics (8 decks)

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