Gene Cloning + Plasmid Vectors Flashcards Preview

AP0511 Molecular Biology and Genetics > Gene Cloning + Plasmid Vectors > Flashcards

Flashcards in Gene Cloning + Plasmid Vectors Deck (15)
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1
Q

What is gene cloning?

A

An in vivo method to amplify DNA fragments

Also used to generate DNA probes, detect mutations and produce recombinant proteins.

2
Q

How are bacteria chemically transformed to become competent to be able to scavenge DNA?

A

Treated with calcium chloride to precipitate and put in ice bath for cold shock process

3
Q

How are bacteria transformed using electroporation?

A

Hit with millisecond electrical pulses which induces temporary pores to form in their cell membrane so plasmids can enter.
Cell membrane reseals after

4
Q

What is a vector?

A

A plasmid

A carrier of DNA

5
Q

What features make a good vector?

A

Small, stable and safe
High copy number
Selectable phenotypic trait
High density of restriction enzyme sites

6
Q

What is the difference between plasmids and bacteriophages?

A

Plasmid- naturally found in bacteria

Bacteriophage- genetically engineered virus vectors

7
Q

What are some advantages of small plasmids?

A

resistant to damage by mechanical shearing
ease of isolation
high copy number per cell

8
Q

Restriction Endonucleases grouped in one small region is called a Polylinker or a Multiple Cloning Site. What does this allow for?

A

Allows linearisation of the plasmid rather than fragmentation

9
Q

How do we select and identify for cells/bacteria that have the recombinant protein in them vs those that don’t?

A
With antibiotic resistance (those that grow are resistant to the antibiotic and contain the vector)
With flourescence (those that glow and flouresce have the vector)
10
Q

What is pBR322?

A

One of the first vectors to be developed, derived from 3 naturally occurring plasmids.
BamHI disrupts the tetracyclin gene so those that grow to clone BamHI are NOT resistant to tetracyclin.

11
Q

What is Replica Plating and how is it used?

A

A technique used to select for colonies of bacteria that are resistant to 2 types of antibiotic (tetracyclin and amplicin). A wooden block is used to transfer colonies between plates containing the different antibiotics, those that grow on both will be the selected pBR322 Recombinants.

12
Q

What are the advantages of pUC-Series Plasmid Vectors?

A

More flexible choices of sticky ends compared to pBR322.
Recombinants identified in a single step using blue white colour selection (not wooden blocks)
Has many unique RES in a MCS

13
Q

Describe how the non-recombinant form of pUC19 comes about?

A

Lac Z is normally turned off by its repressor Lac R
So Lac Z will produce an incomplete enzyme B-galactosidase
But in non-recombinant forms…….pUC contains the missing fragment for Lac Z so complete B-galactosidase is made and this means X-gal is metabolised
Blue product formed

14
Q

Describe how the recombinant form of pUC19 comes about?

A
Lac Z is turned off by Lac R as normal
Inactive B-galactosidase formed
X-gal is not metabolised so no blue product formed
White (normal colour of bacteria) formed
They contain the recombinant plasmid
15
Q

Give an advantage of pZErO-1 vector?

A

Allows for DIRECT selection of inserts by producing a zero-background of non-recombinants.
By the zeocin gene- it disrupts the ccdB gene which is lethal to bacteria so only those that are recombinant will survive.