Gene Cloning + Plasmid Vectors Flashcards
What is gene cloning?
An in vivo method to amplify DNA fragments
Also used to generate DNA probes, detect mutations and produce recombinant proteins.
How are bacteria chemically transformed to become competent to be able to scavenge DNA?
Treated with calcium chloride to precipitate and put in ice bath for cold shock process
How are bacteria transformed using electroporation?
Hit with millisecond electrical pulses which induces temporary pores to form in their cell membrane so plasmids can enter.
Cell membrane reseals after
What is a vector?
A plasmid
A carrier of DNA
What features make a good vector?
Small, stable and safe
High copy number
Selectable phenotypic trait
High density of restriction enzyme sites
What is the difference between plasmids and bacteriophages?
Plasmid- naturally found in bacteria
Bacteriophage- genetically engineered virus vectors
What are some advantages of small plasmids?
resistant to damage by mechanical shearing
ease of isolation
high copy number per cell
Restriction Endonucleases grouped in one small region is called a Polylinker or a Multiple Cloning Site. What does this allow for?
Allows linearisation of the plasmid rather than fragmentation
How do we select and identify for cells/bacteria that have the recombinant protein in them vs those that don’t?
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)
What is pBR322?
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.
What is Replica Plating and how is it used?
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.
What are the advantages of pUC-Series Plasmid Vectors?
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
Describe how the non-recombinant form of pUC19 comes about?
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
Describe how the recombinant form of pUC19 comes about?
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
Give an advantage of pZErO-1 vector?
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.
