Lecture

11 Summary
Wednesday, November 15, 2017 2:27 PM

Plasmid cloning strategy: (6 steps total)

1. Choose an appropriate plasmid vector
pUC19: for generating large quantities of target DNA
Has LacZ w/ MCS inserted in it, ori site, AMPr gene
2.7kB
Use alpha complementation (blue & white colonies on lb/AMP agar plate)
pGEX vectors: for fusion protein expression in bacteria cells
Ori site, LaciQ gene, Ampr gene, glutathione S-transferase, MCS
Size 4900bp
Foreign proteins expressed as fusions to C-terminus of glutathione S-
transferase (GST)
pGEX has Ptac promoter (for inducible expression of GST-tagged recomb
protein)
The LacI gene encodes for LacI Repressor > it represses Ptac promoter
IPTG: inducer that inactivates LacI repressor protein!
Fusion protein can be purified from lysed bacterial cells (GST moiety has
affinity for glutathione)
To purify, immobilize glutathion onto agarose beads > GST moiety
will bind to these!
Fusion protein is then incubated w/ site specific protease (factor Xa) to
remove the GST portion
pcDNA3: target gene expression in mammalian cells
Ampicillin, ColE1ori, Neomycin, multiple other ori sites (3 total), Pcmn,
BGH pA?
Size 5.4kb
Contains CMV (ctomegalovirus) = early promoter
Recognized by mammalian transcription machinery
Has eukaryotic transcription termination/polyadenylation site
Polyadneylation machinery adds polyA tail to nascent RNA molecule
Aids in transcription termination and protects mRNA from
degradation
Neomycin resistance used as a selection marker in mammalian cells
T7 promoter: recognized by T7 RNA polymerase
PURPOSE: add purified T7 RNA pol to test Tube containing pcDNA3,
dNTPs, right buffer = get RNA transcript of DNA insert!
RNA transcription used as strand-specific probe or in vitro
 degradation
Neomycin resistance used as a selection marker in mammalian cells
T7 promoter: recognized by T7 RNA polymerase
PURPOSE: add purified T7 RNA pol to test Tube containing pcDNA3,
dNTPs, right buffer = get RNA transcript of DNA insert!
RNA transcription used as strand-specific probe or in vitro
translation of protein: yield target proteins in shorter amt of
time compared to in vivo system
In vitro translation advantage over in vivo system when
over-expression of product is toxic to host cell OR
protein undergoes rapid proteolytic degradation by
intracellular proteases

2. Restriction digest target DNA & plasmid vector to generate compatible ends
Target DNA & plasmid vector should have internal sites for restriction enzymes
chosen for cloning
3. Ligate target DNA w/ plasmid vector
Cloning target DNA w/ homologous ends: (aka same restriction site on each
end!)
1st cleave plasmid w/ RE = generation linear plasmid
2nd inactivate or remove RE after cleavage rxn completed
Use phenol/chloroform extraction OR column purification
PROBLEM: mixing linearized DNA plasmid w/ ligase = high vector self-
ligation product
SOLUTION: treat linearized vector w/ phosphatase = it removes the
5phosphate on 5end of DNA
Now the vector can no longer serve as a substrate for self-ligation by
ligase!!
Now the 5phosphate on the target DNA will allow ligase to ligate
target DNA to vector
NOTE: remove phosphatase before adding target to vector (or the 5P on
target DNA will also be removed)
PROBLEM #2: target DNA can also self-ligate
NOTE: self-ligated target DNA cannot propagate cuze it doesnt have
an ori site
So it wont make undesired, non-recombinant bacterial clones
yayyyy
Cloning target DNA w/ heterologous ends (aka different restriction sites on one
end, ex. EcoRI & BamHI)
Cleaves plasmids w/ these enzymes
Delete small polylinker DNA piece (lies btw the two sites)
Dont need a phosphatase treatment in this case! (BUT WHY THOUGH?)
4. Transform E. Coli w/ ligation products
Introduce recombinant molecule into bacterial host!
 Cleaves plasmids w/ these enzymes
Delete small polylinker DNA piece (lies btw the two sites)
Dont need a phosphatase treatment in this case! (BUT WHY THOUGH?)
4. Transform E. Coli w/ ligation products
Introduce recombinant molecule into bacterial host!
Chemical treatment makes E. Coli cells more competent
Treat w/ divalent cation (ex. Ca2+, Rb2+) since surface is (-) charged, DNA
is also (-) charged
These cations allow DNA to adhere to cell surface!
Cations can also perturb cell walls & membrane = lets exogenous
DNA be taken up easier
1st competent bacterial cells are incubated w/ DNA ligation products on ice!
Then cells are heat shocked (42C for 30 sec) = generate thermo imbalance
= increase DNA uptake
Another possible method: electroporation (use high voltage to poke temporary
holes in membranes/walls)
10-20kv/cm for few milliseconds = DNA enters cell & cell membrane/walls
are rapidly repaired :)
Finally, choose proper strain based on your experiemnt
Ex. Choose E. Coli w/ 5region in LacZ deleted to allow for alpha
complementation to occur with pUC-vector
5. Grow on agar plates w/ selection for antibiotic resistance
Even w/ great transforming procedures, only a few cells will be transformed AND
not all plasmids taken up will have the targetDNA attached to it
Blue-White Color screening w/ pUC19 vector (aka alpha complementation from
lecture 10)
Plate: x-gal/amp agar plate
Bacteria w/out plasmid wont grow AND bacteria w/out insert will
grow but be blue
You are selecting for white colonies (have recombinant vector)
Isolate plasmid DNA from transformed bacterial cells ==> cleave w/ RE ==> do
gel electrophoresis
To ID that the cells have the right orientation (for homologous ends)
Check orientation of insert using restriction enzyme (or enzymes) = cut
asymmetrically w/in insert & in vector)
Isolate plasmid DNA from bact cells = use specific primers & do PCR
Get appropirate size PCR products ONLY IF plasmid has insert in right
orientation!
Isolate plasmid DNA from transformed bacteria cells (sequence the junction of
vector-insert

6. ID bacterium clones carrying correct plasmid-target DNA recombinants

In E.coli, plasmid DNA is usually Closed-circular and supercoiled
Alkaline lysis extraction: most frequently used method for isolating circular
 Isolate plasmid DNA from transformed bacteria cells (sequence the junction of
vector-insert

6. ID bacterium clones carrying correct plasmid-target DNA recombinants

In E.coli, plasmid DNA is usually Closed-circular and supercoiled
Alkaline lysis extraction: most frequently used method for isolating circular
plasmid DNA from e.coli
NaOH and SDS are used to lyse E.coli
NaOH also denatures DNA (both bacterial & plasmid genome DNA)
Follow alkaline lysis, plasmid DNA is no longer closed circular & supercoiled ==>
still constrained & remain associated together
Genomic DN exists as nicked circular form in E. coli
Do denaturation = 2 physically separated strands from each other =
become large pieces of ssDNA
Following alkaline lysis, potassium acetate (used to neutralize alkaline) =
neutralization
Neutralization = leads to quick renaturation of plasmid DNA
Ds plasmid DNA remains in liquid solution
Genomic DNA (already fully separated) doesn't renature quickly = remains ss
Now have large ssDNAs = these have low solubility in high salt
E.coli genomic DNAs are associated w/ cell membrane (insoluble) at multiple
places
SOOOOO: genomic DNAs percipitate out of solution
Potassium acetate ALSO: reacts w/ SDS to form KDS
KDS: is insoluble & precipitates from the solution
Allows for easy removal of SDS from the solution

Centrifuge step: remove all insoluble garbage and only leave plasmid DNA in
solution
Plasmi DNA can then be precipitated out by alcohol precipitation followed by
centrifugation