Value for money? Yes! #PostTransformationalVectorAmplification would give you that! #Pichia pastoris has many advantages over bacterial and non-Methylotrophic #yeasts in terms of #ProteinExpression. With #PTVA a lot more protein can be obtained by inducing transformants to make more copies of the recombinant #DNA. Lets understand how!
Principle
Although the actual molecular mechanism of #PTVA is not well understood, the hypothesis that is widely accepted states that it happens because of #SpontaneousAmplification of the resistance marker. Now #Zeocin™ resistance marker produces a protein that sequesters the antibiotic (rather than an enzyme which catalyzes its degradation). So #PTVA generates increased copies of the entire vector, producing increased levels of resistance protein and thus increased sensitivity to #Zeocin™ .
Now, this #SpontaneousAmplification stems from initial integration of the #vector part in the #Yeast genome, which is subsequently followed by a second round of integration with more free #vectors. This is supported by strong evidence the event can be readily stimulated in vivo.
Method
Transformation: Up to 5 ug of #SacI-digested plasmid #DNA is transformed into ~40uL of #Pichia pastoris competent cells by electroporation in a 2-mm gap cuvette (a charging voltage of 1500 V, a resistance of 129 O, a field strength of 7500 kV cm1 and a pulse length of 5 ms). After pulse, 500 uL of cold 1M #sorbitol was added to the cells, followed by 500 uL of #YPD medium. The entire suspension was then transferred into a 1.5mL centrifuge tube and incubated at 30C for about 4 h,100 r.p.m.
Plating: The whole mix was plated on either #Zeocin (100 ug/mL) or another selective plate and incubated at 30C.
Master Plate: Single colonies from that plate were then streaked/ spotted onto a #YPD master plate (100 ug/mL Zeocin) and incubated for 3-5 days.
The same process was repeated for the spots with 200, 300, 500, 1000, 2000 ug/mL Zeocin.
Selection of PTVA enriched clones: The colonies growing with 2000 ug/mL #Zeocin were spotted on #YNBmethanol plates for about 14 h and beta-lactamase activity solution was spotted on each colony. Beta-lactamase activity corresponds to expression of #GOI and manifests as pink to red color development. Select the darkest red colony and you have the #PTVA enriched clone with highest number of copies.
Merits
#PTVA has been carried out in two most common antibiotic-based systems: #Zeocin and #G418, as well as for a large number of proteins, suggesting #replicability.
#PTVA could yield even 52 copies of a gene in an experimental attempt pointing at its #scalability and #yield.
Demerits
This takes at least a week to get perfect clones, not time-efficient.
Only ~6% of the clones are #JackpotClones harboring >10 repeats of the GOI.
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