The monoclonal myth

 Abstract

-Addenda to: Quantifying and  resolving multiple vector transformants in S. cerevisiae  plasmid libraries

Most researchers confidently assume that transformation of recombinant plasmid libraries into
microbial hosts followed by outgrowth of isolated colonies results in a “one cell – one mutant gene –
one protein variant” paradigm. Indeed, this assumption is supported by the overwhelming majority of
published studies employing bacterial expression hosts. In stark contrast, we recently reported on
Saccharomyces cerevisiae libraries containing unexpectedly high frequencies of cells harboring
heterogeneous mixtures of plasmids, so called Multiple Vector Transformants (MVT). Intriguingly,
we observed that yeast MVT persist as a significant proportion of populations for multiple
generations. MVT can lead to misidentification of isolated mutants, loss of functionally enhanced
clones, and unwitting propagation of false positives derived from contaminating control sequences.
Such experimental complications can have devastating outcomes in the context of protein
engineering by combinatorial library screening. Herein, we demonstrate that the phenomenon of
MVT is not restricted to vectors bearing the CEN/ARSH origin of replication, but may be an even
greater concern when using high copy 2 µm plasmids. To mitigate the risks associated with MVT,
we have developed an optimized sequencing procedure that facilitates rapid and reliable
identification of MVT among clones of interest. In our experience, MVT and their associated risks
can be virtually eliminated by employing extended liquid outgrowths of transformed populations and
archiving sequence-verified, monoclonal, mutant genes from cell-templated PCR amplicons.

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Pages
225 - 228
doi
10.4161/bbug.1.3.11724
Type
Addendum
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The monoclonal myth