BACKGROUND
a key aspect of combinatorial protein engineering is the absolute requirement that each protein variant remain physically associated with its encoding gene .
while there are numerous strategies for establishing and maintaining this critical link, the dominant approach remains recombinant microbial expression.
the compartmentalized nature of microbial cells ensures the physical association of mutant genes with their cognate variant proteins, and also provides the molecular machinery and a regulated environment for efficient transcription and translation of library members.
generally, it is assumed that transformation of microbial hosts with a recombinant plasmid library results in a "one cell - one mutant gene - one protein variant" paradigm.
this assumption motivates clonal high throughput library screens as a strategy for identifying functionally enhanced proteins and their encoding genes.
although the first description of high efficiency escherichia coli electroporation suggested that co-transformation with multiple plasmids may occur at high dna concentrations  <cit> , the potential for simultaneous transformation of more than one plasmid into any given microbial cell during library construction has typically been dismissed as an improbable event  <cit> .
furthermore, it has been assumed that rare occurrences of multiple vector transformants  are quickly resolved into clonal progeny by plasmid partitioning during cell division  <cit> .
thus, protein engineers have long been comfortable in the assumption that sequenced genes from selected clones do in fact encode for the proteins producing the observed clonal phenotype.
while the one cell - one mutant gene - one protein variant paradigm has proven sufficiently accurate in the vast majority of reported experimental outcomes, there have been several high profile retractions wherein confounding results and inaccurate conclusions were specifically traced to contamination from mvt  <cit>  and other cases in which mvt could have contributed to unexplained in vivo results  <cit> .
these examples underscore the liabilities associated with mvt in recombinant library populations.
to examine the issue of mvt in bacterial libraries,  <dig> and 3-plasmid model systems based on orthogonal selectable markers have been characterized in detail.
one report demonstrated that multiply transformed cells can represent up to  <dig> % of electroporated e. coli populations under the conditions tested  <cit> .
a second study showed that mvt resulting from phagmid infection can persist as a significant proportion of cell populations even after five consecutive overnight outgrowths  <cit> .
these reports have significantly advanced knowledge regarding mvt in the gram-negative bacterial host e. coli, but no analogous detailed information regarding yeast libraries is currently available.
early studies of saccharomyces cerevisiae plasmid transformation indicated that this common yeast host may exhibit a higher than expected frequency of mvt  <cit> , although these preliminary studies involved low efficiency spheroplast-cacl <dig> transformation, which is not suitable for construction of large libraries.
subsequent studies using low copy number plasmids bearing centromeric origins of replication revealed toxicity associated with mvt  <cit> , suggesting that their persistence was unlikely.
thus, while it has long been known that s. cerevisiae are prone to acquisition of multiple plasmids during transformation, there remains a dearth of quantitative information describing the prevalence and maintenance of mvt events in conditions representative of recombinant library screening experiments.
furthermore, no previous studies have addressed methods for reducing the occurrence of mvt.
given the widespread use of this microorganism as a screening platform for combinatorial protein engineering, two hybrid protein-protein interactome mapping, and other library screening protocols, a detailed characterization of s. cerevisiae mvt was undertaken in the context of low copy number plasmid libraries constructed by yeast mediated homologous recombination.
experiments were designed to answer three key questions regarding the nature of mvt in recombinant s. cerevisiae libraries:
1) what fraction of successfully transformed library members bear two or more plasmids?
2) among these mvt, how many unique mutant genes does the average clone harbor?
3) under standard liquid growth conditions, how long do mvt persist as a significant proportion of the library population?
the answers to these questions have wide ranging implications for laboratories using yeast as an expression and screening platform for recombinant dna libraries.
RESULTS
proportion of multiple vector transformants in initial library populations
previous studies using a selection based, two plasmid model system in e. coli demonstrated that transformation of this bacterial host with  <dig> ng of plasmid dna resulted in a  <dig> % double transformant frequency  <cit> .
note that these experiments were designed to model parental plasmid contamination and therefore used a skewed 1: <dig> ratio of the two model plasmids.
to assess the proportion of mvt in yeast libraries generated using similar total plasmid concentrations, site-directed lysozyme gene libraries were constructed using the isor strategy  <cit> , and freshly prepared electrocompetent s. cerevisiae were transformed with  <dig> ng of vector backbone and  <dig>  μg of gene insert yielding library l-1: <dig> .
control transformations with linear vector alone yielded approximately 1000-fold fewer transformants than did the transformations with both vector and gene insert .
in the studies presented below, all mutations mapped to one of eight codons that were specifically targeted by combinatorial mutagenesis.
this observation provides strong evidence that the sequenced mutations resulted from library construction and not from downstream pcr processing.
to assess the frequency of mvt in l-1: <dig> , an aliquot of the transformation mixture was diluted and plated on selective media at a concentration appropriate to yield spatially segregated, spherical colonies upon outgrowth.
the mutant gene encoded by each yeast colony were amplified using an optimized procedure for yeast-templated, high fidelity pcr as described in the materials and methods .
the resulting pcr amplicons from  <dig> randomly selected colonies were subsequently sequenced.
based on visual inspection of the sequencing chromatograms , greater than 30% of the sampled colonies were mvt .
while the estimated  <dig>  population proportion of mvt in library l-1: <dig>   was almost 100-fold greater than that previously reported for e. coli, the dna concentration in this transformation solution was relatively low compared to typical transformation protocols designed to yield large yeast libraries.
* library generated using low efficiency, freeze-thawed electrocompetent cells.
to assess the frequency of mvt in an s. cerevisiae library generated using a standard protocol  <cit> , freshly prepared electrocompetent yeast were transformed with  <dig> μg of vector backbone and  <dig> μg of mutagenized dna insert yielding library h-1: <dig>  following dna sequence analysis of  <dig> randomly selected colonies, the observed sample proportion of mvt was  <dig>  , and the estimated population proportion was  <dig>  .
the difference between mvt proportions in h-1: <dig> and l-1: <dig>  was clearly significant , but it was unclear whether the increased mvt frequency in h-1: <dig> was a result of the 2-fold increased concentration of vector backbone, the 2-fold increased ratio of insert to vector, or some combination thereof.
therefore, a third library  was generated by transforming freshly prepared yeast with  <dig> μg of vector backbone  and  <dig>  μg of gene insert .
a total of  <dig> mvt were found amongst  <dig> randomly sampled colonies , and the estimated h-1: <dig>  population proportion of mvt was  <dig>  .
this proportion was significantly different from that of l-1: <dig>   and h-1: <dig> .
these results suggest that both vector backbone concentration as well as gene insert to vector ratio are determinants of yeast mvt frequency.
a closer inspection of the above results suggested a linear correlation between mvt frequency and library size .
to determine if mvt frequency did in fact exhibit a simple correlation with transformation efficiency, library h-1: <dig>  was remade by low efficiency transformation of freeze-thawed electrocompetent yeast yielding library h-1: <dig> *.
while the size of h-1: <dig> * was 10-fold smaller than h-1: <dig> , the proportion of mvt in the small freeze-thaw library was essentially identical to that of the corresponding larger library .
thus, while dna concentration plays a critical role determining library proportion of mvt , it appears that mvt proportions are independent of overall transformation efficiency .
average number of unique genes in multiple vector transformants
in addition to knowing the fraction of mvt in a library population, it would also be advantageous to know the average number of unique plasmids harbored by multiply transformed cells.
this information is particularly relevant given the dominance of mvt in the  <dig> μg vector backbone libraries.
to estimate this value, aqueous suspensions of  <dig> randomly selected mvt from both library h-1: <dig> and h-1: <dig>  were restreaked to single cell resolution on dextrose growth agar yielding isolated, spherical, progeny colonies upon outgrowth .
lysozyme genes from  <dig> to  <dig> individual colonies of each plate were sequenced .
the number of unique gene sequences harbored by each mvt was extrapolated from these data using a first order jackknife estimator  <cit> , and the population median for each library was derived from the distribution of values for the  <dig> corresponding clones .
mvt from library h-1: <dig> were estimated to harbor a median of  <dig>  unique plasmids and those of h-1: <dig>  carried  <dig>  unique plasmids.
because data from h-1: <dig>  did not appear to be normally distributed , a non-parametric wilcoxon-mann-whitney test was used to determine that the distributions of unique plasmids among mvt of h-1: <dig>  and h-1: <dig> were not significantly different .
thus it is interesting to note that, while library proportions of mvt appear to be a function of gene insert concentration , the average plasmid content of mvt populations is independent of this variable.
persistence of multiple vector transformants during liquid culture outgrowth
one approach to eliminate mvt from library populations is to perform liquid culture outgrowth leading to segregation and copy number control of cen-based plasmids during cell division  <cit> .
in an effort to determine the time necessary to eliminate mvt as a significant subpopulation, their frequency in the h-1: <dig>  library was assessed as a function of growth time.
following electroporation, the transformed library was subcultured into dextrose growth media and grown with aeration at 30°c for up to  <dig> hours.
cultures were serial diluted to prevent saturation during outgrowth.
aliquots were spread to single cell resolution on dextrose growth agar every two hours for the first  <dig> hours.
an additional aliquot was plated at  <dig> hours.
following incubation of the plates to yield spatially resolved single colonies, 10- <dig> randomly selected spherical colonies from various time points were sequenced as described in the materials and methods.
the observed proportion of mvt in the library population was plotted as a function of outgrowth time .
surprisingly, 20% of the sampled clones continued to harbor more than one unique plasmid even after  <dig> hours of outgrowth.
not until the  <dig> hour time point did the mvt subpopulation decrease below the detection threshold for the experimental sample size.
discussion
while the prevalence and persistence of mvt in recombinant e. coli libraries has been studied previously, the issue of mvt in s. cerevisiae libraries has received only cursory and indirect attention.
to shed light on this important issue, mvt were quantitatively analyzed in experimental libraries constructed by yeast homologous recombination.
unlike model systems based on selective genetic markers that produce readily observed phenotypes , the practical nature of the libraries studied here necessitated direct dna sequencing to identify and characterize mvt.
as such, the sample sizes used in this study are smaller than those that would have been possible using orthogonal antibiotic or auxotrophic markers as a model system.
however, analysis of a realistic library, currently in use for practical protein engineering, provided insights that would have been inaccessible using any convenient model.
for example, an estimation of average unique plasmids harbored by mvt cells would not be accessible a priori using a model system with a small number of selectable markers; an accurate estimate requires that the mean number of unique sequences per mvt be exceeded by the total number of unique sequences in the library population.
furthermore, due to the particularly high degree of similarity among genes in mutant libraries, yeast mediated homologous recombination between two or more unique genes in any given mvt could produce additional library diversity not present in the original transformation mixture  <cit> .
the lack of similarity between genes for different antibiotic or auxotrophic selection markers dictates that such post-transformation diversification would not be captured by convenient model systems.
therefore, analysis of an actual mutant gene library was employed in order to provide data with more direct relevance to practical library screening experiments.
underscoring the potential impact of yeast mvt, it was observed that cells bearing multiple plasmids persisted in library populations at ~20% frequency for more than  <dig> hours, despite the stringently regulated low copy cen/arsh <dig> origin employed here.
given the potential for mvt to interfere with accurate interpretation of experimental results, the staggering proportions of mvt observed in these studies advocates for careful examination of this issue as a part of any yeast library screening protocol.
it is emphasized that the studies described here employed a plasmid bearing the ura <dig> selectable marker, and alternative markers were not examined.
in previous studies, however, cen6/arsh <dig> plasmids bearing either the ura <dig>  leu <dig>  his <dig> or trp <dig> markers were shown to exhibit less than a 2-fold difference in transformation efficiency, and the mitotic stabilities of these markers were similar under selective pressure  <cit> .
these results suggest that a plasmid's auxotrophic marker would not be a dominant factor relating to mvt in library populations.
our results suggest two alternative strategies for limiting mvt in recombinant s. cerevisiae libraries.
in one approach, s. cerevisiae libraries can be outgrown for extended time periods, i.e.
>  <dig> hours, in media repressing recombinant gene expression.
importantly, it is possible that yeast vectors bearing higher copy number origins, for example the  <dig> μm origin, would require even longer outgrowths for complete segregation of plasmids to meet the one cell-one mutant gene-one protein variant paradigm.
as an alternative to long outgrowths, lower concentrations of dna may be used to transform yeast hosts thereby reducing the frequency of mvt.
compare for example the fraction of mvt in libraries l-1: <dig>  and h-1: <dig>  .
however, the benefit of reduced mvt frequency must be weighed against the smaller size of libraries obtained with lower dna concentrations.
although the hypercompetent nature of s. cerevisiae represents a potential liability for clonal library screening, it may also constitute a previously unrecognized advantage to the protein engineer.
the size of library populations is typically determined by plating serial dilutions of transformed cells onto selective media shortly after the transformation procedure.
colony forming units are enumerated following outgrowth, and the total library size is back calculated.
based on the results reported here, this established methodology may under represent the true diversity of analogously constructed yeast libraries by 4-fold or more.
this additional diversity may be accessed by allowing sufficiently long outgrowths to completely segregate mvt plasmids prior to clonal library screening.
alternatively, a single cell variation of "pooled" clone screening  <cit>  could be implemented to take advantage of the additional diversity.
from this perspective, mvt in a library represent a diversity advantage, provided that one is fully aware of the potential pitfalls and remedies for addressing them.
ultimately, great care must be taken by the protein engineer to ensure that any observed improvement in protein function is correlated with the correct mutant gene sequence.
conventional validation of the genotype-phenotype link in selected s. cerevisiae library members requires outgrowth of the isolated yeast cells, isolation of low quality/quantity yeast plasmid dna, transformation into an e. coli host, outgrowth of the transformed bacteria on solid media, outgrowth of selected bacterial transformants in liquid media, isolation of high quality/quantity plasmid dna from bacterial cells, sequencing gene insert from several colonies, retransformation of the sequenced plasmid into the yeast host, and revalidation of the originally observed phenotype.
due to slow growth rates of yeast, this process typically results in inconveniently long timeframes for clone validation , and it is particularly arduous when validating large numbers of clones.
this time, labor, and resource intensive process limits productivity, and can easily become intractable for small labs examining large libraries.
in contrast to the conventional, laborious, shuttle vector strategy described above, the yeast-templated pcr and direct amplicon sequencing approach developed for these studies yields robust sequencing results in roughly  <dig> day.
importantly, this technique was shown to be highly reliable as verified by extensive characterization of a combinatorial, site-directed mutant library in which all sequenced mutations mapped to the target sites.
it is anticipated that other researchers may find this procedure useful in speeding analysis and preliminary validation of clones selected from recombinant yeast libraries, although genes of particular interest should ultimately be confirmed via sequencing of high quality plasmid template.
CONCLUSIONS
using carefully designed dna sequencing studies of a site-directed mutant gene library, the frequency and persistence of mvt in s. cerevisiae has been quantitatively evaluated with cen/arsh <dig> based plasmids bearing a ura <dig> selectable marker.
in libraries constructed by electroporation and homologous recombination, both the vector backbone concentration and the vector to gene insert ratio were found to influence the population proportion of mvt.
however, mvt frequency was shown to be independent of transformation efficiency.
an awareness of the potential for mvt to dominate yeast libraries will help researchers avoid associated pitfalls.
to facilitate rapid validation of genotype-phenotype linkage in yeast clones selected from recombinant libraries, an optimized procedure for yeast-templated, high fidelity pcr and direct amplicon sequencing has been described.

 a key aspect of combinatorial protein engineering is the absolute requirement that each protein variant remain physically associated with its encoding gene. while there are numerous strategies for establishing and maintaining this critical link, the dominant approach remains recombinant microbial expression. the compartmentalized nature of microbial cells ensures the physical association of mutant genes with their cognate variant proteins, and also provides the molecular machinery and a regulated environment for efficient transcription and translation of library members. generally, it is assumed that transformation of microbial hosts with a recombinant plasmid library results in a "one cell - one mutant gene - one protein variant" paradigm. this assumption motivates clonal high throughput library screens as a strategy for identifying functionally enhanced proteins and their encoding genes. although the first description of high efficiency escherichia coli electroporation suggested that co-transformation with multiple plasmids may occur at high dna concentrations, the potential for simultaneous transformation of more than one plasmid into any given microbial cell during library construction has typically been dismissed as an improbable event. furthermore, it has been assumed that rare occurrences of multiple vector transformants are quickly resolved into clonal progeny by plasmid partitioning during cell division. thus, protein engineers have long been comfortable in the assumption that sequenced genes from selected clones do in fact encode for the proteins producing the observed clonal phenotype. while the one cell - one mutant gene - one protein variant paradigm has proven sufficiently accurate in the vast majority of reported experimental outcomes, there have been several high profile retractions wherein confounding results and inaccurate conclusions were specifically traced to contamination from mvt and other cases in which mvt could have contributed to unexplained in vivo results. these examples underscore the liabilities associated with mvt in recombinant library populations. to examine the issue of mvt in bacterial libraries, and 3-plasmid model systems based on orthogonal selectable markers have been characterized in detail. one report demonstrated that multiply transformed cells can represent up to % of electroporated e. coli populations under the conditions tested. a second study showed that mvt resulting from phagmid infection can persist as a significant proportion of cell populations even after five consecutive overnight outgrowths. these reports have significantly advanced knowledge regarding mvt in the gram-negative bacterial host e. coli, but no analogous detailed information regarding yeast libraries is currently available. early studies of saccharomyces cerevisiae plasmid transformation indicated that this common yeast host may exhibit a higher than expected frequency of mvt, although these preliminary studies involved low efficiency spheroplast-cacl transformation, which is not suitable for construction of large libraries. subsequent studies using low copy number plasmids bearing centromeric origins of replication revealed toxicity associated with mvt, suggesting that their persistence was unlikely. thus, while it has long been known that s. cerevisiae are prone to acquisition of multiple plasmids during transformation, there remains a dearth of quantitative information describing the prevalence and maintenance of mvt events in conditions representative of recombinant library screening experiments. furthermore, no previous studies have addressed methods for reducing the occurrence of mvt. given the widespread use of this microorganism as a screening platform for combinatorial protein engineering, two hybrid protein-protein interactome mapping, and other library screening protocols, a detailed characterization of s. cerevisiae mvt was undertaken in the context of low copy number plasmid libraries constructed by yeast mediated homologous recombination. experiments were designed to answer three key questions regarding the nature of mvt in recombinant s. cerevisiae libraries: 1) what fraction of successfully transformed library members bear two or more plasmids? 2) among these mvt, how many unique mutant genes does the average clone harbor? 3) under standard liquid growth conditions, how long do mvt persist as a significant proportion of the library population? the answers to these questions have wide ranging implications for laboratories using yeast as an expression and screening platform for recombinant dna libraries.  proportion of multiple vector transformants in initial library populations previous studies using a selection based, two plasmid model system in e. coli demonstrated that transformation of this bacterial host with ng of plasmid dna resulted in a % double transformant frequency. note that these experiments were designed to model parental plasmid contamination and therefore used a skewed 1: ratio of the two model plasmids. to assess the proportion of mvt in yeast libraries generated using similar total plasmid concentrations, site-directed lysozyme gene libraries were constructed using the isor strategy, and freshly prepared electrocompetent s. cerevisiae were transformed with ng of vector backbone and μg of gene insert yielding library l-1:. control transformations with linear vector alone yielded approximately 1000-fold fewer transformants than did the transformations with both vector and gene insert. in the studies presented below, all mutations mapped to one of eight codons that were specifically targeted by combinatorial mutagenesis. this observation provides strong evidence that the sequenced mutations resulted from library construction and not from downstream pcr processing. to assess the frequency of mvt in l-1:, an aliquot of the transformation mixture was diluted and plated on selective media at a concentration appropriate to yield spatially segregated, spherical colonies upon outgrowth. the mutant gene encoded by each yeast colony were amplified using an optimized procedure for yeast-templated, high fidelity pcr as described in the materials and methods. the resulting pcr amplicons from randomly selected colonies were subsequently sequenced. based on visual inspection of the sequencing chromatograms, greater than 30% of the sampled colonies were mvt. while the estimated population proportion of mvt in library l-1: was almost 100-fold greater than that previously reported for e. coli, the dna concentration in this transformation solution was relatively low compared to typical transformation protocols designed to yield large yeast libraries. * library generated using low efficiency, freeze-thawed electrocompetent cells. to assess the frequency of mvt in an s. cerevisiae library generated using a standard protocol, freshly prepared electrocompetent yeast were transformed with μg of vector backbone and μg of mutagenized dna insert yielding library h-1: following dna sequence analysis of randomly selected colonies, the observed sample proportion of mvt was, and the estimated population proportion was. the difference between mvt proportions in h-1: and l-1: was clearly significant, but it was unclear whether the increased mvt frequency in h-1: was a result of the 2-fold increased concentration of vector backbone, the 2-fold increased ratio of insert to vector, or some combination thereof. therefore, a third library was generated by transforming freshly prepared yeast with μg of vector backbone and μg of gene insert. a total of mvt were found amongst randomly sampled colonies, and the estimated h-1: population proportion of mvt was. this proportion was significantly different from that of l-1: and h-1:. these results suggest that both vector backbone concentration as well as gene insert to vector ratio are determinants of yeast mvt frequency. a closer inspection of the above results suggested a linear correlation between mvt frequency and library size. to determine if mvt frequency did in fact exhibit a simple correlation with transformation efficiency, library h-1: was remade by low efficiency transformation of freeze-thawed electrocompetent yeast yielding library h-1: *. while the size of h-1: * was 10-fold smaller than h-1:, the proportion of mvt in the small freeze-thaw library was essentially identical to that of the corresponding larger library. thus, while dna concentration plays a critical role determining library proportion of mvt, it appears that mvt proportions are independent of overall transformation efficiency. average number of unique genes in multiple vector transformants in addition to knowing the fraction of mvt in a library population, it would also be advantageous to know the average number of unique plasmids harbored by multiply transformed cells. this information is particularly relevant given the dominance of mvt in the μg vector backbone libraries. to estimate this value, aqueous suspensions of randomly selected mvt from both library h-1: and h-1: were restreaked to single cell resolution on dextrose growth agar yielding isolated, spherical, progeny colonies upon outgrowth. lysozyme genes from to individual colonies of each plate were sequenced. the number of unique gene sequences harbored by each mvt was extrapolated from these data using a first order jackknife estimator, and the population median for each library was derived from the distribution of values for the corresponding clones. mvt from library h-1: were estimated to harbor a median of unique plasmids and those of h-1: carried unique plasmids. because data from h-1: did not appear to be normally distributed, a non-parametric wilcoxon-mann-whitney test was used to determine that the distributions of unique plasmids among mvt of h-1: and h-1: were not significantly different. thus it is interesting to note that, while library proportions of mvt appear to be a function of gene insert concentration, the average plasmid content of mvt populations is independent of this variable. persistence of multiple vector transformants during liquid culture outgrowth one approach to eliminate mvt from library populations is to perform liquid culture outgrowth leading to segregation and copy number control of cen-based plasmids during cell division. in an effort to determine the time necessary to eliminate mvt as a significant subpopulation, their frequency in the h-1: library was assessed as a function of growth time. following electroporation, the transformed library was subcultured into dextrose growth media and grown with aeration at 30°c for up to hours. cultures were serial diluted to prevent saturation during outgrowth. aliquots were spread to single cell resolution on dextrose growth agar every two hours for the first hours. an additional aliquot was plated at hours. following incubation of the plates to yield spatially resolved single colonies, 10- randomly selected spherical colonies from various time points were sequenced as described in the materials and methods. the observed proportion of mvt in the library population was plotted as a function of outgrowth time. surprisingly, 20% of the sampled clones continued to harbor more than one unique plasmid even after hours of outgrowth. not until the hour time point did the mvt subpopulation decrease below the detection threshold for the experimental sample size. discussion while the prevalence and persistence of mvt in recombinant e. coli libraries has been studied previously, the issue of mvt in s. cerevisiae libraries has received only cursory and indirect attention. to shed light on this important issue, mvt were quantitatively analyzed in experimental libraries constructed by yeast homologous recombination. unlike model systems based on selective genetic markers that produce readily observed phenotypes, the practical nature of the libraries studied here necessitated direct dna sequencing to identify and characterize mvt. as such, the sample sizes used in this study are smaller than those that would have been possible using orthogonal antibiotic or auxotrophic markers as a model system. however, analysis of a realistic library, currently in use for practical protein engineering, provided insights that would have been inaccessible using any convenient model. for example, an estimation of average unique plasmids harbored by mvt cells would not be accessible a priori using a model system with a small number of selectable markers; an accurate estimate requires that the mean number of unique sequences per mvt be exceeded by the total number of unique sequences in the library population. furthermore, due to the particularly high degree of similarity among genes in mutant libraries, yeast mediated homologous recombination between two or more unique genes in any given mvt could produce additional library diversity not present in the original transformation mixture. the lack of similarity between genes for different antibiotic or auxotrophic selection markers dictates that such post-transformation diversification would not be captured by convenient model systems. therefore, analysis of an actual mutant gene library was employed in order to provide data with more direct relevance to practical library screening experiments. underscoring the potential impact of yeast mvt, it was observed that cells bearing multiple plasmids persisted in library populations at ~20% frequency for more than hours, despite the stringently regulated low copy cen/arsh origin employed here. given the potential for mvt to interfere with accurate interpretation of experimental results, the staggering proportions of mvt observed in these studies advocates for careful examination of this issue as a part of any yeast library screening protocol. it is emphasized that the studies described here employed a plasmid bearing the ura selectable marker, and alternative markers were not examined. in previous studies, however, cen6/arsh plasmids bearing either the ura leu his or trp markers were shown to exhibit less than a 2-fold difference in transformation efficiency, and the mitotic stabilities of these markers were similar under selective pressure. these results suggest that a plasmid's auxotrophic marker would not be a dominant factor relating to mvt in library populations. our results suggest two alternative strategies for limiting mvt in recombinant s. cerevisiae libraries. in one approach, s. cerevisiae libraries can be outgrown for extended time periods, i.e. > hours, in media repressing recombinant gene expression. importantly, it is possible that yeast vectors bearing higher copy number origins, for example the μm origin, would require even longer outgrowths for complete segregation of plasmids to meet the one cell-one mutant gene-one protein variant paradigm. as an alternative to long outgrowths, lower concentrations of dna may be used to transform yeast hosts thereby reducing the frequency of mvt. compare for example the fraction of mvt in libraries l-1: and h-1:. however, the benefit of reduced mvt frequency must be weighed against the smaller size of libraries obtained with lower dna concentrations. although the hypercompetent nature of s. cerevisiae represents a potential liability for clonal library screening, it may also constitute a previously unrecognized advantage to the protein engineer. the size of library populations is typically determined by plating serial dilutions of transformed cells onto selective media shortly after the transformation procedure. colony forming units are enumerated following outgrowth, and the total library size is back calculated. based on the results reported here, this established methodology may under represent the true diversity of analogously constructed yeast libraries by 4-fold or more. this additional diversity may be accessed by allowing sufficiently long outgrowths to completely segregate mvt plasmids prior to clonal library screening. alternatively, a single cell variation of "pooled" clone screening could be implemented to take advantage of the additional diversity. from this perspective, mvt in a library represent a diversity advantage, provided that one is fully aware of the potential pitfalls and remedies for addressing them. ultimately, great care must be taken by the protein engineer to ensure that any observed improvement in protein function is correlated with the correct mutant gene sequence. conventional validation of the genotype-phenotype link in selected s. cerevisiae library members requires outgrowth of the isolated yeast cells, isolation of low quality/quantity yeast plasmid dna, transformation into an e. coli host, outgrowth of the transformed bacteria on solid media, outgrowth of selected bacterial transformants in liquid media, isolation of high quality/quantity plasmid dna from bacterial cells, sequencing gene insert from several colonies, retransformation of the sequenced plasmid into the yeast host, and revalidation of the originally observed phenotype. due to slow growth rates of yeast, this process typically results in inconveniently long timeframes for clone validation, and it is particularly arduous when validating large numbers of clones. this time, labor, and resource intensive process limits productivity, and can easily become intractable for small labs examining large libraries. in contrast to the conventional, laborious, shuttle vector strategy described above, the yeast-templated pcr and direct amplicon sequencing approach developed for these studies yields robust sequencing results in roughly day. importantly, this technique was shown to be highly reliable as verified by extensive characterization of a combinatorial, site-directed mutant library in which all sequenced mutations mapped to the target sites. it is anticipated that other researchers may find this procedure useful in speeding analysis and preliminary validation of clones selected from recombinant yeast libraries, although genes of particular interest should ultimately be confirmed via sequencing of high quality plasmid template.  using carefully designed dna sequencing studies of a site-directed mutant gene library, the frequency and persistence of mvt in s. cerevisiae has been quantitatively evaluated with cen/arsh based plasmids bearing a ura selectable marker. in libraries constructed by electroporation and homologous recombination, both the vector backbone concentration and the vector to gene insert ratio were found to influence the population proportion of mvt. however, mvt frequency was shown to be independent of transformation efficiency. an awareness of the potential for mvt to dominate yeast libraries will help researchers avoid associated pitfalls. to facilitate rapid validation of genotype-phenotype linkage in yeast clones selected from recombinant libraries, an optimized procedure for yeast-templated, high fidelity pcr and direct amplicon sequencing has been described. 
in addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. it was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than hours during liquid outgrowth. in order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by dna sequencing. as a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. in contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. these results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. the quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid. the previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. it is known that escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments.
 in addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. it is known that escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. as a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. in contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. in order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by dna sequencing.  it was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than hours during liquid outgrowth. both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion.  these results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. the previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. the quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid. 