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Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

Overview of attention for article published in Microbial Cell Factories, August 2016
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  • Above-average Attention Score compared to outputs of the same age (63rd percentile)
  • High Attention Score compared to outputs of the same age and source (82nd percentile)

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5 tweeters

Citations

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29 Dimensions

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136 Mendeley
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Title
Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications
Published in
Microbial Cell Factories, August 2016
DOI 10.1186/s12934-016-0540-5
Pubmed ID
Authors

Andrea Camattari, Amelia Goh, Lian Yee Yip, Andy Hee Meng Tan, Sze Wai Ng, Anthony Tran, Gaowen Liu, Ivan Liachko, Maitreya J. Dunham, Giulia Rancati

Abstract

Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence ("panARS") in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells. A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest. An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.

Twitter Demographics

The data shown below were collected from the profiles of 5 tweeters who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 136 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Spain 1 <1%
China 1 <1%
Austria 1 <1%
Unknown 133 98%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 26 19%
Researcher 22 16%
Student > Master 22 16%
Student > Bachelor 14 10%
Other 6 4%
Other 11 8%
Unknown 35 26%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 46 34%
Agricultural and Biological Sciences 43 32%
Engineering 3 2%
Social Sciences 3 2%
Immunology and Microbiology 2 1%
Other 5 4%
Unknown 34 25%

Attention Score in Context

This research output has an Altmetric Attention Score of 3. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 17 August 2016.
All research outputs
#3,324,702
of 8,229,276 outputs
Outputs from Microbial Cell Factories
#140
of 576 outputs
Outputs of similar age
#85,001
of 233,219 outputs
Outputs of similar age from Microbial Cell Factories
#5
of 28 outputs
Altmetric has tracked 8,229,276 research outputs across all sources so far. This one has received more attention than most of these and is in the 59th percentile.
So far Altmetric has tracked 576 research outputs from this source. They receive a mean Attention Score of 2.8. This one has done well, scoring higher than 75% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 233,219 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 63% of its contemporaries.
We're also able to compare this research output to 28 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 82% of its contemporaries.