Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial c...

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Autores principales:	Aditya Sarnaik, Apurv Mhatre, Muhammad Faisal, Dylan Smith, Ryan Davis, Arul M Varman
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c7001b5c8c774f04a0dee45f8c97b919
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spelling	oai:doaj.org-article:c7001b5c8c774f04a0dee45f8c97b9192021-11-25T06:19:11ZNovel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.1932-620310.1371/journal.pone.0251640https://doaj.org/article/c7001b5c8c774f04a0dee45f8c97b9192021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0251640https://doaj.org/toc/1932-6203Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.Aditya SarnaikApurv MhatreMuhammad FaisalDylan SmithRyan DavisArul M VarmanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 5, p e0251640 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Aditya Sarnaik Apurv Mhatre Muhammad Faisal Dylan Smith Ryan Davis Arul M Varman Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
description	Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.
format	article
author	Aditya Sarnaik Apurv Mhatre Muhammad Faisal Dylan Smith Ryan Davis Arul M Varman
author_facet	Aditya Sarnaik Apurv Mhatre Muhammad Faisal Dylan Smith Ryan Davis Arul M Varman
author_sort	Aditya Sarnaik
title	Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
title_short	Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
title_full	Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
title_fullStr	Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
title_full_unstemmed	Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.
title_sort	novel perspective on a conventional technique: impact of ultra-low temperature on bacterial viability and protein extraction.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/c7001b5c8c774f04a0dee45f8c97b919
work_keys_str_mv	AT adityasarnaik novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction AT apurvmhatre novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction AT muhammadfaisal novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction AT dylansmith novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction AT ryandavis novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction AT arulmvarman novelperspectiveonaconventionaltechniqueimpactofultralowtemperatureonbacterialviabilityandproteinextraction
_version_	1718413952359071744

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.

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