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Title: Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

Abstract

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefitmore » from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.« less

Authors:
 [1];  [1];  [2];  [1];  [1]
  1. University of Arizona
  2. Bellevue University
Publication Date:
Research Org.:
Univ. of Arizona, Tucson, AZ (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1093679
Report Number(s):
DOE-DANF-0003046-Q2
Journal ID: ISSN 0921-8971; 28302-Q
DOE Contract Number:  
EE0003046
Resource Type:
Journal Article
Journal Name:
Journal of Applied Phycology
Additional Journal Information:
Journal Volume: 25; Journal Issue: 5; Journal ID: ISSN 0921-8971
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Chloroplast isolation; oleaginous algae; sonication

Citation Formats

Angelova, Angelina, Park, Sang-Hycuk, Kyndt, John, Fitzsimmons, Kevin, and Brown, Judith K. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing. United States: N. p., 2013. Web. doi:10.1007/s10811-013-0125-1.
Angelova, Angelina, Park, Sang-Hycuk, Kyndt, John, Fitzsimmons, Kevin, & Brown, Judith K. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing. United States. https://doi.org/10.1007/s10811-013-0125-1
Angelova, Angelina, Park, Sang-Hycuk, Kyndt, John, Fitzsimmons, Kevin, and Brown, Judith K. 2013. "Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing". United States. https://doi.org/10.1007/s10811-013-0125-1. https://www.osti.gov/servlets/purl/1093679.
@article{osti_1093679,
title = {Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing},
author = {Angelova, Angelina and Park, Sang-Hycuk and Kyndt, John and Fitzsimmons, Kevin and Brown, Judith K},
abstractNote = {With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.},
doi = {10.1007/s10811-013-0125-1},
url = {https://www.osti.gov/biblio/1093679}, journal = {Journal of Applied Phycology},
issn = {0921-8971},
number = 5,
volume = 25,
place = {United States},
year = {Sun Sep 01 00:00:00 EDT 2013},
month = {Sun Sep 01 00:00:00 EDT 2013}
}