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Biotechnol Lett (2011) 33:16151619 DOI 10.1007/s10529-011-0596-6
ORIGINAL RESEARCH PAPER
An improved colony PCR procedure for genetic screening of Chlorella and related microalgae
Minxi Wan Julian N. Rosenberg
Junaid Faruq Michael J. Betenbaugh
Jinlan Xia
Received: 14 December 2010 / Accepted: 10 March 2011 / Published online: 24 March 2011 Springer Science+Business Media B.V. 2011
Abstract A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of ve different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efcacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.
Keywords Chelex-100 Chlorella Colony
polymerase chain reaction Genetic screening
Microalgae
Introduction
Green microalgae of the Chlorella genus are widely utilized in commercial applications due to their high growth rate, ease of cultivation, photosynthetic efciency and signicant oil content (Chisti 2007; Grs et al. 2010; Liang et al. 2009; Lv et al. 2010). As eukaryotic photosynthetic autotrophs, Chlorella spp. (species) share similar metabolic pathways with higher plants, indicating that Chlorella algae have the potential to produce commercially relevant heterologous proteins as an alternative to conventional mammalian or yeast expression platforms (Rosenberg et al. 2008). However, a large number of microalgal transformants are usually obtained during transformation. Using PCR to identify successful transformants of Chlorella is simple; but preparing DNA templates are laborious and time consuming as a sufcient quantity of cells cultured from a single transformed colony are required for the extraction of DNA.
Colony PCR is a method that utilizes a minimal quantity of cells from the medium or a single colony from a plate to amplify only the expected fragments for
M. Wan J. Xia
School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China
M. Wane-mail: [email protected]
J. Xiae-mail: [email protected]
M. Wan J. N. Rosenberg J. Faruq
M. J. Betenbaugh (&)
Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA e-mail: [email protected]
J. N. Rosenberge-mail:...