Construction of genomic library for leucosporidium antarcticum

• Main Author: Muhd Khairul Luqman, Muhd Sakaff
• Format: Book
• Language: English
• Published: Universiti Sains Malaysia 2008
• Subjects: R Medicine (General); Antarc*; Antarctica; Leucosporidium antarcticum
• Online Access: http://eprints.usm.my/48855/; http://eprints.usm.my/48855/1/MUHD%20KHAIRUL%20LUQMAN%20BIN%20MUHD%20SAKAFF%20-%2024%20Pages.pdf

L. antarcticum is a yeast species found in Antarctica. With special adaptation to survive in cold harsh environment of Antarctica, L. antarcticum have various potential applications like antifreeze proteins and useful features. The genome size of L. antarcticum is approximately 14 Mb. In this research, we tried to construct a gene library for L. antarcticum by using plasmid vector. L. antarcticum was cultivated in YPD broth before harvested. Three methods were evaluated for DNA extraction from L. antarcticum (Proteinaise K proteolytic lysis method, Proteinaise K + Pronase proteolytic lysis method and also liquid nitrogen lysis method). We found that liquid nitrogen lysis method was the best method to extract and fragmentize the L. antarcticum DNA at the required size of 1000 bp to 1500 bp. They were then purified to obtain DNA fragment of 1000 bp to 1500 bp. The purified DNA were repaired, dephosphorylated and inserted into pCR®4Blunt-TOPO® DNA vector. The recombinant plasmids were fmally transformed into Top Ten E. coli strain using heat shock before plated on LB agar. Plasmid extractions were carried out on clones to screen for presence of DNA insert. In conclusion, this study managed to construct a gene library containing approximately 2976 E. coli clones which cover 31.9 % of the whole L. antarcticum genome.