Cloning of candida antarctica lipase a gene in to kluveromyces lactis


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Lipase A is an enzyme from Candida antarctica strain isolated from Lake vanda in Antarctica. The gene coding for Lipase A is CALA gene. Lipase A is the most thermostable lipase known and is used as a biocatalyst in food and pharmaceutical industry. Lipases (triacylglycerol acylhydrolases EC are ubiquitous enzymes of considerable physiological significance and industrial potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. Lipases are serine hydrolases. Lipases display little activity in aqueous solutions containing soluble substrates.
Lipase A gene was from Candida antarctica was amplified using gene specific primers Xho1 restriction site. The gene was cloned unidirectionally into Xho1 site in pKLAC2 vector. pKLAC2 with lipase A gene insert was transformed into E.Coli TOP 10F’ strain and the transformed colonies were screened for positive transformants. Future prospects involve expressing lipase A gene in Kluveromyces lactis expression system and conducting expression studies in a fermenter.

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Cloning of candida antarctica lipase a gene in to kluveromyces lactis

  1. 1. CLONING OF Candida antarctica LIPASE A GENE IN Kluveromyceslactis EXPRESSION SYSTEM <br />Submitted by<br />KARTHIKEYAN.T <br />RAJARAJAN.P <br />RAMKUMAR.M <br />
  2. 2. Lipase A is an enzyme from Candida antarctica strain.<br />The gene coding for Lipase A is CALA gene.<br />Lipase A is the most thermostable Lipase known and is used in food and pharmaceutical industry as a biocatalyst.<br />Lipases catalyze the hydrolysis of triacylglycerols to glycerol.<br />Rationale<br />
  3. 3. To extract CALA gene from Candida antarctica.<br />To purify CALA gene.<br />To insert purified CALA gene in to pKLAC2 Vector restricted at xho sites.<br />To transform E.coli TOP10F’ strain with unidirectionally cloned CALA/pKLAC2 recombinant vector.<br />Objective<br />
  4. 4. Maintenance Host – E.coli TOP10F’<br />Expression Host – Kluveromyceslactis<br />Vectors – pKLAC2<br />Vector and Host Systems<br />
  5. 5. Ampicillin (100μg/ml) is the antibiotic which is used in the selection process. <br />LB Medium (Luria Bertain Medium)is used to grow E.coli.<br />Antibiotics and Culture medium<br />
  6. 6. CALA gene obtained from PlamidpPICZαB having CALA insert.<br />CALA gene in pPICZαB is amplified using PCR.<br />The amplified CALA gene is then restricted at xho1 site.<br />CALA gene was purified to remove excess salts and to increase CALA concentration.<br />CALA gene Isolation, Amplification and purification<br />
  7. 7. Analysis of PCR amplified lipase A gene<br />
  8. 8. Analysis of purified samples of Lipase A gene<br />
  9. 9. pKLAC2 was extracted from E.coli DH5α maintenance host.<br />pKLAC2 was restricted at xho1 sites using xho1 restriction enzyme.<br />pKLAC2 EXTRACTION AND RESTRICTION<br />
  10. 10. Analysis of pKLAC2 plasmid extracted by standard Kit method <br /> 1 2 3 4<br />1 – 1kb marker<br />2 – First Elution Sample<br />3 – Second Elution Sample<br />4 – Third Elution Sample <br />
  11. 11. Analysis of restricted pKLAC2 plasmid <br />
  12. 12. Since two sticky ends in the restricted pKLAC2 plasmid are the same, one of the end has to be dephosphorylated.<br />Antarctic phosphatase is used for this purpose. It catalyzes removal of phosphate groups from DNA and RNA.<br />This is used to reduce vector background in cloning strategies.<br />The restricted plasmid is then purified before continuing on to ligation<br />Dephosphorylation of Restricted pKLAC2 plasmid<br />
  13. 13. Ligation involves the formation of four phosphodiester bond.<br />Both the plasmid and lipase A were restricted using same restriction enzyme Xho1. <br />They were ligated with T4 ligase enzyme .<br />Ligation<br />
  14. 14. Analysis of ligation mixtures of Lipase A gene with PKLAC2 plasmid using pKLAC2 forward and lipase reverse as primers. <br />
  15. 15. Competent cell preparation and Transformation<br />
  16. 16.  <br /> After the plates are checked for the presence of colonies they are to be patched in new LB/amp+ agar plates. This is done in the following way:<br />Check the plate for presence of colonies<br />Take a loop and with it take a colony form the transformed plates and streak on a new LB/amp+ agar plates.<br />Mark the streaked places and allow it grow overnight<br />After 12 hours check for the growth of colonies.<br />Patching<br />
  17. 17. Take the 10 colonies from the patched and suspend it in 50 µl distilled water<br />Heat at 100˚C for 10 min<br />Immediately place it in ice for 5 min<br />Centrifuge at 10000 rpm for 2 min and take only the supernatant for the PCR<br />The PCR reaction was done.<br />Lysate PCR analysis of E. coli transformantshaving the plasmids with the inserts lipase A gene using pKLAC2 forward and pKLAC 2 TT reverse as primers <br />
  18. 18. Lysate PCR analysis of E. coli transformants (7-10) having the plasmids with the inserts lipase A gene using pKLAC2 forward and Lip A reverse as primers <br />
  19. 19. Analysis of plasmids extracted by manual method from patches of 7, 8, 9, and 10 Top 10 E.coliPositive transformants<br />
  20. 20. The recombinant plasmid pKLAC2/CALA from the positive transformantE.coli TOP10F’ cells can be linearised.<br />The linearised DNA is then transformed into the expression host K.lactis for further expression studies. <br />Conclusion<br />