This document describes cloning the lipase A (CALA) gene from Candida antarctica into the Kluveromyces lactis expression system using the pKLAC2 vector. Key steps include: 1. Amplifying the CALA gene from C. antarctica and restricting it with XhoI. 2. Extracting and restricting pKLAC2 with XhoI. 3. Ligating the CALA insert into linearized pKLAC2. 4. Transforming E. coli with the ligation mixture and confirming transformants contain the CALA insert through colony PCR and plasmid extraction.

1. CLONING OF Candida antarctica LIPASE A GENE IN Kluveromyceslactis EXPRESSION SYSTEM Submitted by KARTHIKEYAN.T RAJARAJAN.P RAMKUMAR.M

2. Lipase A is an enzyme from Candida antarctica strain. The gene coding for Lipase A is CALA gene. Lipase A is the most thermostable Lipase known and is used in food and pharmaceutical industry as a biocatalyst. Lipases catalyze the hydrolysis of triacylglycerols to glycerol. Rationale

3. To extract CALA gene from Candida antarctica. To purify CALA gene. To insert purified CALA gene in to pKLAC2 Vector restricted at xho sites. To transform E.coli TOP10F’ strain with unidirectionally cloned CALA/pKLAC2 recombinant vector. Objective

4. Maintenance Host – E.coli TOP10F’ Expression Host – Kluveromyceslactis Vectors – pKLAC2 Vector and Host Systems

5. Ampicillin (100μg/ml) is the antibiotic which is used in the selection process. LB Medium (Luria Bertain Medium)is used to grow E.coli. Antibiotics and Culture medium

6. CALA gene obtained from PlamidpPICZαB having CALA insert. CALA gene in pPICZαB is amplified using PCR. The amplified CALA gene is then restricted at xho1 site. CALA gene was purified to remove excess salts and to increase CALA concentration. CALA gene Isolation, Amplification and purification

7. Analysis of PCR amplified lipase A gene

8. Analysis of purified samples of Lipase A gene

9. pKLAC2 was extracted from E.coli DH5α maintenance host. pKLAC2 was restricted at xho1 sites using xho1 restriction enzyme. pKLAC2 EXTRACTION AND RESTRICTION

10. Analysis of pKLAC2 plasmid extracted by standard Kit method 1 2 3 4 1 – 1kb marker 2 – First Elution Sample 3 – Second Elution Sample 4 – Third Elution Sample

11. Analysis of restricted pKLAC2 plasmid

12. Since two sticky ends in the restricted pKLAC2 plasmid are the same, one of the end has to be dephosphorylated. Antarctic phosphatase is used for this purpose. It catalyzes removal of phosphate groups from DNA and RNA. This is used to reduce vector background in cloning strategies. The restricted plasmid is then purified before continuing on to ligation Dephosphorylation of Restricted pKLAC2 plasmid

13. Ligation involves the formation of four phosphodiester bond. Both the plasmid and lipase A were restricted using same restriction enzyme Xho1. They were ligated with T4 ligase enzyme . Ligation

14. Analysis of ligation mixtures of Lipase A gene with PKLAC2 plasmid using pKLAC2 forward and lipase reverse as primers.

15. Competent cell preparation and Transformation

16. 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: Check the plate for presence of colonies Take a loop and with it take a colony form the transformed plates and streak on a new LB/amp+ agar plates. Mark the streaked places and allow it grow overnight After 12 hours check for the growth of colonies. Patching

17. Take the 10 colonies from the patched and suspend it in 50 µl distilled water Heat at 100˚C for 10 min Immediately place it in ice for 5 min Centrifuge at 10000 rpm for 2 min and take only the supernatant for the PCR The PCR reaction was done. 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

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

19. Analysis of plasmids extracted by manual method from patches of 7, 8, 9, and 10 Top 10 E.coliPositive transformants

20. The recombinant plasmid pKLAC2/CALA from the positive transformantE.coli TOP10F’ cells can be linearised. The linearised DNA is then transformed into the expression host K.lactis for further expression studies. Conclusion