Cloning of candida antarctica lipase a gene in to kluveromyces lactis
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Transcript of Cloning of candida antarctica lipase a gene in to kluveromyces lactis
CLONING OF Candida antarctica LIPASE A GENE IN Kluveromyces
lactis EXPRESSION SYSTEM
KARTHIKEYAN.T RAJARAJAN.P
RAMKUMAR.M
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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
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
Maintenance Host – E.coli TOP10F’ Expression Host – Kluveromyces lactis Vectors – pKLAC2
Vector and Host Systems
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
CALA gene obtained from Plamid pPICZαB 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
Analysis of PCR amplified lipase A gene
Analysis of purified samples of Lipase A gene
pKLAC2 was extracted from E.coli DH5α maintenance host.
pKLAC2 was restricted at xho1 sites using xho1 restriction enzyme.
pKLAC2 EXTRACTION AND RESTRICTION
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
Analysis of restricted pKLAC2 plasmid
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
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
Analysis of ligation mixtures of Lipase A gene with PKLAC2
plasmid using pKLAC2 forward and lipase reverse as primers.
Competent cell preparation and Transformation
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
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 transformants having the plasmids with the inserts lipase A gene using pKLAC2
forward and pKLAC 2 TT reverse as primers
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
Analysis of plasmids extracted by manual method from patches of 7, 8, 9, and 10 Top 10 E.coli Positive
transformants
The recombinant plasmid pKLAC2/CALA from the positive transformant E.coli TOP10F’ cells can be linearised .
The linearised DNA is then transformed into the expression host K.lactis for further expression studies.
Conclusion