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Utilizing Ligation Independent Cloning in an Undergraduate Biochemistry Laboratory Course
Author(s) -
Hazzard James,
Forte Brittany,
Nelp Micah,
Young Anthony,
Hausrath Andrew
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.559.27
Subject(s) - cloning (programming) , moiety , expression vector , transformation (genetics) , biochemistry , ligation , fusion protein , computational biology , chemistry , biology , microbiology and biotechnology , gene , computer science , stereochemistry , recombinant dna , programming language
As a continuation of the transformation of our laboratory course to a more research focused experience we tested whether the Ligation Independent Cloning (LIC) technique would be suitable for students with little to no prior experience in molecular biology methodologies. To produce an expression system for for E. coli alkaline phosphatase (PhoA) we cloned its gene into the pETHSUL vector kindly donated by Dr. Patrick Loll. The construct expresses a fusion protein where a His‐tagged SUMO protein is fused to PhoA. The sumo moiety can be proteolytically released from PhoA following affinity column purification. We compare the ease of building the pETHSUL‐PhoA construct and enzyme activity data for the purified enzyme with an expression system developed previously in our class using a classical double restriction enzyme sub‐cloning into a pET29a vector that incorporates a His‐tag at the C‐terminus of PhoA.