A gene encoding a lipase enzyme was transfered from $\underline{Pseudomonas}$ $\underline{fluorescens}$ to $\underline{Escherichia}$ $\underline{coli}$ JM83 by a molecular cloning using pUC19 as a vector.
The chromosomal DNA was isolated from $\underline{Pseudomonas}$ $\underline{fluorescens}$ and partially digested with a restriction \ndonuclease Sau3A I. The pUC19 vector was digested with BamH I and dephosphorylated by calf intestinal alkaline phosphatase. The restriction fragments of chromosomal DNA were joined into the linearized pUC19 DNA and the resulting recombinant plasmids were used to transform $\underline{Escherichia}$ $\underline{coli}$ JM83, a strain with a negative lipase activity.
To select the positive clones containing the DNA fragments coding for lipase enzyme, about 12,000 transformants were toothpicked adn transfered onto LAT agar in which tributyrine is emulsified at LB medium, and 21 transformants showed clear halos, which means that thesecolonies have the activity of esterase(EC 3.1.). To screen the bacterial clone carrying the lipase (EC 3.1.1.3) gene from these clones indicating esterase activity, these transformants were transfered onto the ROM agar plate which includes olive oil and rhodamine-B. Only one out of the esterase positive 21 colonies exhibited lipase activity and the clone carried a plamid pJH9, which has the size of 13 kb. The lipase gene in the plasmid pJH9 was subcloned into pUC19 vector to yield recombinant plasmid pJH91. The plasmid pJH91 has the insert size 2.0 kb and the lipase gene was located within the 1.65 kb Pst I-EcoR I fragment of the plasmid pJH91.
