Improvement
Overview
Lipase is widespread in yeast, bacteria, fungi, animals, plants, and the human body, and it is a kind of catalytic
material such as triglyceride hydrolysis into glycerol the floorboard of the enzyme. In order to carry forward the
spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library
for related lipase and picked a biological part BBa_K258006, Thermostable lipase (TliA) of Pseudomonas fluorescens
SIK W1, submitted by iGEM09_METU-Gene in 2009, and they measured spectrophotometrically using p-nitrophenyl
palmitate (pNPP) as a substrate with lipase, and they measured the efficiency of TilA at different concentrations of
pNPO.
This part was improved by the team: Sheffield in 2014, the team chose rhodamine B to act as a reporter to measure the activity of lipase.
To further improve this part, our team carried out a lactobacillus Plantarum lipase gene W1-ligase of this part in the laboratory, adding data on available lipases. This information can be a good reference for future iGEM teams working on lipases.
This part was improved by the team: Sheffield in 2014, the team chose rhodamine B to act as a reporter to measure the activity of lipase.
To further improve this part, our team carried out a lactobacillus Plantarum lipase gene W1-ligase of this part in the laboratory, adding data on available lipases. This information can be a good reference for future iGEM teams working on lipases.
Introduction
Lipase catalyzed triglyceride hydrolysis, mainly produces glycerol, fatty acid, glycerin monoester, and diester.
Because of microbial diversity and rapid proliferation, compared with the plant, animal, and human body lipase, the
source of microbial lipase to secrete richer, and microorganisms secreting lipase form is usually extracellular
enzymes.
a) Construction of W1-lipase expression plasmids
We let the synthetic company synthesize the DNA fragments of W1-lipase, and inserted the fragment into the XhoI and
HindIII sites of the pET28a vector. The certificate of recombinant plasmid sequencing results is as Figure1.
Figure 1. The sequencing data maps to the recombinant plasmid pET28a-W1-lipase.
b) W1-lipase protein expression and purification
In order to obtain the W1-lipase protein, we transferred the recombinant plasmids into E.coli BL21(DE3), expanded
the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.4-0.5. After
overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the
intracellular proteins. Next, we used the Ni-NTA column to purify the protein we wanted.
Figure 2. The process of purification of W1-lipase protein by Ni-NTA column.