Contribution
Overview
Lipase is widespread in yeast, bacteria, fungi, animals, plants, and the human body, 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, we specially searched the iGEM Biological Parts library for related projects and picked BBa_K1671001, Lipase.
This is a biological part submitted by iGEM15_Hangzhou-H14Z in 2015, they only provided a lipase expression plasmid.
However, the activity of the lipase didn’t be verified. So it is really important to detect the activity of the
lipase and optimize the in vitro reaction system.
In this project, our team carried out two lipases for this part in the laboratory, adding data for testing its function in vitro. What’s more, we also optimized the reaction system by changing the pH value and the reaction temperatures, which also provides more ideas for future iGEM teams. This information can be a good reference for future iGEM teams working on lipases.
In order to verify if our lipases work well, we developed an in vitro verification platform. we transformed the recombinant plasmid into E. coli BL21(DE3) to purify the protein, mixed it with substrates, and set up the in vitro verification platform.
In this project, our team carried out two lipases for this part in the laboratory, adding data for testing its function in vitro. What’s more, we also optimized the reaction system by changing the pH value and the reaction temperatures, which also provides more ideas for future iGEM teams. This information can be a good reference for future iGEM teams working on lipases.
In order to verify if our lipases work well, we developed an in vitro verification platform. we transformed the recombinant plasmid into E. coli BL21(DE3) to purify the protein, mixed it with substrates, and set up the in vitro verification platform.
Add new experimental data to an existing Part BBa_K1671001, Lipase
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 SP-lipase expression plasmids
We amplified the lipase gene from pseudomonas lipase gene SP-ligase and inserted the fragment in the XhoI and
HindIII sites of pET28a (Figure 1).
Figure 1. SP-ligase expression plasmids in this project
In order to build our plasmids, we let the synthetic company synthesize the target gene fragment (SP-lipase). We
digested the target fragments and the pET28a vector with XhoI and HindIII (Figure 2), and we used T4 DNA ligase to
ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α competent cells
and coated on the LB (Kanamycin) solid plates.
Figure 2. Gel electrophoresis results of target gene fragments. A. double-enzyme digested withthe SP-lipase,
B. double-enzyme digested with the pET28a vector.
We verified the colonies through colony-PCR, and then we inoculated single colonies and we send the constructed
recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that
there were no mutations in the ORF region, and the plasmid was successfully constructed. So far, we have
successfully obtained two recombinant plasmids, which were respectively on the pET28a vector, which can be used to
express lipase proteins.
As a result, the amplified target gene SP-lipase and the double digested vector pET28a were ligated with T4 ligase to obtain recombinant plasmid pET28a-SP-lipase, so that the recombinant protein had 6-His tags at the carboxyl terminus which could be used to purify the corresponding proteins.
As a result, the amplified target gene SP-lipase and the double digested vector pET28a were ligated with T4 ligase to obtain recombinant plasmid pET28a-SP-lipase, so that the recombinant protein had 6-His tags at the carboxyl terminus which could be used to purify the corresponding proteins.
b)Protein expression and purification
The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and cultured overnight in the medium
containing resistance. When the OD600 was around 0.4-0.5, the IPTG was added to induce the expression of recombinant
protein W1-lipase/SP-lipase, and then the strains were cultured at 16℃ for 20h. After that, the collected bacterial
solution was cracked by Ultrasonic crushing and then transferred to a pre-balanced Ni-NTA affinity column. The
recombinant protein was bound to a nickel column and then eluted by imidazole. SDS-PAGE was used to analyze the
recombinant proteins. Figure 4 showed the electrophoretic results of the protein gel.
Figure 2. SDS-PAGE detection of lipase protein.
Add new information to the Part BBa_K4279000, BBa_K4279001, and BBa_K4279002
a)BBa_K4279000, SP-lipase
Lipase is a primary lipase critical for triacylglyceride digestion in humans and is considered a promising target
for the treatment of obesity, triacylglycerol lipase is the primary lipase secreted by the pancreas, and is
responsible for breaking down dietary lipids into unesterified fatty acids (FAs) and monoglycerides (MGs). The
SP-lipase is obtained from the Pseudomonas nanhaiensis sp. nov., which is a lipase-producing bacterium isolated from
deep-sea sediment of the South China Sea, and it is assembled by 618aa.
b)BBa_K4279001, W1-lipase
W1-lipase is a lipase amplified from Lactiplantibacillus Plantarum (LP1406), which is a gram-positive lactic acid
bacteria species and exhibits ecological and metabolic adaptability and is capable of inhabiting a range of
ecological niches including fermented foods, meats, plants, and the mammalian gastrointestinal tract. The W1-lipase
is made up of 265aa and it is a smaller protein compared with SP-lipase.
c)BBa_K4279002, pET28a-SP-lipase
pET28a-SP-lipase is a composite part that could be used for SP-lipase protein expression in the E. coli system.
pET28a-vector is one of the most commonly used E. coli protein expression vectors, which uses the T7 promoter to
regulate the expression of exogenous genes. The vector is a high-copy-number plasmid. When expressed in the
prokaryotic system, the Kana+ resistance can be used to screen the right colony and can insert target genes in MCS.
This plasmid backbone can be used to express different proteins in the future.
Our team has carried out another candidate for available lipases. To construct the protein expression plasmids, both
the DNA fragments and the vector were digested by double enzymes, verified by agarose gel electrophoresis, and the
correct recombinant plasmid was extracted and transformed into BL21(DE3). And the protein was purified through the
Ni-NTA method. Overall, our study provides a foundation for lipase research and could be applied for industrial
applications.
Reference
1. Winkler, F. K., d'Arcy, A., & Hunziker, W. (1990). Structure of human pancreatic lipase. Nature, 343(6260),
771-774.
2. Lombardo, D. (2001). Bile salt-dependent lipase: its pathophysiological implications. Biochimica et biophysica acta, 1533(1), 1-28.
3. Diaz, B.L.; J. P. Arm. (2003). Phospholipase A(2). Prostaglandins Leukot Essent Fatty Acids. 69 (2–3): 87–97.
4. Siener, R., Machaka, I., Alteheld, B., Bitterlich, N., & Metzner, C. (2020). Effect of fat-soluble vitamins A, D, E and K on vitamin status and metabolic profile in patients with fat malabsorption with and without urolithiasis. Nutrients, 12(10), 3110.
5. Seth, S., Chakravorty, D., Dubey, V. K., & Patra, S. (2014). An insight into plant lipase research–challenges encountered. Protein Expression and Purification, 95, 13-21.
6. Chepyshko, H., Lai, C. P., Huang, L. M., Liu, J. H., & Shaw, J. F. (2012). Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis. BMC genomics, 13(1), 1-19.
7. Verma, S., Kumar, R., Kumar, P., Sharma, D., Gahlot, H., Sharma, P. K., & Meghwanshi, G. K. (2020). Cloning, characterization, and structural modeling of an extremophilic bacterial lipase isolated from saline habitats of the Thar desert. Applied Biochemistry and Biotechnology, 192(2), 557-572.
8. US gov (Feburary 4, 2008). A computer-generated image of a type of pancreatic lipase (PLRP2) from the guinea pig [photograph]. Protein Data Base.
9. Jaeger, K. E., & Reetz, M. T. (1998). Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16(9), 396-403.
2. Lombardo, D. (2001). Bile salt-dependent lipase: its pathophysiological implications. Biochimica et biophysica acta, 1533(1), 1-28.
3. Diaz, B.L.; J. P. Arm. (2003). Phospholipase A(2). Prostaglandins Leukot Essent Fatty Acids. 69 (2–3): 87–97.
4. Siener, R., Machaka, I., Alteheld, B., Bitterlich, N., & Metzner, C. (2020). Effect of fat-soluble vitamins A, D, E and K on vitamin status and metabolic profile in patients with fat malabsorption with and without urolithiasis. Nutrients, 12(10), 3110.
5. Seth, S., Chakravorty, D., Dubey, V. K., & Patra, S. (2014). An insight into plant lipase research–challenges encountered. Protein Expression and Purification, 95, 13-21.
6. Chepyshko, H., Lai, C. P., Huang, L. M., Liu, J. H., & Shaw, J. F. (2012). Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis. BMC genomics, 13(1), 1-19.
7. Verma, S., Kumar, R., Kumar, P., Sharma, D., Gahlot, H., Sharma, P. K., & Meghwanshi, G. K. (2020). Cloning, characterization, and structural modeling of an extremophilic bacterial lipase isolated from saline habitats of the Thar desert. Applied Biochemistry and Biotechnology, 192(2), 557-572.
8. US gov (Feburary 4, 2008). A computer-generated image of a type of pancreatic lipase (PLRP2) from the guinea pig [photograph]. Protein Data Base.
9. Jaeger, K. E., & Reetz, M. T. (1998). Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16(9), 396-403.