Difference between revisions of "Part:BBa K258006:Experience"

Latest revision as of 03:09, 18 October 2014

Experiment

Tributyrin : It is a triglyceride naturally present in butter. It is an ester composed of butyric acid and glycerol.

Chemical Formula: C15-H26-O6
Molecular Weight: 302.37 g/mole

Lipase : It is a soluble enzyme that catalyzes the hydrolysis of ester bonds in water–insoluble, lipid substrates.

ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) is secreted through the ABC transporter. TliA has four glycine rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. Whole TliA is the longest LARD. Lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins. Result of our experiment There was TliA-AbC in the LB Agar+Chm+Amp+Try plate so lipase reacted with tributyrin and butyric acid was formed. As a result of this, we have seen the zone formation.

We holed 3mm zone in the plate and we poured the supernatant. 2 of them was including E.coli with ABC-transporter and 2 of them did not contain. After that we left these plates in incubation at 25 C for 2 days.

As we expected, area of zone in plate with ABC transporter is larger than the without ABC transporter.

Enzyme Kinetic Assay Lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate. Ten millimolar pNPP dissolved in acetonitrile was mixed with ethanol and 50 mM Tris-HCl (pH 8.5) to a final ratio of acetonitrile:ethanol:Tris-HCl of 1:4:95 (v/v/v). The reaction was started by adding 50 μl of culture supernatant to 200 μl of reaction mixture at 42°C, and absorbance at 420 nm was monitored with a Hitachi spectrophotometer (Enzyme Kinetic Activity Assay)

Rhodamine B assay

Lipase activity of secreted enzyme was shown on a 1.5% agar plate, containing 0.02% w/v rhodamine B and 6.6% v/v olive oil. The sterile filtered supernatant of cells expressing BBa_K258006 under the control of BBa_J23110 and the ABC transporter (BBa_K258008) was applied to wells in the rhodamine B plates. Sterile Luria-Bertani broth and the supernatant of cells expressing BBa_J23110 only were loaded as negative controls. Following incubation at 37°c overnight a halo was seen around the lipase containing supernatant (See left hand image: left: secreted lipase, middle:BBa_J23110 only, right: LB broth only . Under UV light fluorescent halo was observed (see right hand image: left: LB broth only, middle:BBa_J23110 only, right: secreted lipase.)

Applications of BBa_K258006

While the secretory phenotype of fusion proteins with TliA was evident based on lipase activity, secretion of fusion proteins with LARDs could be detected by Western blotting.

Lipase activity To identify a secretion phenotype on solid medium, E. coli was grown at 25°C for 48 h on LAT (LB medium, 1.5% Bacto Agar, 0.5% tributylin). The phenotype was evident by the development of a halo due to the secreted lipase [12]. In addition, lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate [12]. Ten millimolar pNPP dissolved in acetonitrile was mixed with ethanol and 50 mM Tris-HCl (pH 8.5) to a final ratio of acetonitrile:ethanol:Tris-HCl of 1:4:95 (v/v/v). The reaction was started by adding 50 μl of culture supernatant to 200 μl of reaction mixture at 42°C, and absorbance at 420 nm was monitored with Hitachi Spectrophotometry for 20 min. The activity was measured by the increase of optical density (OD).

procedure from:

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD) Chan Woo Chung, Jinsun You, Kyeongyeon Kim, Yuseok Moon, Hoeon Kim and Jung Hoon Ahn* Published: 29 January 2009 Microbial Cell Factories 2009, 8:11 doi:10.1186/1475-2859-8-11 Received: 18 November 2008 Accepted: 29 January 2009 This article is available from: http://www.microbialcellfactories.com/content/8/1/11

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UNIQ9bd45c3733c099bf-partinfo-00000005-QINU UNIQ9bd45c3733c099bf-partinfo-00000006-QINU In our experiment, we observed that TliA fused proteins were excreted

to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection

with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-

fold more with ABC transporter PrtDEF of Erwinia chrysanthemi. Same results took place lipase activity at size of zone around supernatant on tributyrin mixed agar plate