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

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===Applications of BBa_K258006===
 
===Applications of BBa_K258006===
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Lipase activity
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While the secretory phenotype of fusion proteins with
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TliA was evident based on lipase activity, secretion of
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fusion proteins with LARDs could be detected by Western
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blotting.
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'''Lipase activity'''
 
To identify a secretion phenotype on solid medium, E. coli
 
To identify a secretion phenotype on solid medium, E. coli
 
was grown at 25°C for 48 h on LAT (LB medium, 1.5%
 
was grown at 25°C for 48 h on LAT (LB medium, 1.5%
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for 20 min. The activity was measured by the increase of
 
for 20 min. The activity was measured by the increase of
 
optical density (OD).
 
optical density (OD).
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procedure from:  
 
procedure from:  
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transporter with an attached lipase ABC transporter recognition
 
transporter with an attached lipase ABC transporter recognition
 
domain (LARD)
 
domain (LARD)
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Chan Woo Chung1, Jinsun You1, Kyeongyeon Kim1, Yuseok Moon2,
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Chan Woo Chung, Jinsun You, Kyeongyeon Kim, Yuseok Moon,
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Hoeon Kim3 and Jung Hoon Ahn*
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Hoeon Kim and Jung Hoon Ahn*
 
Published: 29 January 2009
 
Published: 29 January 2009
 
Microbial Cell Factories 2009, 8:11 doi:10.1186/1475-2859-8-11
 
Microbial Cell Factories 2009, 8:11 doi:10.1186/1475-2859-8-11

Revision as of 12:04, 17 October 2009

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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

User Reviews

UNIQ2a16b4bf782299ce-partinfo-00000000-QINU UNIQ2a16b4bf782299ce-partinfo-00000001-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