Difference between revisions of "Part:BBa K1498001:Experience"
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===Applications of BBa_K1498001===
 
===Applications of BBa_K1498001===
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Lipase activity was observed following consultative expression in ''E. coli'' (DH5a) . This required co-expression of the ABC transporter BBa_K258008, to implement secretion of lipase into the extracellualr environment. ''E. coli'' harbouring BBa_K1498001 and BBa_K258008 were plated onto Rhodamine B (a fluorescent dye) plates supplemented with olive oil. Following overnight incubation a halo was seen around BBa_K1498001 and BBa_K258008 positive cells. Under UV light this effect was more apparent. In contrast this was not seen in Dh5a alone, which was used as a negative control.
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'''Rhodamine B assay'''
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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.)
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'''Reference.'''
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Kouker, G., & Jaeger, K. E. (1987). Specific and sensitive plate assay for bacterial lipases. Applied and environmental microbiology, 53(1), 211–3
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 03:10, 18 October 2014

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Applications of BBa_K1498001

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.)

Reference.

Kouker, G., & Jaeger, K. E. (1987). Specific and sensitive plate assay for bacterial lipases. Applied and environmental microbiology, 53(1), 211–3

User Reviews

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