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Establishment of a real-time PCR protocol for expression studies of secondary metabolite biosynthetic gene clusters in the G/C-rich myxobacterium Sorangium cellulosum So ce56

Paper ID Volume ID Publish Year Pages File Format Full-Text
25732 43594 2006 12 PDF Available
Title
Establishment of a real-time PCR protocol for expression studies of secondary metabolite biosynthetic gene clusters in the G/C-rich myxobacterium Sorangium cellulosum So ce56
Abstract

In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90 kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between chiA and chiB was examined by using the developed real-time PCR protocol.

Keywords
kbp, kilobasepairs; RT, reverse transcription; So ce56, Sorangium cellulosum So ce56Sorangium cellulosum; Real-time PCR; Secondary metabolism; Chivosazol; Etnangien
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Establishment of a real-time PCR protocol for expression studies of secondary metabolite biosynthetic gene clusters in the G/C-rich myxobacterium Sorangium cellulosum So ce56
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 121, Issue 2, 24 January 2006, Pages 201–212
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us