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| Status |
Public on Jan 27, 2010 |
| Title |
Vinyl Chloride Degradation by Mixed Microbial Community KB1 |
| Organism |
dechlorinating enrichment culture KB-1 |
| Experiment type |
Expression profiling by array
|
| Summary |
A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes.
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| Overall design |
2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.
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| Contributor(s) |
Waller AS, Edwards EA |
| Citation(s) |
22179237 |
| |
| Submission date |
Jan 26, 2010 |
| Last update date |
Mar 22, 2012 |
| Contact name |
Elizabeth A Edwards |
| E-mail(s) |
elizabeth.edwards@utoronto.ca
|
| Organization name |
University of toronto
|
| Department |
Department of Chemical Engineering and Applied Chemistry
|
| Street address |
200 College st.
|
| City |
Toronto |
| ZIP/Postal code |
M5S3E5 |
| Country |
Canada |
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| Platforms (1) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA124301 |
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