Research Projects

Logos GenomEnviron and Ampere
Laboratoire Ampere
Ecole Centrale de Lyon . France
(33) 472 18 65 14

     

 


 

GENEFISH
Coordinator:
Pascal Simonet and Laure Franqueville
Funding Source:

 Ingénierie écologique CNRS / METAEXPLORE: SEVENTH FRAMEWORK PROGRAMME; THEME [#] KBBE-2007-3-3-05
ACTIVITY MINING IN METAGENOMES

Period:

2006-2008 + 2010-2012 / 2009-2013

Partners:

 L. Philippot, INRA, UMR 1347 Agroecology, 17 rue Sully 21065 Dijon Cedex France

JC. Lazzaroni, Unité de Microbiologie, Adaptatation et Pathogénie, UMR5240, Université de Lyon, 69622 Villeurbanne cedex, France

Xiaojun Zhang, School of Life Science and Biotechnology, Shanghai Jiaotong University Dongchuan Rd. 800, Shanghai, 200240, China

Abstracts:

The metagenomic approach, defined as the direct recovery and cloning of bacterial DNA from the environment in domesticated bacterial hosts has been widely used to study bacterial populations and their functional genes in numerous environments. The advantage of this approach over conventional culture based techniques is that it encompasses a wider range of bacteria by bypassing the bias of uncultivability of more than 99% of the bacteria in soil. However, in complex and rich environments such as soils, the huge level of bacterial diversity requires construction, handling and screening of several million clones in order to cover a significant proportion of bacterial genes in the indigenous community. These methods are time and money consuming, and require access to specialized robots that are unavailable to most microbial ecology laboratories. Our objectives were to develop an alternative metagenomic approach in which only bacterial recombinant clones harbouring inserts with sequence based selected genes could develop on growth media. This positive screening technology, called “Genefish” is based on homeologous recombination to extract specific genes from the metagenome into the specifically engineered recipient E. coli strain. The key characteristic of this approach is the use of two inducible lethal genes to kill non recombinant bacteria.

Objectives:

·The Genefish approach can be considered as a simpler alternative to the conventional metagenomic techniques in order to recover specific genes or DNA fragments from a metagenome.


Genefish prospection on biotechnologically relevant enzymes involved in the biodegradation of recalcitrant and xenobiotic molecules with a wide range of DNA including PCR products, genomic DNA and soil or wastewater bioreactor metagenome.

Description
of the work:

The so-called “Genefish” method is based on the use of specific sequences cloned into the recipient strain to serve as template for homeologous recombination with corresponding DNA present in the metagenome. In addition, the double cross-over event involving the targeted genes leads to the replacement of two inducible lethal genes that kill non-recombinant bacteria.

The feasibility of the “Genefish” project was confirmed by using the experimental model of the nitrate reductase (narGH) genes. However the amount of the recovered recombinant clones was lower than expected probably due to the presence of non-recombinant toxic capture plasmids into the Genefish strain (10-15 copies). An appropriate co-electroporation procedure was tested in order to control the number of the suicide plasmid copies during the transformation/recombination/selection steps and this hypothesis was confirmed.

Deliverables:

Publications & Communications:

Genefish : an alternate metagenomic approach for capturing targeted bacterial diversity in an engineered recipient E. coli strainN.Lombard, A.Faugier, C.Lavire, S.Jacquiod, L.Philippot, X.Zhang, J.C.Lazzaroni, P.Simonet and L.Franqueville. (2009) 10th symposium on bacterial genetics and ecology (BAGECO-10), Uppsala, Sweden.
Genefish or how to capture targeted bacterial diversity
Lombard.N, Jacquiod.S, Yuan.J, Faugier.A, Lavire.C, Zhang.X, Philippot.L, Lazzaroni.JC, Simonet and Franqueville.L. (2011) Integrative Ecological Genomics, Jacques Monod Conferences, Roscoff, France.
Genefish: a window into targeted bacterial diversity
Jacquiod.S, Franqueville.L,  Lombard.N, Yuan.J, Faugier.A, Lavire.C, Zhang.X, Philippot.L, Lazzaroni.JC, Simonet.P. (2010) ISME13, Seattle, USA.
Prospecting specific genes involved in the biodegradation of aromatic compounds through classical and innovative metagenomic approaches
 J. Yuan, S.Jacquiod, N.Lombard, A.Faugier, C.Lavire, L.Philippot, X.Zhang, J.C.Lazzaroni, P.Simonet and L. Franqueville. (2012) ISME14, Copenhagen , Denmark.

 

 
     

PROJECT