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Team:UNAM-Genomics Mexico/Project/RhizobialKit
From 2011.igem.org
Contents |
General Overview
Abstract
Expanding our ability to manipulate and engineer biological parts in new organisms is an ever present goal of synthetic biology. With this idea in mind we set out to develop a kit of biological tools to enable working with BioBricks in Rhizobial species. We developed the tools in this kit in our model chasis is Rhizobium etli, a member of the Rhizobiaceae family of nitrogen fixing bacteria, that establishes a symbiotic relationship with legumes within the root nodules.
Rhizobial Kit Description
The Rhizobial Kit will set the ground for the use of Rhizobial species, especially R. etli, as organisms available for the design and construction of novel synthetic biological machines. The kit will be composed of parts with distinct molecular functions that will increase the repertoire of possible design and engineering approaches in Rhizobial species. The parts that we will develop are the following:
- The nifH promoter. This promoter sequence comes from the promoter region of the nifHa gene in R. etli. It functions in conditions of low oxygen concentration. (¿también en condiciones de bajo nitrógeno fijado? referencia). This promoter region comprises a contains a σ54-dependent promoter located between position -24 and -12 relative to the transcription start site, as well as a binding site for NifA, a positive transcriptional regulator (Beat Thöny, Hauke Hennecke, 1989; Valderrama, B., et. al., 1996).
- A re-characterization of the Anderson Promoter Collection in R. etli. These are constitutive promoters already characterized in E. coli [falta referencia de la caracterización de estos promotores en E. coli], but the aim is to investigate the strength of these promoters in R. etli as the chasis. This will hopefully give a spectrum of promoters with different strengths that will allow the fine-tuning of the expression of devices in this model.
- We developed a standardized plasmid (pBBR1MCS-5) that is able to integrate BioBrick constructions into Rhizobial species after conjugation with transformed E. coli cells. This plasmid is a modification of the pBBR1MCS-5 plasmid, it is a broad-host-range (bhr) vector that has been designed to assist genetic anaylsis in prokaryotes, specifically for Gram- bacteria that are naturally CmR (chloramphenicol resistant). We used to transfer genes from E. coli to R. etli by first transforming E. coli S17 competent cells with this vector containing an RFP reporter and later performing a conjugation between the transformed E. coli cells and R. etli CFN42.
- A repC “plasmid replication device” will also be included, with the purpose of rendering a plasmid capable of repC-dependent replication in R. etli. The repC region contains only two genes: one encoding the initiator protein RepC (46.8 kDa) and other, an antisense RNA (67 nt). repC is the minimal region required for stable replication of a member of the repC plasmid family.
Rhizobial Kit Details
The kit is composed by a group of promoters including the Anderson promoter collection as well as the nifH promoter that functions in low oxygen conditions. It also has a plasmid named pBBR1MCS-5 which is a derivative of the broad-host-range cloning vector pBBR1MCS. (Kovach et al., 1995).
References
- Brenda Valderrama, Araceli Dávalos, Lourdes Girard, Enrique Morett. 1996. Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated NifH genes. Journal of Bacteorology, 178, 3119-3126.
- Beat Thöny, Hauke Hennecke. 1989. The -24/-12 promoter comes of age. Microbiology Reviews 63, 341-358.
- Michael E. Kovach, Philip H. Elzer, D. Steven Hill, Gregory T. Robertson, Michael A. Farris,R. Martin Roop, Kenneth M. Peterson (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176
