Team:Utah State/Broad-HostVectors


USU iGem

Untitled Document

PROJECT Abstract
Broad-Host Vectors
Future Work
Broad-Host Range Vectors

Advantages for Using a Broad Host Range Vector

A multi-host vector allows for genetic manipulation to occur in one organism, and the ultimate application of the vector to be served in another. Genetic manipulation is ideally done in E. coli, due to its fast growth, ease of use, and availability of transformable cells. However, it does not always represent the best choice for production of recombinant proteins or other compounds, and thus it is ideal to be able to transfer genetic information into other organisms once manipulation and testing of the construct is complete.

Most broad host range vectors are naturally occurring or a derivative of a natural vector. They tend to be large, around 10 kbp, although some commercial versions have been optimized to a much shorter length ( They can be self-transmissible (presence of tra genes) and mobilizable (mob genes), but desirable vectors are both mobilizable and non-transmissible (Haller, & Dichristina, 2002). This allows for more control over conjugation in the laboratory through use of a helper plasmid (Haller, & Dichristina, 2002). A helper plasmid is a conjugative plasmid, that is it contains both transmission and mobilization genes. While a broad-host range plasmid can be conjugated into another organism, its copy number will remain undetectably low unless a fully functioning helper plasmid is present (Haller, & Dichristina, 2002).. If a helper plasmid shares the same origin of transfer (oriT), mob genes are no longer necessary (Snyder and Champness 2007). Due to this property, the mob genes of commercial plasmids are often removed, thereby resulting in vectors that are significantly shorter than their natural counterparts (Snyder and Champness 2007). Use of a helper plasmid becomes necessary if the self-transmission genes are not present to achieve any detectable degree of replication in the recipient organism (Haller, & Dichristina, 2002).

Genetic Characteristics of Broad-Host Range Vectors

Broad host range vectors are a class of mobilizable plasmids, that is they lack the complete tra-genes necessary for conjugation but can still transfer and replicate at high copy number in the presence of a conjugative plasmid. Mobilizable vectors still contain some of the genes necessary for transfer. The mob genes code proteins that aid the vector in transferring from one organism to another. One protein produced in the region, nickase-helicase, nics the DNA at the origin of transfer (oriT). As the envelopes of the two cells meet, the mobility proteins synthesize a new strand of DNA from the plasmid parent strand as it enters the recipient cell. A new strand is also synthesized in the donor cell simultaneously. In this way, the plasmid is transferred from one cell to another (Porter, 2002).

Team USU
Figure 1. Mechanism for bacterial conjugation

The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in E. coli. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).