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Development of BioBrick parts relies heavily on the use of a set of plasmid vectors that are only replicative in Escherichia coli and the enterics. While this approach has been successful for many projects, there are a number of concerns that we believe should be addressed in order to augment the palette of possibilities that can be exploited in Synthetic biology based on standard parts.
Plasmid stability. Although many plasmids are relatively stable in a culture setting, it is customary to maintain antibiotic selection to prevent plasmid loss, but this is often not possible in an industrial setting or in the environment. A small subpopulation lacking the plasmid may quickly overtake the culture, especially after repeated subculturing, because of the relief of the energetic burden of plasmid replication.
Plasmid copy number. Plasmids are extrachromosomal genetic elements that occur in a variable number of copies. Plasmids generally used in iGEM range from 15-20 to nearly 2000 copies per chromosome. In addition, the plasmid copy number may fluctuate from cell to cell due to drift in the replication process. On the other hand, many Synthetic biology projects require rewiring regulatory circuits with components that are not meant to be present in multiple copies. This may provoke unwanted effects, such as the titration of trans-acting elements, that lead to a decrease in the robustness of the resulting circuit.
Potential for horizontal transfer. Plasmids have been shown to mediate horizontal transfer by conjugation or transformation. The possibility of horizontal transfer of plasmids, including drug resistance markers, make plasmid-bearing strains unsuitable for applications involving the release of bacterial strains to the environment.
E. coli as a host. E. coli is the workhorse of genetic engineering. Because of its easy and fast growth in culture and the extensive knowledge accumulated on its genetics, metabolism and physiology, it is appropriate for many applications. However, there are others for which it is a better choice to use a different microbial host. For example, E. coli does not survive well in the environment, particularly in soils, and it is therefore unsuitable for many environmental applications.
For the iGEM 2011 competition, the UPO-Sevilla team intends to develop the miniTn7 BioBrick toolkit, a set of fully biobrick-compatible vectors that intend to overcome the concerns listed above. These vectors contain the prefix/suffix biobrick multi-cloning site within the ends of defective Tn7 transposons. Some of the features of our miniTn7 BioBrick toolbox are:
Stable integration at a single site in the chromosome. Tn7 has the ability to integrate at a single target in the bacterial chromosome downstream from the conserved gene glmS. The insertion site is neutral, as it is located at a convergent intergenic region (Gringauz, 1988; Waddell, 1989). Transposition is exerted by Tn7 transposase provided transiently from a suicide plasmid, granting a single transposition event and the impossibility of secondary transposition (Peters, 2001). Stable transposon insertion generates safer organisms, as horizontal gene transfer is minimized in this way.
Antibiotic selection not required. MiniTn7 insertions do not require antibiotic selection, as the transposon is faithfully replicated as part of the bacterial chromosome. Furthermore, all our miniTn7 vectors will be equipped with FRT-flanked antibiotic resistance that allows precise excision of the markers by Flp-mediated recombination (Cherepanov, 1995). Stability of the insertion and the lack of drug resistance markers make strains with miniTn7 insertions more appropriate for environmental release than plasmid-bearing strains
Suitability to multiple bacterial hosts. Most likely, Tn7 transposition is functional in a large array of Gram-negative bacteria. So far, Tn7 has been shown to transpose in over 20 different Gram-negative bacterial species, including several enterics, members of the genus Pseudomonas, Caulobacter crescentus, Desulfovibrio desulfuricans and others (Craig, 1996). Delivery of the transposon by mating or electroporation is possible in all of these organisms.
The parts in the miniTn7 BioBrick toolkit are based on the excellent set of miniTn7 derivatives developed by Choi et al. (2005). All our derivatives contain the Tn7-L and Tn7-R ends, an antibiotic resistance gene flanked by FRTs and the prefix/suffix BioBrick-compatible multi-cloning site (Figure 1a). Constructs will be available in derivatives of pUC18-Sfi, (high copy number, replicative in any E. coli strain), or derivatives of pUC18-R6KT (medium copy number, replicative only in E. coli strains expressing the pir gene). Specialized miniTn7 vectors containing promoters for gene expression or reporters for expression measurement will also be constructed (Figure 1b, 1c and 1d). In addition, we will provide a portable attTn7 (Tn7 attachment site) to allow using the miniTn7 BioBrick toolkit in any organism as well as in plasmids.
Figure 1. Basic architecture of the miniTn7 derivatives. a. General purpose cloning vectors; b. Promoter characterization vectors; c. Expression vectors; d. Strain labelling vectors.