Table S4 of Additional File 1 provides more details of the GFP fusions generated. As discussed before, the selection conditions for the mutagenesis experiment just mentioned were such that they ruled
out inactivation of essential and metabolic genes necessary for growth in minimal medium. Also, GFP fusions may conceal the original localization of the inserted protein Blasticidin S (as just seen with FliC). However, random generation of fluorescent fusions of the sort discussed above pinpoints proteins that are highly expressed under physiologically relevant conditions. We argue that this may become a phenomenal tool to tackle the still standing question of gene expression Combretastatin A4 mw sites vs. chromosomal localization [50, 51], an important issue that is beyond the scope
of this paper. Conclusion We have created a synthetic plasmid composed of multiple formatted and optimized functional parts that behave as predicted -both individually and as an integrated system. To the best of our knowledge this is the first report since the early 90s that describes a fully edited genetic tool optimized and streamlined for its final applications -rather than relying on cutting and pasting naturally occurring sequences [52]. In a nutshell, non-functional DNA sequences were trimmed-off, common restriction sites present AZD1480 cost outside the multiple cloning site inside the mobile element were eliminated and the Immune system plasmid was designed following a modular pattern in which each business sequence was flanked by non-frequent restriction sites. In this respect, the key features of pBAM1 include not only the removal of many bottlenecks that flawed utilization of many of its predecessors, but also the incorporation of a fixed
standard for physical assembly and exchange, where required, of new DNA pieces while maintaining its overall layout. The modularity of the design and the origin of the parts in mobile elements, which are endowed with considerable orthogonality, enable pBAM1 for two specific applications. The first, straightforward application is the use of pBAM1 as a high-throughput mutational analysis tool [41]. Second, more important, the new vector allows exploitation of the cargo site (Figure 1 and 2) for placing a whole collection of extra genetic gadgets for expression of heterologous genes, reporter systems and environmental markers at user’s will. This includes the possibility of cloning large DNA fragments inside the mobile element for a final implantation of new traits into the chromosome of the target strain. Given the randomness and the high frequencies of such insertions, one can then select the insertion out of a large collection, which adjusts expression of the desired feature to the right level under the required operation conditions [53, 54].