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Construction of additional miniTn7BB derivatives

The modular nature of the miniTn7BB-Gm minitransposon and the presence of appropriate restriction sites allows the easy generation of variants with new traits. We have focused on two lines: the construction of variants with different drug resistance markers and the development of miniTn7BB derivatives containing BioBrick parts suitable for specialized functions.

Derivatives with alternative drug resistance markers

Bacterial species show diverse degrees of sensitivity to antibiotics. Drug resistance markers that support efficient selection in one bacterium may not be suitable for others. To facilitate the generation of miniTn7BB transposons bearing a variety of antibiotic resistance genes, the Gm resistance cassette in miniTn7BB-Gm was flanked with duplicated NcoI and SphI sites, which are absent in the rest of the transposon and the delivery vectors. Marker replacement is achieved by (i) digestion of a miniTn7BB-Gm delivery plasmid with either SphI or NcoI, (ii) PCR amplification of the new drug resistance marker with primers bearing sites for the same restriction enzyme, (iii) digestion of the PCR product with the indicated enzyme, (iv) ligation of the cleaved plasmid and PCR product, (v) transformation in a suitable E. coli strain, selecting resistance to the new antibiotic, and (vi) scoring for loss of the Gm^r marker. Using this method, we have successfully constructed pUC18Sfi-miniTn7BB-Km, a kanamycin-resistant (Km^r) variant of pUC18Sfi-miniTn7BB-Gm; obtaining the Km^r cassette from pSB4K5. A Km^r derivative in pUC18R6KT and chloramphenicol-resistant miniTn7BB derivatives in both pUC18Sfi and pUC18R6KT are currently under construction (Figure 5).

Figure 5. Schematic of the kanamycin-resistant and chloramphenicol-resistant minitransposons miniTn7BB-Km (top) and miniTn7BB-Cm (bottom). Transposon ends (red boxes), relevant restriction sites (vertical lines), FRT elements (blue circles), transcriptional terminators (red hexagons), drug resistance markers (green boxes), BioBrick cloning sites (cyan boxes) and primer annealing targets (blue arrows) are indicated

Specialized miniTn7BB-Gm derivatives

In order to provide the iGEM community with a wider range of Tn7-based tools, we undertook the construction of a number of composite parts in which pre-existing BioBricks are cloned into the BioBrick cloning site, thus providing new functionality to the transposon. The specialized miniTn7BB transposons planned include (i) gene fusion vectors for single-copy promoter analysis, such as miniTn7BB-Gm-RBS+RFP, containing the promoterless mRFP gene (part BBa_K093005) and miniTn7BB-Gm-lacZ-GFP, containing the promoterless lacZα fragment and a gfp gene encoding the unstable GFP-AAV variant (part BBa_I732094); (ii) expression vectors for controlled expression of BioBrick-encoded proteins, such as miniTn7BB-Gm-pBAD/araC, harboring araC (encoding the arabinose activator-repressor) and the tightly controlled pBAD promoter (part BBa_I0500), and miniTn7BB-Gm-tetR harboring constitutively expressed tetR (encoding the tetracycline repressor) and the TetR-regulated ptetR promoter (part BBa_I732083); and (iii) vectors for stable fluorescent or bioluminiscent labeling of bacteria, such as miniTn7BB-Gm-RFP, constitutively producing mRFP (part BBa_I13521), and miniTn7BB-Gm-Lux, expressing the lux operon from the arabinose-inducible pBAD promoter (part BBa_K325909). The structures of these constructs are shown in Figure 6.

Figure 6. Schematic of the specialized miniTn7BB-Gm derived minitransposons. miniTn7BB-Gm-RFP+RFP (a), miniTn7BB-Gm-lacZ-GFP (b), miniTn7BB-Gm-pBAD/araC (c), miniTn7BB-Gm-tetR (d), miniTn7BB-Gm-RFP (e), and miniTn7BB-Gm-Lux (f)

We have already constructed pUC18Sfi-miniTn7BB-Gm-RBS+RFP (BBa_K510037), pUC18Sfi-miniTn7BB-Gm-LacZ+GFP (BBa_K510040), and pUC18Sfi-miniTn7BB-Gm-RFP (BBa_K510041), by digesting the corresponding Biobrick parts with EcoRI and PstI and ligating into EcoRI+PstI-cleaved pUC18Sfi-miniTn7BB-Gm (Figure 7). The remaining specialized minitransposons in the set are currently under construction, and we hope to make them available to the community soon.

Figure 7. EcoRI and PstI digest of the specialized miniTn7BB-Gm derivatives. pUC18Sfi-miniTn7BB-Gm-RBS+RFP (BBa_K510037) (a), pUC18Sfi-miniTn7BB-Gm-LacZ+GFP (BBa_K510040) (b), and pUC18Sfi-miniTn7BB-Gm-RFP (BBa_K510041) (c)