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Team:UPO-Sevilla/Foundational Advances/MiniTn7/Experimental Results/miniTn7BB derivatives
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| - | <h1>miniTn7BB derivatives</h1> | + | <h1>Construction of additional miniTn7BB derivatives</h1> |
| + | <p>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.</p> | ||
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| + | <h3>Derivatives with alternative drug resistance markers</h3> | ||
| + | <p>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 Gmr marker. Using this method, we have successfully constructed pMPO18Sfi-miniTn7BB-Km, a kanamycin-resistant (Kmr) variant of pMPO18Sfi-miniTn7BB-Gm. A Kmr derivative in pMPO18R6KT and chloramphenicol-resistant miniTn7BB derivatives in both pMPO18Sfi and pMPO18R6KT are currently under construction (Figure 3).</p> | ||
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| + | <img width="600px" src="https://static.igem.org/mediawiki/2011/c/cc/UPOSevilla-Mini-Tn7-Km.png" alt="Schematic representation of miniTn7BB-Km module"/> </div> | ||
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| + | <img width="600px" src="https://static.igem.org/mediawiki/2011/4/4b/Mini-Tn7-Cm.png" alt="Schematic representation of miniTn7BB-Cm module"/> </div> | ||
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| + | <p class caption><strong>Figure 3. Schematic of the kanamycin-resistant and chlormphenicol-resistant minitransposons miniTn7BB-Km (top) and miniTn7BB-Cm (bottom)</strong>. 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</p> | ||
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Revision as of 23:01, 20 September 2011
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 Gmr marker. Using this method, we have successfully constructed pMPO18Sfi-miniTn7BB-Km, a kanamycin-resistant (Kmr) variant of pMPO18Sfi-miniTn7BB-Gm. A Kmr derivative in pMPO18R6KT and chloramphenicol-resistant miniTn7BB derivatives in both pMPO18Sfi and pMPO18R6KT are currently under construction (Figure 3).
Figure 3. Schematic of the kanamycin-resistant and chlormphenicol-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
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