Difference between revisions of "Part:BBa K4729003"
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The results presented in figure 2 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of A. rhizogenes ARqua1 cultures in liquid medium was inhibited by sodium salicylate (Figure 2). In Agrobacterium tumefacies C58, PnttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021). | The results presented in figure 2 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of A. rhizogenes ARqua1 cultures in liquid medium was inhibited by sodium salicylate (Figure 2). In Agrobacterium tumefacies C58, PnttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021). | ||
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| + | The next step was to evaluate the specificity of the selected promoters and their inducers in an independent assay. Overall, we observed a good degree of orthogonality in the promoters tested. The PnahR promoter showed the strongest activation by other inducers, mainly by vanillin and taurine, while Ptau showed some activation by salicylic acid and vanillin (Figure 3). While the molecular similarity between sodium salicylate and vanillin could be the cause of the non-specific activation of PnahR by vanillin, the same is not the case for taurine. In <i>E. coli</i>, PnahR remained unresponsive to vanillin (Meyer et al., 2019). | ||
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| + | <img | ||
| + | class="img-inline" | ||
| + | src="https://static.igem.wiki/teams/4729/wiki/results/cross-talk-inducible-systems.png" | ||
| + | alt="Cross-talk between inducible systems." | ||
| + | /> | ||
| + | <figcaption> | ||
| + | <strong>Figure 3</strong>: | ||
| + | </figcaption> | ||
| + | </figure> | ||
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taken from:<br><br> | taken from:<br><br> | ||
Seiler, V. N. (2023). Improved inducible switches for the implementation of genetic tools in Sinorhizobium meliloti and Ensifer adhaerens (Master's thesis, Philipps-University Marburg) | Seiler, V. N. (2023). Improved inducible switches for the implementation of genetic tools in Sinorhizobium meliloti and Ensifer adhaerens (Master's thesis, Philipps-University Marburg) | ||
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Jacob, F., & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology, 3, 318–356. https://doi.org/10.1016/s0022-2836(61)80072-7 | Jacob, F., & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology, 3, 318–356. https://doi.org/10.1016/s0022-2836(61)80072-7 | ||
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Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), Article 2. https://doi.org/10.1038/s41589-018-0168-3 | Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), Article 2. https://doi.org/10.1038/s41589-018-0168-3 | ||
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Döhlemann, J., Wagner, M., Happel, C., Carrillo, M., Sobetzko, P., Erb, T. J., Thanbichler, M., & Becker, A. (2017). A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti. ACS Synthetic Biology, 6(6), 968–984. https://doi.org/10.1021/acssynbio.6b00320 | Döhlemann, J., Wagner, M., Happel, C., Carrillo, M., Sobetzko, P., Erb, T. J., Thanbichler, M., & Becker, A. (2017). A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti. ACS Synthetic Biology, 6(6), 968–984. https://doi.org/10.1021/acssynbio.6b00320 | ||
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Qian, Y., Kong, W., & Lu, T. (2021). Precise and reliable control of gene expression in Agrobacterium tumefaciens. Biotechnology and Bioengineering, 118(10), 3962–3972. https://doi.org/10.1002/bit.27872 | Qian, Y., Kong, W., & Lu, T. (2021). Precise and reliable control of gene expression in Agrobacterium tumefaciens. Biotechnology and Bioengineering, 118(10), 3962–3972. https://doi.org/10.1002/bit.27872 | ||
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Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Research, 49(12), 7189–7202. https://doi.org/10.1093/nar/gkab496 | Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Research, 49(12), 7189–7202. https://doi.org/10.1093/nar/gkab496 | ||
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Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765 | Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765 | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 14:22, 12 October 2023
Ptac - inducible promoter system
The IPTG/lacI promoter, one of the most widely used inducible systems in molecular biology, was originally described by Jacques Monod in 1961, for which he was awarded the Nobel Prize in 1965 (Jacob & Monod, 1961). As such, it is also popular in the iGEM community. More recently, a E. coli optimized version of this construct was published, displaying excellent orthogonality and dynamic range (Meyer et al., 2019). The research group one of our PIs created a improved version of the Ptac “Marionette” promoter for use in the Alphaproteobacteria Sinorhizobium meliloti, which we now present as a characterized part in A. rhizogenes and use in our Plant Trafo Master Switch construct <a href=”https://parts.igem.org/Part:BBa_K4729703”>BBa_K4729703</a>.
Characterization & Measurement
Measurements of expression strength were done using luminescence measurements and the LUX operon. Luminescence measurement are significantly more sensitive in comparison to fluorescence, allowing the characterization of weaker regulatory elements.The strains used in measurement experiments were grown in MOPS minimal medium liquid cultures containing the appropriate antibiotic. The use of minimal medium reduces possible variations due to inconsistencies in rich medium components. In order to maximize homogeneity between sample, the optical density of liquid cultures was measured before each measurement and diluted down to the same value using lab automation Platforms.
Figure 1: Characterization construct for basic parts in A. rhizogenes
This construct was designed for portability between E. coli and Alphaproteobacteria and compatibility with the MoClo Golden Gate standard as a level 1 entry vector (Döhlemann et al., 2017; Weber et al., 2011)
Results
Figure 2: Testing different inducible promoter systems in A. rhizogenes ARqua1.
The results presented in figure 2 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of A. rhizogenes ARqua1 cultures in liquid medium was inhibited by sodium salicylate (Figure 2). In Agrobacterium tumefacies C58, PnttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021).
The next step was to evaluate the specificity of the selected promoters and their inducers in an independent assay. Overall, we observed a good degree of orthogonality in the promoters tested. The PnahR promoter showed the strongest activation by other inducers, mainly by vanillin and taurine, while Ptau showed some activation by salicylic acid and vanillin (Figure 3). While the molecular similarity between sodium salicylate and vanillin could be the cause of the non-specific activation of PnahR by vanillin, the same is not the case for taurine. In E. coli, PnahR remained unresponsive to vanillin (Meyer et al., 2019).
