Difference between revisions of "Part:BBa K1493000"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | A fusaric acid efflux pump within ''Pseudomonas putida'' is encoded by an operon consisting of four genes. | + | A fusaric acid efflux pump within ''Pseudomonas putida'' is encoded by an operon consisting of four genes. We found that this operon is controlled by a LysR-type gene (pp1262) which is located upstream of the operon. This gene inhibits the binding of RNA polymerase to the promoter in the intergenic region between pp1262 and the operon. Fusaric acid blocks this inhibition, allowing activity of the operon. (See figure 1) Hence, pp1262 and the intergenic region are isolated and put into BioBrick form, effectively acting as a Fusaric Acid inducible Promoter (FAiP). |
| − | https://static.igem.org/mediawiki/2014/ | + | https://static.igem.org/mediawiki/2014/a/a2/Wageningen_UR_sensings_Faip10.jpg<br> |
| − | + | <html><p><i><center>Figure 1. Fusaric acid efflux pump operon present in the genome of KT2440 Pseudomonas putida.</i></center></p></html> | |
| + | <br><br> | ||
| + | A growth experiment was set up, using M9 medium. In this experiment both the WT and transformed ''P. putida'' containing the BBa_K1493000 BioBrick are grown in a 96-wells plate with 0, 85, 170, 255, 340 and 425µM (0, 15, 30, 45, 60 and 75µg/ml) fusaric acid. Furthermore, ''E. coli'' DH5α cells (WT and BBa_K741002) were also grown in the same plate, but without fusaric acid. The transformed ''E. coli'' has a well characterized promoter with the same GFP gene downstream. By comparing the fluorescence of our fusaric acid induced promoter at different fusaric acid concentrations to this constitutive promoter, a characterization can be performed.<br> | ||
| + | <html><img src="https://static.igem.org/mediawiki/2014/7/7e/Wageningen_UR_sensing_Faip20.jpg" width="80%"><br> | ||
| + | <p><center><i>Figure 2. <br> | ||
| + | *Significantly different from WT with p<0.05<br> | ||
| + | The measurement is based on GFP fluorescence in P. putida at increased concentrations of fusaric acid to prove and characterize the activity of the fusaric acid induced promoter, BBa_K1493000. For comparison, the well characterized pLac promoter (BBa_K741002, uninduced by IPTG) was used to quantify the activity of this promoter at different concentrations of fusaric acid. Our fusaric acid inducible promoter does not respond to low concentrations up to 170µM. From 255µM and up, the activity increases. The maximum measured activity of the promoter is 0.21 RPU at 425µM. </i></center></p></html> | ||
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| + | <!-- --> | ||
| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K1493000 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K1493000 parameters</partinfo> | ||
| + | <!-- --> | ||
===Features=== | ===Features=== | ||
| − | This | + | This BioBrick can only be used in ''Pseudomonas putida'', since the regulatory gene has been disrupted. In ''P. putida'', the suppressing gene is still expressed, which will still inhibit the binding of the RNA polymerase to this part, resulting in a functioning BioBrick. This disruption will also stop overexpression of the inhibiting gene, which also inhibits the efflux pump in the original genome and thus decreases its host's immunity to fusaric acid. The BioBrick with an intact inhibitor gene can be found here: https://parts.igem.org/Part:BBa_K1493002<br><br> |
| + | For more information on the functioning of this BioBrick, please check: http://2014.igem.org/Team:Wageningen_UR/project/fungal_sensing | ||
===References=== | ===References=== | ||
Latest revision as of 11:51, 21 October 2014
Fusaric acid induced regulatory promoter
Promoter fusaric acid inducible
Usage and Biology
A fusaric acid efflux pump within Pseudomonas putida is encoded by an operon consisting of four genes. We found that this operon is controlled by a LysR-type gene (pp1262) which is located upstream of the operon. This gene inhibits the binding of RNA polymerase to the promoter in the intergenic region between pp1262 and the operon. Fusaric acid blocks this inhibition, allowing activity of the operon. (See figure 1) Hence, pp1262 and the intergenic region are isolated and put into BioBrick form, effectively acting as a Fusaric Acid inducible Promoter (FAiP).
A growth experiment was set up, using M9 medium. In this experiment both the WT and transformed P. putida containing the BBa_K1493000 BioBrick are grown in a 96-wells plate with 0, 85, 170, 255, 340 and 425µM (0, 15, 30, 45, 60 and 75µg/ml) fusaric acid. Furthermore, E. coli DH5α cells (WT and BBa_K741002) were also grown in the same plate, but without fusaric acid. The transformed E. coli has a well characterized promoter with the same GFP gene downstream. By comparing the fluorescence of our fusaric acid induced promoter at different fusaric acid concentrations to this constitutive promoter, a characterization can be performed.
*Significantly different from WT with p<0.05
The measurement is based on GFP fluorescence in P. putida at increased concentrations of fusaric acid to prove and characterize the activity of the fusaric acid induced promoter, BBa_K1493000. For comparison, the well characterized pLac promoter (BBa_K741002, uninduced by IPTG) was used to quantify the activity of this promoter at different concentrations of fusaric acid. Our fusaric acid inducible promoter does not respond to low concentrations up to 170µM. From 255µM and up, the activity increases. The maximum measured activity of the promoter is 0.21 RPU at 425µM.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1049
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 855
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 928
Features
This BioBrick can only be used in Pseudomonas putida, since the regulatory gene has been disrupted. In P. putida, the suppressing gene is still expressed, which will still inhibit the binding of the RNA polymerase to this part, resulting in a functioning BioBrick. This disruption will also stop overexpression of the inhibiting gene, which also inhibits the efflux pump in the original genome and thus decreases its host's immunity to fusaric acid. The BioBrick with an intact inhibitor gene can be found here: https://parts.igem.org/Part:BBa_K1493002
For more information on the functioning of this BioBrick, please check: http://2014.igem.org/Team:Wageningen_UR/project/fungal_sensing
References
Hu, R.-M., et al., An Inducible Fusaric Acid Tripartite Efflux Pump Contributes to the Fusaric Acid Resistance in Stenotrophomonas maltophilia. PLoS ONE, 2012. 7(12): p. e51053.
Nelson, K.E., et al., Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environmental microbiology, 2002. 4(12): p. 799-808.