Difference between revisions of "Part:BBa K4591002"
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===Usage and Biology=== | ===Usage and Biology=== | ||
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| − | <p><i>XylS</i> is an archetype transcriptional activator of <i>AraC/XylS</i> family, mined from the TOL plasmid pWW0 of the bacterium <i>Pseudomonas putida</i>. It is composed of a C-terminal domain (CTD) involved in DNA binding, and an N-terminal domain required for effector binding and protein dimerization. <sup>[1]</sup></p> | + | <p><i>XylS</i> is an archetype transcriptional activator of <i>AraC/XylS</i> family, mined from the TOL plasmid <i>pWW0</i> of the bacterium <i>Pseudomonas putida</i>. It is composed of a C-terminal domain (CTD) involved in DNA binding, and an N-terminal domain required for effector binding and protein dimerization. <sup>[1]</sup></p> |
<p>XylS can bind benzoic acid and various derivatives, but it cannot recognize PA and TPA.<sup>[2-5]</sup> So the Jiawei Li and Mario Roque Huanca Nina successfully make the directed evolution of XylS to generate new TFs. Among these new TFs, Xylsmut<sub>K32R-L224Q</sub> and Xylsmut<sub>W88C-L224Q</sub> have showed the capacity of binding and responding to TPA and PA. Such XylS mutants could be used to construct whole-cell biosensors for fluorometric detection of PA and TPA. </p> | <p>XylS can bind benzoic acid and various derivatives, but it cannot recognize PA and TPA.<sup>[2-5]</sup> So the Jiawei Li and Mario Roque Huanca Nina successfully make the directed evolution of XylS to generate new TFs. Among these new TFs, Xylsmut<sub>K32R-L224Q</sub> and Xylsmut<sub>W88C-L224Q</sub> have showed the capacity of binding and responding to TPA and PA. Such XylS mutants could be used to construct whole-cell biosensors for fluorometric detection of PA and TPA. </p> | ||
<p>When the TPA was detected by the Xylsmut, the P<sub>m</sub> promotor would be actived and transcript the downstream fragments.<sup>[2-5,6]</sup></p> | <p>When the TPA was detected by the Xylsmut, the P<sub>m</sub> promotor would be actived and transcript the downstream fragments.<sup>[2-5,6]</sup></p> | ||
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<img src="https://static.igem.wiki/teams/4591/wiki/parts/tpa.svg" width="900" height="auto"> | <img src="https://static.igem.wiki/teams/4591/wiki/parts/tpa.svg" width="900" height="auto"> | ||
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| − | <p style="text-align: center;">Fig 1. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA</p> | + | <p style="text-align: center;">Fig.1. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA</p> |
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<p>In this year, we have designed a system of PET detection, attachment and degradation.In order to detect the TPA, the degradation products of the PET. The Xylsmut was chosen as the "sensor" of the TPA concentration, and the sfGFP was also constructed in our circuit as the indicator of TPA.</p> | <p>In this year, we have designed a system of PET detection, attachment and degradation.In order to detect the TPA, the degradation products of the PET. The Xylsmut was chosen as the "sensor" of the TPA concentration, and the sfGFP was also constructed in our circuit as the indicator of TPA.</p> | ||
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===Characterization=== | ===Characterization=== | ||
| − | <p>We have constructed a test circuit <i>P<sub>HpaII<sub></i>-<i> | + | <p>We have constructed a test circuit <i>P<sub>HpaII</sub></i>-<i>XylSmut</i>-<i>T</i><sub>500</sub>-<i>P<sub>m</sub></i>-<i>sfGFP-T</i><sub>500</sub> to characterize the Xylsmut. After incubating the engineered bacteria with 50 ml LB medium in 37℃,200RPM for 2 hours, we add 200ul 1g/100ml TPA solution and keep incubating. Test the OD200 and Fluorescence intensity in 488-520nm after 4 hours by Microplate reader.</p> |
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<p>The results is in the table and photo below.</p> | <p>The results is in the table and photo below.</p> | ||
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| − | <p style="text-align: center;"> <p>Fig 2. The result of the | + | <p style="text-align: center;"> <p>Fig.2. The result of the XylSmut testing.</p><p> A) The WT without TPA B) Engineered bacteria with TPA added C) WT with TPA added D) Engineered bacteria without TPA added</p> |
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| − | <img src="https://static.igem.wiki/teams/4591/wiki/parts/002- | + | <img src="https://static.igem.wiki/teams/4591/wiki/parts/002-x.jpg" width="600" height="auto"> |
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| − | <p style="text-align: center;"> Fig 3. The original data of the Xylsmut testing. | + | <p style="text-align: center;"> Fig.3. The original data of the Xylsmut testing. |
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https://static.igem.wiki/teams/4591/wiki/parts/2023xylsmutpig3.jpg | https://static.igem.wiki/teams/4591/wiki/parts/2023xylsmutpig3.jpg | ||
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| − | <p style="text-align: center;"> Fig 4. The time-course fluorescence changes of <i>pUC</i>57<i>-XylS-sfGFP</i> in response | + | <p style="text-align: center;"> Fig.4. The time-course fluorescence changes of <i>pUC</i>57<i>-XylS-sfGFP</i> in response |
to different inducers. </p><p> a) 2-hydroxybenzoic acid, b) 3-chlorobenzoic acid, c) PA, and d) | to different inducers. </p><p> a) 2-hydroxybenzoic acid, b) 3-chlorobenzoic acid, c) PA, and d) | ||
TPA.<sup>[6]</sup></p> | TPA.<sup>[6]</sup></p> | ||
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https://static.igem.wiki/teams/4591/wiki/parts/2023xylsmut-pig2.jpg | https://static.igem.wiki/teams/4591/wiki/parts/2023xylsmut-pig2.jpg | ||
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| − | <p style="text-align: center;">Fig 5. The time-course fluorescence of whole-cell biosensors containing different | + | <p style="text-align: center;">Fig.5. The time-course fluorescence of whole-cell biosensors containing different |
XylS mutants in response to PA and TPA.</p><p> (a) XylS-K38R-L224Q, PA; (b) XylS-K38R-L224Q, TPA; (c) XylS-W88C-L224Q, PA; (d) W88C-L224Q, TPA. <sup>[6]</sup></p> | XylS mutants in response to PA and TPA.</p><p> (a) XylS-K38R-L224Q, PA; (b) XylS-K38R-L224Q, TPA; (c) XylS-W88C-L224Q, PA; (d) W88C-L224Q, TPA. <sup>[6]</sup></p> | ||
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Latest revision as of 15:36, 12 October 2023
XylSmut
iGEM23_ZJUT-China's Contribution
Usage and Biology
XylS is an archetype transcriptional activator of AraC/XylS family, mined from the TOL plasmid pWW0 of the bacterium Pseudomonas putida. It is composed of a C-terminal domain (CTD) involved in DNA binding, and an N-terminal domain required for effector binding and protein dimerization. [1]
XylS can bind benzoic acid and various derivatives, but it cannot recognize PA and TPA.[2-5] So the Jiawei Li and Mario Roque Huanca Nina successfully make the directed evolution of XylS to generate new TFs. Among these new TFs, XylsmutK32R-L224Q and XylsmutW88C-L224Q have showed the capacity of binding and responding to TPA and PA. Such XylS mutants could be used to construct whole-cell biosensors for fluorometric detection of PA and TPA.
When the TPA was detected by the Xylsmut, the Pm promotor would be actived and transcript the downstream fragments.[2-5,6]
Depends on this, previews researchers have construct a simple XylSmut-based fluorometric biosensors which can detect the TPA.It contains a Pm promotor, a constitutive promotor, a fluorometric protein such as the GFP, and a terminator. [7]
Fig.1. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA
In this year, we have designed a system of PET detection, attachment and degradation.In order to detect the TPA, the degradation products of the PET. The Xylsmut was chosen as the "sensor" of the TPA concentration, and the sfGFP was also constructed in our circuit as the indicator of TPA.
Characterization
We have constructed a test circuit PHpaII-XylSmut-T500-Pm-sfGFP-T500 to characterize the Xylsmut. After incubating the engineered bacteria with 50 ml LB medium in 37℃,200RPM for 2 hours, we add 200ul 1g/100ml TPA solution and keep incubating. Test the OD200 and Fluorescence intensity in 488-520nm after 4 hours by Microplate reader.
The results is in the table and photo below.
Fig.2. The result of the XylSmut testing.
A) The WT without TPA B) Engineered bacteria with TPA added C) WT with TPA added D) Engineered bacteria without TPA added
Fig.3. The original data of the Xylsmut testing.
(The following experimental data are from the literature and modeling, not the actual data made by the experiment)
Limited by the Labotory equipment, some of the data was collected from the literature.
Fig.4. The time-course fluorescence changes of pUC57-XylS-sfGFP in response to different inducers.
a) 2-hydroxybenzoic acid, b) 3-chlorobenzoic acid, c) PA, and d) TPA.[6]
Fig.5. The time-course fluorescence of whole-cell biosensors containing different XylS mutants in response to PA and TPA.
(a) XylS-K38R-L224Q, PA; (b) XylS-K38R-L224Q, TPA; (c) XylS-W88C-L224Q, PA; (d) W88C-L224Q, TPA. [6]
The data shows that the TF XylSK38R-L224Q have higher sensitivity of the TPA. So we choose the XylSK38R-L224Q as our new part for the TPA detection.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]