Difference between revisions of "Part:BBa K1031941"
 
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<partinfo>BBa_K1031941 short</partinfo>
 
<partinfo>BBa_K1031941 short</partinfo>
 
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<p>For detailed information concerning XylS and Pm promoter, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/HbpR">2013 Peking iGEM Biosensor HbpR</a></p>
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<p>For detailed information concerning XylS and Pm promoter, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/XylS">2013 Peking iGEM Biosensor XylS</a></p>
  
 
<img src="https://static.igem.org/mediawiki/igem.org/c/c9/Peking_Logo.jpg" style="width:960px;"/>
 
<img src="https://static.igem.org/mediawiki/igem.org/c/c9/Peking_Logo.jpg" style="width:960px;"/>
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HbpR is the activator of two promoters, Pc and Pd, they are σ-54 dependent. HbpR binds to UAS C-1 and UAS C-2 on Pc, UAS D-1 and D-2 on Pd. The 32-bp space between the centers of UASs C-1 and C-2 is critical for cooperative interactions. However, when the UASs C-1/C-2 are deleted and UASs C-3/C-4 are placed in an appropriate position with respect to the promoter region, the Pc promoter is still inducible with 2-HBP, albeit at a lower level. It shows that the presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR('''Fig 1 a, b''')
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XylS is the activator of ''Pm'' promoter, which is σ-54 dependent. XylS recognizes two 15-bp repeats (TGCA-N6-GGNTA) in ''Pm'' promoter, each featured by box A1/A2 (TGCA) and box B1/B2 (GGNTA), respectively. The arrangement of the two repeats is deposited as shown in '''Fig 1'''; the proximal XylS binding site overlaps the -35 box by 2 bp (the sequence for the binding of RNA polymerase)('''Fig 1 a, b''')
  
 
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<img src="https://static.igem.org/mediawiki/igem.org/a/a3/HbpR_Figure4_2013Peking_WH1.png"  style="width:700px; margin-left:130px" />
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<img src="https://static.igem.org/mediawiki/igem.org/6/66/Peking2013_Xyls_figure2.png"   
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<p style="text-align:center"><b>Fig 1</b>  The sequences preceding hbpC and hbpD promoter contains the binding sites for HbpR (UAS). The UASs are boxed in red. (<b>a</b>) HbpR binding sites on <i>Pc</i> promoter, there are 4 UASs responsible for binding with HbpR. (<b>b</b>) HbpR binding sites on <i>Pd</i> promoter, there are 2 UASs for binding with HbpR.  
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<p style="text-align:center"><b>Fig 1</b>  Sequence features of <i>Pm</i> promoter. The orange arrows indicate the two XylS binding sites (proximal and distal), each consisting of conserved A1/A2 and B1/B2 boxes. The -10 and -35 hexamers are in blue. A right-angled arrow indicates the transcription start site (+1).  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1031302 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1031941 SequenceAndFeatures</partinfo>
  
  
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K1031302 is composed of three elements, the inducible promoter ''Pc'', RBS (Ribosome Binding Site) <html><a href="https://parts.igem.org/Part:BBa_B0032">B0032</a></html>, and reporter gene sfGFP with terminator <html><a href="https://parts.igem.org/Part:BBa_B0015">B0015</a></html>.  ('''Fig 2''')  
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K1031941 is composed of three elements, the inducible promoter ''Pm'', RBS (Ribosome Binding Site) <html><a href="https://parts.igem.org/Part:BBa_B0034">B0034</a></html>, and reporter gene eGFP with terminator <html><a href="https://parts.igem.org/Part:BBa_B0015">B0015</a></html>.  ('''Fig 2''')  
  
 
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<img src="https://static.igem.org/mediawiki/igem.org/4/4c/Peking2013_part_32-Pc_HbpR.png"  style="width:400px; margin-left:250px" />
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<img src="https://static.igem.org/mediawiki/2013/thumb/7/71/Peking2013_part_Pm-B0034-eGFP.png/800px-Peking2013_part_Pm-B0034-eGFP.png"  style="width:450px; margin-left:220px" />
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<p style="text-align:center"><b>Fig 2</b> Construction of reporter circuit. The orange arrow represents <i>Pc</i> promoter for HbpR. The green oval stands for RBS B0032. sfGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.  
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<p style="text-align:center"><b>Fig 2</b> Construction of reporter circuit. The orange arrow represents <i>Pm</i> promoter for XylS. The green oval stands for RBS B0034. eGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.  
 
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We created a library for the RBS of sfGFP, including B0031, B0032 and B0034. Dose response curve were tested at the presence of 2-ABP and 2-HBP respectively. Experiment results showed that for inducers 2-ABP and 2-HBP, RBS B0032 had a superior performance compared with B0031 and B0034('''Fig 3 a, b''')
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We tested this reporter circuit with different expression intensity of XylS coding sequence and selected the optimal-performed biosensor circuit composed of J23114-XylS and Pm-B0034-eGFP, then this circuit is subjected to ON/OFF test with 78 aromatic compounds, characterizing XylS detecting profile.(Fig 3)
  
 
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/3/39/Peking2013_part_RBS_library_HbpR.png/800px-Peking2013_part_RBS_library_HbpR.png" style="width:850px margin-left:110px"  />
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<img src="https://static.igem.org/mediawiki/igem.org/f/f4/Peking2013_Xyls_figure5.1.png"  
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<p style="text-align:center"><b>Fig 3</b> (<b>a</b>) Dose response curve of HbpR when exposed to 2-ABP at the concentration of 0.5µM, 1µM, 5µM, 10µM, 50µM, 100µM respectively. The reporter circuit consists of Pc promoter, RBS B0031, and reporter gene sfGFP. The curve with dark orange represents the circuit with B0032. Three lines represent circuit adopting different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio. (<b>b</b>) Dose response curve of HbpR when exposed to 2-HBP at the concentration of 0.5µM, 1µM, 5µM, 10µM, 50µM, 100µM respectively.
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<p style="text-align:center"><b>Fig 3</b> The induction ratios of all 78 typical aromatic compounds in the ON/OFF test following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives.  
 
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Dose-response curve is plotted to further characterize XylS performance with efficiency inducers at different concentration, proving that biosensor circuit ''Pm''/J23114-XylS performs like Hill function.
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<img src="https://static.igem.org/mediawiki/igem.org/9/93/Peking2013_Xyls_figure6.png"
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<p style="text-align:center"><b>Fig 4</b> Dose-response curves of XylS biosensor induced by benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratios. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1031302
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<partinfo>BBa_K1031941
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== Use Experience ==
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<!-- TPR_China iGEM2020 review -->
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_K1031941 AddReview 1</partinfo>
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<I>TPR_China iGEM2020</I>
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This part is not such good in our own experiment. However, we changed the reporter from eGFP ([[Part:BBa_K3599016|BBa_K3599016]]) to sfGFP ([[Part:BBa_K3599017|BBa_K3599017]]), and the induction is rescued. The new part we made is [[Part:BBa_K3599034|BBa_K3599034]].
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To characterize the property of the device itself, the original aromatic compond m-Toluic acid (mTa) was used as inducer. According to the experment results provided by [http://2013.igem.org/Team:Peking Peking iGEM2013], the mTa could cause the largest dynamic range and the most sensity of the Pm-eGFP sensor.
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====The fluorescence of the sensor induced by small molecules====
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Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on its corresponding aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control. <b>And here we can see that Xyls-sfGFP is 18 times of the Xyls-eGFP</b>. And what surprises us is that Paax sensor also has a good response effect to small molecules, which has not been detected before.<br>
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[[File:T--TPR China--Sensor1.png|600px|thumb|center|The fluorescence of the sensor induced by small molecules]]<br>
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[[File:T--TPR China--Sensor2.png|600px|thumb|center|The fluorescence of the NahR sensor induced by small molecules]]<br>
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====The fluorescence of the sensor induced by PAN====
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And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.
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[[File:T--TPR China--Sensor3.jpg|600px|thumb|center|The fluorescence of the sensor induced by PAN]]<br>
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|}

Latest revision as of 15:51, 27 October 2020

Pm-B0034-eGFP (XylS)

For detailed information concerning XylS and Pm promoter, please visit 2013 Peking iGEM Biosensor XylS


Structure

XylS is the activator of Pm promoter, which is σ-54 dependent. XylS recognizes two 15-bp repeats (TGCA-N6-GGNTA) in Pm promoter, each featured by box A1/A2 (TGCA) and box B1/B2 (GGNTA), respectively. The arrangement of the two repeats is deposited as shown in Fig 1; the proximal XylS binding site overlaps the -35 box by 2 bp (the sequence for the binding of RNA polymerase)(Fig 1 a, b)

Fig 1 Sequence features of Pm promoter. The orange arrows indicate the two XylS binding sites (proximal and distal), each consisting of conserved A1/A2 and B1/B2 boxes. The -10 and -35 hexamers are in blue. A right-angled arrow indicates the transcription start site (+1).


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 38
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 750


Construction and data

K1031941 is composed of three elements, the inducible promoter Pm, RBS (Ribosome Binding Site) B0034, and reporter gene eGFP with terminator B0015. (Fig 2)

Fig 2 Construction of reporter circuit. The orange arrow represents Pm promoter for XylS. The green oval stands for RBS B0034. eGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.


We tested this reporter circuit with different expression intensity of XylS coding sequence and selected the optimal-performed biosensor circuit composed of J23114-XylS and Pm-B0034-eGFP, then this circuit is subjected to ON/OFF test with 78 aromatic compounds, characterizing XylS detecting profile.(Fig 3)

Fig 3 The induction ratios of all 78 typical aromatic compounds in the ON/OFF test following Test Protocol 1. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives.


Dose-response curve is plotted to further characterize XylS performance with efficiency inducers at different concentration, proving that biosensor circuit Pm/J23114-XylS performs like Hill function.

Fig 4 Dose-response curves of XylS biosensor induced by benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratios. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

•

TPR_China iGEM2020

This part is not such good in our own experiment. However, we changed the reporter from eGFP (BBa_K3599016) to sfGFP (BBa_K3599017), and the induction is rescued. The new part we made is BBa_K3599034.

To characterize the property of the device itself, the original aromatic compond m-Toluic acid (mTa) was used as inducer. According to the experment results provided by [http://2013.igem.org/Team:Peking Peking iGEM2013], the mTa could cause the largest dynamic range and the most sensity of the Pm-eGFP sensor.

The fluorescence of the sensor induced by small molecules

Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on its corresponding aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control. And here we can see that Xyls-sfGFP is 18 times of the Xyls-eGFP. And what surprises us is that Paax sensor also has a good response effect to small molecules, which has not been detected before.

The fluorescence of the sensor induced by small molecules

The fluorescence of the NahR sensor induced by small molecules

The fluorescence of the sensor induced by PAN

And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.

The fluorescence of the sensor induced by PAN