Difference between revisions of "Part:BBa K2771000"

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<h2>Aim of experiment </h2>
 
<h2>Aim of experiment </h2>
Based on K2771000 part, a genetic circuit LuxR-pLux-GFP was constructed to characterize the function of this modified pLux promoter and contributed to this part.
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Based on K2771000 part, a genetic circuit LuxR-pLux-GFP was constructed to characterize the function of this modified pLux promoter.
  
  
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1. Construction of pLuxR-pLux-GFP  
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1. Construction of pSB1C3-pLuxR-pLux-GFP  
We designed our genetic circuit based on the synthetic biology principle, which is shown in Fig1. The circuit was then constructed into plasmid backbone pSB1C3 by DNA synthesis. The pLuxR promoter was identified by PCR and enzyme digestion.
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We designed our genetic circuit based on the synthetic biology principle in Fig 1. The circuit was then constructed into plasmid backbone pSB1C3 by DNA synthesis. This plasmid was identified by PCR and enzyme digestion.
  
  
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2. GFP expression of E.coli under the control of K2771000 induced by different concentration of AHL (3OC6HSL)
 
2. GFP expression of E.coli under the control of K2771000 induced by different concentration of AHL (3OC6HSL)
  
Constructed plasmid pSB1C3-pLuxR-pLux-GFP containing K2771000 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of 3OC6HSL solutions were added into the test tube. The final concentration of 3OC6HSL was 0, 0.01, 0.1, 1, 10, 100, 1,000, 10,000, 100,000nM respectively. Samples were taken every one hour. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.
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The plasmid (pSB1C3-pLuxR-pLux-GFP) containing K2771000 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing chloramphenicol at 1:50 to expand the culture. When OD600 reached 0.4-0.6, we performed the experiment. Different concentrations of 3OC6HSL solutions were added into the test tube. The final concentration of 3OC6HSL was 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1,000 nM, 10,000 nM, and 100,000 nM respectively. Samples were collected every one hour. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.
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<h2> Results</h2>
 
<h2> Results</h2>
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https://2019.igem.org/wiki/images/0/0d/T--XHD-WS-Wuhan-A--Cu2.jpeg
 
https://2019.igem.org/wiki/images/0/0d/T--XHD-WS-Wuhan-A--Cu2.jpeg
  
Fig2. Map of pLux + GFP fragment after PCR and plasmid pSB1C3-pLuxR-pLux-GFP double-digested with Sph I and Pst I in gel electrophoresis.
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Fig 2. Gel electrophoresis of pLux + GFP fragment after PCR and pSB1C3-pLuxR-pLux-GFP double-digested with Sph I and Pst I.
 
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The standard fluorescence intensity(FL/OD600) was obtained by dividing the fluorescence intensity by OD600. As show in Fig.3, there are basically no difference in the first 6 hours of different 3OC6HSL concentrations. From 7hours, the fluorescence intensity was increased compared to the control group (0nM).
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The standard fluorescence intensity (FL/OD600) was obtained by dividing the fluorescence intensity by OD600. As show in Fig.3, there is no difference from 0 h to 6 h in DH5α treated with different 3OC6HSL concentrations. The fluorescence intensity was increased compared to the control group after 7 h treatment (0nM).
  
 
https://2019.igem.org/wiki/images/d/d8/T--XHD-WS-Wuhan-A--Cu3.jpeg
 
https://2019.igem.org/wiki/images/d/d8/T--XHD-WS-Wuhan-A--Cu3.jpeg
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https://2019.igem.org/wiki/images/8/89/T--XHD-WS-Wuhan-A--Cu4.jpeg
 
https://2019.igem.org/wiki/images/8/89/T--XHD-WS-Wuhan-A--Cu4.jpeg
  
Fig.4 GFP expression of E.coli under the control of pLux (K2771000) in different concentrations 3OC6HSL.
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Fig 3. The value of GFP fluorescence in DH5α treated with different 3OC6HSL concentrations.
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As show in Fig.3,  the fluorescence intensity is strongest at 8 h. So 8 h was chosen for further study. As shown in Fig.4, pLux promoter is very sensitive to 3OC6HSL. And 1 nm of 3OC6HSL was required to detect fluorescence signals. The threshold for pLux detection is low and the background of the control is very high, which indicats the leak of this promoter. 
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https://2019.igem.org/wiki/images/8/89/T--XHD-WS-Wuhan-A--Cu4.jpeg
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Fig.4 GFP expression of E.coli under the control of pLux (K2771000) in different concentrations of 3OC6HSL.
  
 
<h2> Summary</h2>
 
<h2> Summary</h2>
The plasmid constructed based on K2771000 is sensitive to 3OC6HSL. The threshold of the pLux promoter is 1nM but the background is very high. Therefore promoters with high thresholds and low leakage need to be developed.
+
 
 +
The plasmid constructed based on K2771000 is sensitive to 3OC6HSL. The threshold of the pLux promoter is 1nM. However, the background is very high. Our results indicated the pLux promoter has high thresholds and low leakage of this promoter need to be developed.
  
  

Revision as of 03:12, 21 October 2019


Modified pLux

Quorum sensing activated promoter that responds natively to the Lux pathway (activated by LuxR + 3OC6HSL). This specific instance of this promoter has a mutation on nucleotide 18 of the consensus sequence, which reportedly diminishes genetic crosstalk with the Las system.



Usage and Biology

XHD-WS-Wuhan-A characterization


Aim of experiment

Based on K2771000 part, a genetic circuit LuxR-pLux-GFP was constructed to characterize the function of this modified pLux promoter.


Methods


1. Construction of pSB1C3-pLuxR-pLux-GFP We designed our genetic circuit based on the synthetic biology principle in Fig 1. The circuit was then constructed into plasmid backbone pSB1C3 by DNA synthesis. This plasmid was identified by PCR and enzyme digestion.



T--XHD-WS-Wuhan-A--Cu1.jpeg

Fig1.Gene circuit of pLuxR-plux-GFP combinations


2. GFP expression of E.coli under the control of K2771000 induced by different concentration of AHL (3OC6HSL)

The plasmid (pSB1C3-pLuxR-pLux-GFP) containing K2771000 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing chloramphenicol at 1:50 to expand the culture. When OD600 reached 0.4-0.6, we performed the experiment. Different concentrations of 3OC6HSL solutions were added into the test tube. The final concentration of 3OC6HSL was 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1,000 nM, 10,000 nM, and 100,000 nM respectively. Samples were collected every one hour. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.


Results

We used the constructed plasmid as template and then took PCR to obtain the corresponding fragment pLux-GFP. As shown in Fig2, the size of the PCR product was as expected. The constructed plasmid was then double digested with Sph I/Pst I. the results indicate that enzyme digestion was as expected.

T--XHD-WS-Wuhan-A--Cu2.jpeg

Fig 2. Gel electrophoresis of pLux + GFP fragment after PCR and pSB1C3-pLuxR-pLux-GFP double-digested with Sph I and Pst I.

The standard fluorescence intensity (FL/OD600) was obtained by dividing the fluorescence intensity by OD600. As show in Fig.3, there is no difference from 0 h to 6 h in DH5α treated with different 3OC6HSL concentrations. The fluorescence intensity was increased compared to the control group after 7 h treatment (0nM).

T--XHD-WS-Wuhan-A--Cu3.jpeg

From Fig.3 we know that the fluorescence intensity is strongest at 8 hours. So 8h was chosen for further study.As shown in Fig.4, pLux promoter is very sensitive to 3OC6HSL and requires only 1nm to detect fluorescence signals. The threshold for pLux detection is low, while the background of the control is very high, indicating the leak of this promoter.

T--XHD-WS-Wuhan-A--Cu4.jpeg


Fig 3. The value of GFP fluorescence in DH5α treated with different 3OC6HSL concentrations.

As show in Fig.3,  the fluorescence intensity is strongest at 8 h. So 8 h was chosen for further study. As shown in Fig.4, pLux promoter is very sensitive to 3OC6HSL. And 1 nm of 3OC6HSL was required to detect fluorescence signals. The threshold for pLux detection is low and the background of the control is very high, which indicats the leak of this promoter.  


T--XHD-WS-Wuhan-A--Cu4.jpeg Fig.4 GFP expression of E.coli under the control of pLux (K2771000) in different concentrations of 3OC6HSL.

Summary

The plasmid constructed based on K2771000 is sensitive to 3OC6HSL. The threshold of the pLux promoter is 1nM. However, the background is very high. Our results indicated the pLux promoter has high thresholds and low leakage of this promoter need to be developed.


Reference:

Grant et al. Orthogonal intercellular signaling for programmed spatial behavior. Mol Syst Biol. (2016) 12: 849


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters