Difference between revisions of "Part:BBa K5206004"

 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5206004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5206004 SequenceAndFeatures</partinfo>
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==Results==
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===（1）Plasmid construction===
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Primers alsr-pd-For-1214 and alsR-0614-Rev were used to amplify the alsR and bidirectional promoter fragment. At the same time, pcdF-P-0614-gj-For and alsr-ZT-REV-1214 were used to amplify the vector. Then Gibson assembly was used to construct the recombinant plasmid. The colony PCR results and sequencing showed that the recombinant plasmid and engineered bacteria were successfully constructed.
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/6.png">
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  <div class="unterschrift"><b> pCDFDuet-1-alsR-PalsI-sfGFP</b>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/7.png">
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  <div class="unterschrift"><b> Principle of single plasmid biosensor</b>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/8.png">
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  <div class="unterschrift"><b> Electrophoretic detection of PCR results </b>
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  </div>
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</p>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/9.png">
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  <div class="unterschrift"><b> Transformation results </b>
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  </div>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/10.png">
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  <div class="unterschrift"><b> Electrophoretic detectionof colony PCR results </b>
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  </div>
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</p>
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===（2）Fluorescence detection===
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In order to verify the effectiveness of the biosensor, we verified the response effects of the single-plasmid biosensor (BBa_K5206004) and the dual-plasmid biosensor (BBa_K5206002 and BBa_K5206003) to different concentrations of D-allose.
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We cultured the original bacterial solution overnight in a shaker at 37℃, and then added different concentrations of D-allose for induction for 16-24 hours. The fluorescence value of the solution under the excitation wavelength of 488 nm and the emission wavelength of 518 nm was measured by a microplate reader to explore the induction expression effect of D-allose at different concentrations.
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We observed that in the biosensor system containing double plasmids, as the concentration of D-allose increased, the fluorescence signal reading showed a graded enhancement, indicating that there is a close positive correlation between the fluorescence intensity and the concentration of D-allose. For the single-plasmid biosensor BL21(DE)3/pCDFDuet-1-alsR-PalsI-sfGFP, as the concentration of D-allose increases, the fluorescence signal reading of the bacterial solution also shows a graded enhancement. The upper limit of its fluorescence value is lower than that of the dual-plasmid system, but the response difference to inducers of different concentrations is more obvious.
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/11.png">
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  <div class="unterschrift"><b> The influence of different concentrations of D-allose on the fluorescence intensity of the dual-plasmid and single-plasmid biosensor. Left: Double-plasmid biosensor; Right: Single-plasmid biosensor. </b>
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The dual-plasmid system may have an advantage in detecting lower concentrations of D-allose because it has a higher fluorescence value at low concentrations. However, when distinguishing different concentrations of D-allose, the single plasmid biosensor may have higher sensitivity or resolution.
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===（3）Performance test of BBa_K5206004===
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The concentration of D-allose, D-allulose, D-fructose, D-glucose stock solution was 100 g/L. The engineering strains containing recombinant plasmids were inoculated into 50 mL of LB medium at 4% inoculum, and the original bacterial solution was cultured overnight in a shaker at 25℃ and 37℃, and also induced by addition of different concentrations of substrates for 16-24 h. In order to accurately assess the effect of different concentrations of D-allose on the biosensors’ induced expression, the solution fluorescence intensity was quantitatively measured with an enzyme marker at 488 nm excitation wavelength and 518 nm emission wavelength. The results indicate that this biosensor performs better at 37 ℃ and has good specificity.
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<html>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/12.png">
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  <div class="unterschrift"><b> Effect of different concentrations of D-allose on the fluorescence intensity of single-plasmid biosensors at different temperature </b>
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  </div>
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</p>
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</html>
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  <img class="bild" src="https://static.igem.wiki/teams/5206/nanjingbiox-parts/13.png">
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  <div class="unterschrift"><b> Effect of different concentrations of various sugars on the fluorescence intensity of single-plasmid biosensors </b>
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  </div>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 06:27, 1 October 2024


alsR-Palsl-sfGFP

This part consists of BBa_K5206001 and BBa_K5206002. We constructed a composite part by fusing the sequence of BBa_K5206001, encoding the transcriptional regulator AlsR, with the expression vector of BBa_K5206002. PalsI is located in the middle of alsR and sfGFP and controls their expression. The AlsR transcriptional regulatory protein is transcriptionally translated from alsR, and it represses sfGFP expression. When D-allose is present in the environment, AlsR binds preferentially to D-allose and the inhibition will be lifted.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2355
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1640

Results

（1）Plasmid construction

Primers alsr-pd-For-1214 and alsR-0614-Rev were used to amplify the alsR and bidirectional promoter fragment. At the same time, pcdF-P-0614-gj-For and alsr-ZT-REV-1214 were used to amplify the vector. Then Gibson assembly was used to construct the recombinant plasmid. The colony PCR results and sequencing showed that the recombinant plasmid and engineered bacteria were successfully constructed.

pCDFDuet-1-alsR-PalsI-sfGFP

Principle of single plasmid biosensor

Electrophoretic detection of PCR results

Transformation results

Electrophoretic detectionof colony PCR results

（2）Fluorescence detection

In order to verify the effectiveness of the biosensor, we verified the response effects of the single-plasmid biosensor (BBa_K5206004) and the dual-plasmid biosensor (BBa_K5206002 and BBa_K5206003) to different concentrations of D-allose. We cultured the original bacterial solution overnight in a shaker at 37℃, and then added different concentrations of D-allose for induction for 16-24 hours. The fluorescence value of the solution under the excitation wavelength of 488 nm and the emission wavelength of 518 nm was measured by a microplate reader to explore the induction expression effect of D-allose at different concentrations. We observed that in the biosensor system containing double plasmids, as the concentration of D-allose increased, the fluorescence signal reading showed a graded enhancement, indicating that there is a close positive correlation between the fluorescence intensity and the concentration of D-allose. For the single-plasmid biosensor BL21(DE)3/pCDFDuet-1-alsR-PalsI-sfGFP, as the concentration of D-allose increases, the fluorescence signal reading of the bacterial solution also shows a graded enhancement. The upper limit of its fluorescence value is lower than that of the dual-plasmid system, but the response difference to inducers of different concentrations is more obvious.

The influence of different concentrations of D-allose on the fluorescence intensity of the dual-plasmid and single-plasmid biosensor. Left: Double-plasmid biosensor; Right: Single-plasmid biosensor.

The dual-plasmid system may have an advantage in detecting lower concentrations of D-allose because it has a higher fluorescence value at low concentrations. However, when distinguishing different concentrations of D-allose, the single plasmid biosensor may have higher sensitivity or resolution.

（3）Performance test of BBa_K5206004

The concentration of D-allose, D-allulose, D-fructose, D-glucose stock solution was 100 g/L. The engineering strains containing recombinant plasmids were inoculated into 50 mL of LB medium at 4% inoculum, and the original bacterial solution was cultured overnight in a shaker at 25℃ and 37℃, and also induced by addition of different concentrations of substrates for 16-24 h. In order to accurately assess the effect of different concentrations of D-allose on the biosensors’ induced expression, the solution fluorescence intensity was quantitatively measured with an enzyme marker at 488 nm excitation wavelength and 518 nm emission wavelength. The results indicate that this biosensor performs better at 37 ℃ and has good specificity.

Effect of different concentrations of D-allose on the fluorescence intensity of single-plasmid biosensors at different temperature

Effect of different concentrations of various sugars on the fluorescence intensity of single-plasmid biosensors