Difference between revisions of "Part:BBa K4691000:Design"
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<partinfo>BBa_K4691000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4691000 SequenceAndFeatures</partinfo>
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===Design Notes===
 
===Design Notes===
 +
We obtained the recA-HRP-eGFP gene circuit by PCR and linked it to pUC19 vector by enzymatic ligation reaction.
 +
https://static.igem.wiki/teams/4691/wiki/result-1.png
 +
Then we transferred this plasmid into BL21 competent cells.We applied 100μL on LB plate which contains 100 μg/mL of ampicillin resistance, cultured it upside down at 37℃ overnight.
 +
We selected the monoclonal colony, and expanded the cultures.After that we sent it to jinweizhi (Suzhou) biochemisty co., ltd for sequencing, and stored the remaining bacteria at -20℃ for reserve.
 +
The HRP bacteria cultured overnight were transferred to 30 ml of fresh LB liquid medium, 30ul ampicillin was added to the logarithmic growth phase, and different concentrations of DNA damaging agent NA were incubated at inducible concentrations for 2 h, and the gradient was set to: 0 μM, 0.3 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM. SFU was calculated to obtain the NA induction curve of HRP type bacteria.
 +
https://static.igem.wiki/teams/4691/wiki/result-7.png
 +
From the curve, the linear interval expressed by HRP bacteria under NA induction was about 1~5μM, and the concentration gradient was refined in the linear interval and characterized again.
 +
The overnight culture of HRP and wild-type bacteria was transferred to 30 ml of fresh LB liquid medium, 30 ul ampicillin was added to the logarithmic growth phase, and different concentrations of DNA damaging agent NA were cultured at an inducible concentration for 2.5 h, and the gradient was set to: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, and induced expression for 2.5 h. Fit a linear equation. According to the formula LOD=3σ/S, the detection limit of the two strains was calculated. The detection limit of the HRP type sensor for NA is 0.01989μM, which is 1.8 times higher than the detection limit of 0.03580μM of the wild type sensor
 +
https://static.igem.wiki/teams/4691/wiki/result-8.png
 
===Source===
 
===Source===
 +
RecA,rbs30,rbs32,hrpR,hrpS,phrpL,eGFP are all synthesized by the Shanghai Sanggong Company.
 
===References===
 
===References===

Revision as of 20:59, 11 October 2023


recA-HRP-eGFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2010
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1220
    Illegal BsaI.rc site found at 2893
    Illegal SapI.rc site found at 1853

Design Notes

We obtained the recA-HRP-eGFP gene circuit by PCR and linked it to pUC19 vector by enzymatic ligation reaction. result-1.png Then we transferred this plasmid into BL21 competent cells.We applied 100μL on LB plate which contains 100 μg/mL of ampicillin resistance, cultured it upside down at 37℃ overnight. We selected the monoclonal colony, and expanded the cultures.After that we sent it to jinweizhi (Suzhou) biochemisty co., ltd for sequencing, and stored the remaining bacteria at -20℃ for reserve. The HRP bacteria cultured overnight were transferred to 30 ml of fresh LB liquid medium, 30ul ampicillin was added to the logarithmic growth phase, and different concentrations of DNA damaging agent NA were incubated at inducible concentrations for 2 h, and the gradient was set to: 0 μM, 0.3 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM. SFU was calculated to obtain the NA induction curve of HRP type bacteria. result-7.png From the curve, the linear interval expressed by HRP bacteria under NA induction was about 1~5μM, and the concentration gradient was refined in the linear interval and characterized again. The overnight culture of HRP and wild-type bacteria was transferred to 30 ml of fresh LB liquid medium, 30 ul ampicillin was added to the logarithmic growth phase, and different concentrations of DNA damaging agent NA were cultured at an inducible concentration for 2.5 h, and the gradient was set to: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, and induced expression for 2.5 h. Fit a linear equation. According to the formula LOD=3σ/S, the detection limit of the two strains was calculated. The detection limit of the HRP type sensor for NA is 0.01989μM, which is 1.8 times higher than the detection limit of 0.03580μM of the wild type sensor result-8.png

Source

RecA,rbs30,rbs32,hrpR,hrpS,phrpL,eGFP are all synthesized by the Shanghai Sanggong Company.

References