Difference between revisions of "Part:BBa K4226004"
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*We first used the promoter of sulA as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR, hrpS and PhrpL , and the eGFP was used as an output module to test the effect of amplifier module. The gene circuits is shown bellow:
 
*We first used the promoter of sulA as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR, hrpS and PhrpL , and the eGFP was used as an output module to test the effect of amplifier module. The gene circuits is shown bellow:
  
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[[File:PSB1C3-sulAp-Amp30E-eGFP.png|600px|thumb|center|Gene circuit: PSB1C3-sulAp-Amp30E-eGFP]]<br>
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[[File:PSB1C3-sulAp-Amp30E-eGFP.png|500px|thumb|center|Gene circuit: PSB1C3-sulAp-Amp30E-eGFP]]<br>
  
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*The recombinant cells were cultured and the parameters were measured using microplate reader. The fluorescence per OD significantly increased after induction with different times of UV exposure (UVC, 254nm) . The results are as follows:  
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*The recombinant cells were cultured to optimum cell growth (OD600) and irradiated under ultraviolet C (UVC, 254nm). The parameters were then measured using microplate reader for 8 hours. The fluorescence per OD significantly increased after induction with UVC exposure. The results are as follows:
 +
 
 +
[[File:The effect of Amp20E.png|500px|thumb|center|Figure: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Competent host cell: BL21. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction]]<br>
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*The results showed that the Amp30E Amplification Device (PSB1C3-sulAp-Amp30E-eGFP) significantly increased the fluorescence expression of eGFP compared with the group of PSB1C3-sulAp-eGFP. This result greatly demonstrates the excellent capability of Amp30E Amplification Device.
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 +
 
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*Further, we use this amplification device to increase the cytotoxic effect of [[Part:BBa_K185047|RelE toxin]] on cells. The genetic circuit that contains sulAp, Amp30E and relE gene was constructed with vector PSB1C3 as follows:  
 +
 
 +
[[File:PSB1C3-sulAp-Amp30E-relE.png|500px|thumb|center|Gene circuit: PSB1C3-sulAp-Amp30E-relE]]<br>
 +
 
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*As expected, the OD600 of the recombinant bacteria (pSB1C3-sulAp-relE) decreased after UVC induction, and the Amp30E Amplification Device significantly increased the inhibitory effect of relE on bacterial growth as shown below:
 +
 
 +
[[File:The effect of Amp30E-relE.png|500px|thumb|center|Figure: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction]]<br>
  
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[[File:The effect of Amp20E.png|600px|thumb|center|Figure: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm).]]<br>
 
  
  

Latest revision as of 07:34, 27 September 2022


Amp30E Amplification Device

The Amp30E Amplification Device is an amplifier module includes hrpR Gene, hrpS Gene and hrpL Promoter, this engineered multi-layered transcriptional amplifiers can be able to sequentially boost output expression level of target genes.


Usage and Biology

Characterized by CAFA_China 2022

  • We applied a typical cellular sensor that can be abstracted as a three-stage processor comprising a sensing module that recognizes and transduces external signals into intracellular transcriptional signals, a computing module that modulates the transduced sensor signals, and an output actuating module that executes physiological responses.
  • We first used the promoter of sulA as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR, hrpS and PhrpL , and the eGFP was used as an output module to test the effect of amplifier module. The gene circuits is shown bellow:
Gene circuit: PSB1C3-sulAp-Amp30E-eGFP

  • The recombinant cells were cultured to optimum cell growth (OD600) and irradiated under ultraviolet C (UVC, 254nm). The parameters were then measured using microplate reader for 8 hours. The fluorescence per OD significantly increased after induction with UVC exposure. The results are as follows:
Figure: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Competent host cell: BL21. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction

  • The results showed that the Amp30E Amplification Device (PSB1C3-sulAp-Amp30E-eGFP) significantly increased the fluorescence expression of eGFP compared with the group of PSB1C3-sulAp-eGFP. This result greatly demonstrates the excellent capability of Amp30E Amplification Device.


  • Further, we use this amplification device to increase the cytotoxic effect of RelE toxin on cells. The genetic circuit that contains sulAp, Amp30E and relE gene was constructed with vector PSB1C3 as follows:
Gene circuit: PSB1C3-sulAp-Amp30E-relE

  • As expected, the OD600 of the recombinant bacteria (pSB1C3-sulAp-relE) decreased after UVC induction, and the Amp30E Amplification Device significantly increased the inhibitory effect of relE on bacterial growth as shown below:
Figure: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1891
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101
    Illegal SapI.rc site found at 1734



Reference
[1] Wan, Xinyi, et al. "Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals." Nature chemical biology 15.5 (2019): 540-548.