Difference between revisions of "Part:BBa Q04510"
 
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[https://parts.igem.org/Part:BBa_K176114 K176114]
 
[https://parts.igem.org/Part:BBa_K176114 K176114]
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=ECUST_China 2019 characterization=
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In ECUST_China 2019 characterization, we added the Plac before the CⅠgene and used mRFP as the reporter gene to measure the effectiveness of this part.
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In the absence of IPTG, CⅠP was constitutively expressed so that the RFP fluorescence could be observed. However, as the concentration of IPTG increasing, Plac was activated to express CⅠprotein which inhibited the transcription of CⅠP ，so the fluorescence of mRFP decreased.
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=Contribution=
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'''Group''': [https://2020.igem.org/Team/XMU-China iGEM Team XMU-China 2020]
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'''Author''': Shi Zhang
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'''Summary''': fluorescence / OD<sub>600</sub>
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===Quantitative Characterization from iGEM20-XMU-China===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inverter is mainly composed of cI repressor from ''E. coli'' phage lambda and pR promoter which is inhibited by cI repressor. It can reverse the inducer action of the upstream  promoter. It was registered in 2003.
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/c/c7/T--XMU-China--XMU-China_2020_inverter.png" width="40%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig.1''' Genetic circuits of Inverter.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We constructed “P<sub>Bad/araC</sub>-RBS-EYFP-pSB1C3”(<partinfo>BBa_K3332082</partinfo>) and “P<sub>Bad/araC</sub>-Inverter-RBS-EYFP-pSB1C3”(<partinfo>BBa_K3332079</partinfo>) in ''E.coli'' BL21（DE3）to characterize its function.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For the latter circuit, cI repressor will not express in the absence of arabinose so that the fluorescence of EYFP can be observed. At the certain concentration of arabinose, PBad/araC can be activated to express cI repressor to inhibit the pR promoter，so the fluorescence of EYFP decreases.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The fluorescence/OD<sub>600</sub> values of these two circuits were measured with or without arabinose and compared with “P<sub>Bad/araC</sub>-pSB1C3” in ''E.coli'' BL21(DE3). We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed. The result proves that inverter can function correctly and reverse the effect of P<sub>Bad/araC</sub>. 
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/3/3d/T--XMU-China--XMU-China_2020-BY_BNY_fluorescence.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig.2''' The results of Fluorescence Intensity/OD.Yellow represents P<sub>Bad/araC</sub>, green represents P<sub>Bad/araC</sub>-EYFP(<partinfo>BBa_K3332082</partinfo>) , and purple represents P<sub>Bad/araC</sub> -Inverter-EYFP(<partinfo>BBa_K3332079</partinfo>) .
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<h1><b>Contribution From CUG-China 2024</b></h1>
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<html>
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Inverter is mainly composed of cI repressor from <i>E. coli</i> phage lambda and <i>P<sub>R</sub></i> promoter which is inhibited by <i>cI</i> repressor. It can reverse the inducer action of the upstream promoter. It was registered in 2003.
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In our project, we expect engineered bacteria to survive and carry out biomining activities only in environments containing gold, so we constructed bacterial suicide switches using tandem gene wiring of gold combined with specific transcription factors, Inverter and <i>mazF</i>.
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<center><img src="https://static.igem.wiki/teams/5323/parts/c5.png" width="50%" alt=""> </center>
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<center>Fig 5. The gene circuit of expression vector “pYDT-<i>golS</i>-<i>P<sub>gol</sub></i>-<i>cI</i>-<i>P<sub>R</sub></i>-<i>mazF</i>” </center>
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In order to quantify the function of inverter and the toxicity of <i>mazF</i>, We designed gene circuits to express inverter and <i>mazF</i> by induction of inducers and reflected the toxicity of MazF to cells by measuring OD<sub>600</sub>.
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<center><img src="https://static.igem.wiki/teams/5323/parts/c6.png" width="50%" alt="">
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<center>Fig 6. Growth profiles of the engineered bacterium <i>E. coli</i> BL21 in the presence of both inducer </center>
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IPTG and Au[III], only inducer IPTG or Au[III], and no inducer IPTG and Au[III].</center>
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In the presence of both Au[III] and the inducer IPTG, the engineered strains grew normally. Whereas, when only Au[III] or inducer IPTG was present and when neither Au[III] nor inducer IPTG was added, the bacteria expressed the toxic protein MazF resulting in failure to grow normally. The effective operation of the suicide module was demonstrated.

Latest revision as of 10:52, 1 October 2024

QPI (B0034.C0051.B0015.R0051)

Lambda cI QPI w/ strong RBS


Usage and Biology

Preliminary data indicates that this inverter functions well. [jb, 5/24/04]

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]


USTC_2009's MEASUREMENT

K176112

K176113

K176114

ECUST_China 2019 characterization

In ECUST_China 2019 characterization, we added the Plac before the CⅠgene and used mRFP as the reporter gene to measure the effectiveness of this part. In the absence of IPTG, CⅠP was constitutively expressed so that the RFP fluorescence could be observed. However, as the concentration of IPTG increasing, Plac was activated to express CⅠprotein which inhibited the transcription of CⅠP ，so the fluorescence of mRFP decreased.


Contribution

Group: iGEM Team XMU-China 2020

Author: Shi Zhang

Summary: fluorescence / OD600

Quantitative Characterization from iGEM20-XMU-China

        Inverter is mainly composed of cI repressor from E. coli phage lambda and pR promoter which is inhibited by cI repressor. It can reverse the inducer action of the upstream promoter. It was registered in 2003.

Fig.1 Genetic circuits of Inverter.


        We constructed “PBad/araC-RBS-EYFP-pSB1C3”(BBa_K3332082) and “PBad/araC-Inverter-RBS-EYFP-pSB1C3”(BBa_K3332079) in E.coli BL21（DE3）to characterize its function.

        For the latter circuit, cI repressor will not express in the absence of arabinose so that the fluorescence of EYFP can be observed. At the certain concentration of arabinose, PBad/araC can be activated to express cI repressor to inhibit the pR promoter，so the fluorescence of EYFP decreases.

        The fluorescence/OD600 values of these two circuits were measured with or without arabinose and compared with “PBad/araC-pSB1C3” in E.coli BL21(DE3). We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed. The result proves that inverter can function correctly and reverse the effect of PBad/araC.

Fig.2 The results of Fluorescence Intensity/OD.Yellow represents PBad/araC, green represents PBad/araC-EYFP(BBa_K3332082) , and purple represents PBad/araC -Inverter-EYFP(BBa_K3332079) .


Contribution From CUG-China 2024

Inverter is mainly composed of cI repressor from E. coli phage lambda and PR promoter which is inhibited by cI repressor. It can reverse the inducer action of the upstream promoter. It was registered in 2003. In our project, we expect engineered bacteria to survive and carry out biomining activities only in environments containing gold, so we constructed bacterial suicide switches using tandem gene wiring of gold combined with specific transcription factors, Inverter and mazF.

Fig 5. The gene circuit of expression vector “pYDT-golS-Pgol-cI-PR-mazF”
In order to quantify the function of inverter and the toxicity of mazF, We designed gene circuits to express inverter and mazF by induction of inducers and reflected the toxicity of MazF to cells by measuring OD600.
Fig 6. Growth profiles of the engineered bacterium E. coli BL21 in the presence of both inducer
IPTG and Au[III], only inducer IPTG or Au[III], and no inducer IPTG and Au[III].
In the presence of both Au[III] and the inducer IPTG, the engineered strains grew normally. Whereas, when only Au[III] or inducer IPTG was present and when neither Au[III] nor inducer IPTG was added, the bacteria expressed the toxic protein MazF resulting in failure to grow normally. The effective operation of the suicide module was demonstrated.