Difference between revisions of "Part:BBa K3332040"

 
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<partinfo>BBa_K3332040 short</partinfo>
 
<partinfo>BBa_K3332040 short</partinfo>
  
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A promoter derived from Ptrc-2 promoter can be strongly repressed by LacI protein.We use it to express mf-lon and MazF in the absence of aTc so as to inhibit the growth of E.coli.
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A promoter derived from Ptrc-2 promoter can be strongly repressed by LacI protein. Ptrc-2 promoter has three lac operators, which means that it has more LacI binding sites so that LacI has a weak inhibitory effect on it.
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===Usage and Biology===
 
===Usage and Biology===
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The Ptrc-2 promoter is used to express mf-lon and MazF in the absence of aTc so as to inhibit the growth of E.coli. It is part of the circut designed to prevent engineered bacteria in the detection instrument from escaping.
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In this circuit, LacI can repress ptrc-2 promoter and ptrc-2 derived promoter while the LacI can repress the pLtetO-1 promoter. When the aTc exits, it can combine tetR, so that the pLtetO-1 promoter can’t be repressed. Then the LacI which is controlled by the pLtetO-1 can repress the ptrc-2 promoter and ptrc-2 derived promoter. As a result, mf-lon and mazF can’t be expressed. As a kind of bacterial toxin, mazF can cause the bacteria death. So there comes the conclusion that as long as the engineered E.coli are cultured in the environment with aTc, it won’t be killed by the mazF, but when the bacteria escape from our testing instrument, the effect can be reversed, that is to say, the bacteria will be killed by the mazF. In the same way, we can conclude that in the presence of IPTG, MazF can be expressed to cause bacterial death.
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<span class='h3bb'>Sequence and Features</span>
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<table><tr><th>[[File:T--XMU-CHINA--circuit--circuit.png|thumb|600px|Fig.1 Circuit.]]</th><th></table>
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===Characterization===
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The agarose gel electrophoresis images are below:
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<table><tr><th>[[File:T--XMU-CHINA-BBa K3332086.png|thumb|300px|Fig.2 pTrc-2_E0420_pSB1C3[BBa_K3332086] digested by <i>Xba</i> I and <i>Pst</i> I.]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|300px|Fig.3 pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by <i>EcoR</i> I and <i>Pst</i> I.]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332088.png|thumb|300px|Fig.4 pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by <i>Pst</i> I.]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|300px|Fig.5 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by <i>Pst</i> I. note: E0420 is equal to B0034_E0020_B0015]]</th><th></table>
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===Protocol===
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1. Preparation of stock solution
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Dissolve IPTG in absolute alcohol to make 1000× stock solution
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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4.Add 100μL IPTG stock solution into the induction group when OD increased to 0.6. 5.Induce for 6 hours and the condition is the same as before.
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6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µl sterile PBS to resuspend.
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7.Measure the fluorescence intensity(ECFP)and corresponding OD600  by 96-well plate reader, then calculate the fluorescence / OD value of each group.
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Here is the result:
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<table><tr><th>[[File:T--XMU-CHINA--figure 14.png|thumb|600px|Fig.6 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.]]</th><th></table>
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The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure below,  pTrc2-derivative are used as the negative control group, the pTrc2-derivative are used as the positive control group while the pLtetO-1-LacI-pTrc2-E0420 (tetR) and pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) are both experience group. We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved and the pLtetO-1-LacI-pTrc2-E0420 (tetR) group the difference of fluorescence intensity is smaller than the pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) group so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter. That’s why after adding IPTG, the fluorescence intensity of pLtetO-1-LacI-pTrc2-E0420 (tetR) group increases faster.
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<table><tr><th>[[File:T--XMU-CHINA--pLtetO-1-LacI-pTrc2-E0420.png|thumb|600px|Fig.7 In each group，the EP tube on the left is without induction while the one on the right is with induction.]]</th><th></table>
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From this figure, the induction effect can be seen more intuitively.
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===Sequence And Features===
 
<partinfo>BBa_K3332040 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332040 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3332040 parameters</partinfo>
 
<partinfo>BBa_K3332040 parameters</partinfo>
 
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===Reference===
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[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. &apos;Deadman&apos; and &apos;Passcode&apos; microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979

Revision as of 03:53, 25 October 2020


pTrc-2 derivative

A promoter derived from Ptrc-2 promoter can be strongly repressed by LacI protein. Ptrc-2 promoter has three lac operators, which means that it has more LacI binding sites so that LacI has a weak inhibitory effect on it.

Usage and Biology

The Ptrc-2 promoter is used to express mf-lon and MazF in the absence of aTc so as to inhibit the growth of E.coli. It is part of the circut designed to prevent engineered bacteria in the detection instrument from escaping. In this circuit, LacI can repress ptrc-2 promoter and ptrc-2 derived promoter while the LacI can repress the pLtetO-1 promoter. When the aTc exits, it can combine tetR, so that the pLtetO-1 promoter can’t be repressed. Then the LacI which is controlled by the pLtetO-1 can repress the ptrc-2 promoter and ptrc-2 derived promoter. As a result, mf-lon and mazF can’t be expressed. As a kind of bacterial toxin, mazF can cause the bacteria death. So there comes the conclusion that as long as the engineered E.coli are cultured in the environment with aTc, it won’t be killed by the mazF, but when the bacteria escape from our testing instrument, the effect can be reversed, that is to say, the bacteria will be killed by the mazF. In the same way, we can conclude that in the presence of IPTG, MazF can be expressed to cause bacterial death.

Fig.1 Circuit.

Characterization

The agarose gel electrophoresis images are below:

Fig.2 pTrc-2_E0420_pSB1C3[BBa_K3332086] digested by Xba I and Pst I.
Fig.3 pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by EcoR I and Pst I.
Fig.4 pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by Pst I.
Fig.5 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by Pst I. note: E0420 is equal to B0034_E0020_B0015

Protocol

1. Preparation of stock solution Dissolve IPTG in absolute alcohol to make 1000× stock solution 2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h. 3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm. 4.Add 100μL IPTG stock solution into the induction group when OD increased to 0.6. 5.Induce for 6 hours and the condition is the same as before. 6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µl sterile PBS to resuspend. 7.Measure the fluorescence intensity(ECFP)and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group. Here is the result:

Fig.6 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure below, pTrc2-derivative are used as the negative control group, the pTrc2-derivative are used as the positive control group while the pLtetO-1-LacI-pTrc2-E0420 (tetR) and pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) are both experience group. We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved and the pLtetO-1-LacI-pTrc2-E0420 (tetR) group the difference of fluorescence intensity is smaller than the pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) group so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter. That’s why after adding IPTG, the fluorescence intensity of pLtetO-1-LacI-pTrc2-E0420 (tetR) group increases faster.

Fig.7 In each group，the EP tube on the left is without induction while the one on the right is with induction.

From this figure, the induction effect can be seen more intuitively.

Sequence And Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 37
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979