Difference between revisions of "Part:BBa K3332038"
 
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<partinfo>BBa_K3332038 short</partinfo>
 
<partinfo>BBa_K3332038 short</partinfo>
  
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A promoter derived from pTrc-2 promoter can be strongly repressed by LacI protein. pTrc-2 promoter has one lac operator, which means that LacI has a weak inhibitory effect on it.
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A promoter can be repressed by LacI protein. It has only one lac operator, which means that it has less LacI binding sites so that LacI has a weak inhibitory effect on it.
  
 
===Usage and Biology===
 
===Usage and Biology===
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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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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 ''E.coli'' in the detection instrument from escaping.
  
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In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter，while tetR can repress pLtetO-1 promoter. When ATc exits, it can combine tetR, so that pLtetO-1 promoter can’t be repressed. Then LacI which is controlled by pLtetO-1 can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, mf-lon and MazF can’t be expressed.
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In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter ，while tetR can repress pLtetO-1 promoter. When ATc exits, it can combine with tetR, so that pLtetO-1 promoter can't be repressed. Then LacI, which is controlled by pLtetO-1, can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, mf-lon and mazF can't be expressed.  
  
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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 MazF, but when the E.coli escape from our testing instrument, the effect can be reversed, that is to say, the E.coli will be killed by 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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As a kind of bacterial toxin, the expression of mazF often leads to the death of the bacteria. So there comes the conclusion that the engineered ''E.coli'' won’t be killed by the mazF as long as it is cultured in the environment with ATc. Therefore, when the ''E.coli'' escapes from our detection instrument, the effects can be reversed. That is to say, the ''E.coli'' will be killed by mazF. In the same way, we can see that mazF can be expressed and kill the ''E.coli'' in the presence of IPTG.
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<html>
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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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    <figure>
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        <img src="https://2020.igem.org/wiki/images/5/56/T--XMU-China--XMU-China_2020-deadman-2.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 1.''' Kill switch of the detection part.
  
 
===Characterization===
 
===Characterization===
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We use pTrc-2 derivative_E0420_pUC57[BBa_K3332087], pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] to characterize pTrc-2 promoter.
 
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The agarose gel electrophoresis images are below:  
 
The agarose gel electrophoresis images are below:  
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|300px|Fig.2 pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by <i>EcoR</i> I and <i>Pst</i> I. (about 1018 bp)]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|500px|'''Fig 2.''' pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by ''EcoR'' I and ''Pst'' I (about 1018 bp).]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332088.png|thumb|300px|Fig.3 pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by <i>Pst</i> I (about 5086 bp).]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332088.png|thumb|500px|'''Fig 3.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by ''Pst'' I (about 5086 bp).]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|300px|Fig.4 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by <i>Pst</i> I (about 5125 bp). note: E0420 is equal to B0034_E0020_B0015]]</th><th></table>
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|500px|'''Fig 4.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by ''Pst'' I (about 5125 bp).]]</th><th></table>
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===Protocol===
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1.Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution
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'''note:''' E0420 is equal to B0034_E0020_B0015.
  
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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'''Protocol:'''
  
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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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1.Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution.
  
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4.Add 100μL IPTG stock solution into the induction group when OD increased to 0.6.  
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2.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.
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3.Add 4 mL 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 100 μL IPTG stock solution into the induction group when OD<sub>600</sub> increased to 0.6.  
  
 
5.Induce for 6 hours and the condition is the same as before.
 
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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6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500 µl 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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7.Measure the fluorescence intensity(ECFP)and corresponding OD<sub>600</sub> by 96-well plate reader, then calculate the fluorescence / OD value of each group.
 
Here is the result:
 
Here is the result:
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<table><tr><th>[[File:T--XMU-CHINA--figure 14.png|thumb|600px|Fig.5 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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<html>
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The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative-E0420(ECFP) are used as the positive control group while pLtetO-1-LacI-pTrc2-E0420 (ECFP) and pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) are both experimental group.  
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    <figure>
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We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than the pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.  
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        <img src="https://2020.igem.org/wiki/images/6/6f/T--XMU-China--XMU-China_2020-pTrc2_2d_%E6%8B%BC%E5%9B%BE.png" width="80%" 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 5.''' Fluorescence intensity/OD for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form．
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The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative_E0420(ECFP) are used as the positive control group while pLtetO-1_LacI_pTrc2_E0420(ECFP) and pLtetO-1_LacI_pTrc2-derivative_E0420(ECFP) are both experimental groups.  
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 +
We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.  
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<table><tr><th>[[File:T--XMU-CHINA-yingguang.png|thumb|600px|Fig.6 In each group，the EP tubes on the left is non-induction group while the one on the right is induction group.]]</th><th></table>
 
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From this figure, the induction effect can be seen more intuitively.
 
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3332038 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332038 SequenceAndFeatures</partinfo>

Latest revision as of 19:58, 27 October 2020


pTrc-2

A promoter can be repressed by LacI protein. It has only one lac operator, which means that it has less LacI binding sites so that LacI has a weak inhibitory effect on it.

Usage and Biology

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 E.coli in the detection instrument from escaping.

In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter ，while tetR can repress pLtetO-1 promoter. When ATc exits, it can combine with tetR, so that pLtetO-1 promoter can't be repressed. Then LacI, which is controlled by pLtetO-1, can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, mf-lon and mazF can't be expressed.

As a kind of bacterial toxin, the expression of mazF often leads to the death of the bacteria. So there comes the conclusion that the engineered E.coli won’t be killed by the mazF as long as it is cultured in the environment with ATc. Therefore, when the E.coli escapes from our detection instrument, the effects can be reversed. That is to say, the E.coli will be killed by mazF. In the same way, we can see that mazF can be expressed and kill the E.coli in the presence of IPTG.

Fig 1. Kill switch of the detection part.

Characterization

The agarose gel electrophoresis images are below:

Fig 2. pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by EcoR I and Pst I (about 1018 bp).
Fig 3. pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by Pst I (about 5086 bp).
Fig 4. pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by Pst I (about 5125 bp).

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 plasmids in test tube for 12h.

3.Add 4 mL of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 100 μL IPTG stock solution into the induction group when OD600 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 500 µl 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 5. Fluorescence intensity/OD for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form．

The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative_E0420(ECFP) are used as the positive control group while pLtetO-1_LacI_pTrc2_E0420(ECFP) and pLtetO-1_LacI_pTrc2-derivative_E0420(ECFP) are both experimental groups.

We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.


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]


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