Difference between revisions of "Part:BBa K3332037"
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===Usage and Biology===
 
===Usage and Biology===
 
LacI is a protein that can repress pTrc-2 and pTrc-2 derivative promoter in absence of IPTG. Especially, the part has ssrAtag(mf-lon), which means it can be degraded by mf-lon.
 
LacI is a protein that can repress pTrc-2 and pTrc-2 derivative promoter in absence of IPTG. Especially, the part has ssrAtag(mf-lon), which means it can be degraded by mf-lon.
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<table><tr><th>[[File:T--XMU-CHINA--circuit--circuit.png|thumb|300px|Fig.2 Circut]]</th><th></table>
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[[File:T--XMU-CHINA--circuit--circuit.png|none|500px|caption]]
 +
'''Fig 1.''' Kill switch of the detection part
  
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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.
+
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 combines with tetR, so that pLtetO-1 promoter can’t be repressed. Then LacI which is controlled by pLtetO-1 can repress the 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, 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.In addition, LacI with ssrAtag(mf-lon) which can be degraded by mf-lon can accelerate the death.
+
As a kind of bacterial toxin, the expression of MazF often lead to the death of bacteria. 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 ''E.coli'' escape from our detection instrument, the effect can be reversed. That is to say, the ''E.coli'' will be killed by MazF. In the same way, we can see that in the presence of IPTG, MazF can be expressed and kill the ''E.coli''.
 +
 
 +
In addition, LacI with ssrAtag(mf-lon) that can be degraded by mf-lon can accelerate the death.
  
 
===Characterization:===
 
===Characterization:===
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We can see the repression of LacI-ssrAtag(mf-lon) on pTrc-2 promoter and pTrc-2 derivative promoter by pLtetO-1_RBS1_lacI-ssrAtag(mf-lon)_B0015[BBa_K3332088] or pLtetO-1_RBS1_lacI-ssrAtag(mf-lon)_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089]
 
  
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The agarose gel electrophoresis images are below:
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[[File:T--XMU-CHINA--BBa_K3332087.png|none|500px|caption]]
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<table><tr><th>[[File:T--XMU-CHINA--BBa_K3332088.png|thumb|300px|Fig.1 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by <i>Pst</i> I (about 5086 bp).]]</th><th></table>
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'''Fig 2.''' pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by ''EcoR '' I and '' Pst '' I (about 1018 bp)
  
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<table><tr><th>[[File:T--XMU-CHINA--BBa_K3332089.png|thumb|300px|Fig.2 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by <i>Pst</i> I(about 5125 bp).]]</th><th></table>
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[[File:T--XMU-CHINA--BBa_K3332088.png|none|500px|caption]]
 +
'''Fig 3.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by '' Pst'' I (about 5086 bp)
  
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Note：E0420 is equal to B0034_E0020_B0015
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[[File:T--XMU-CHINA--BBa_K3332089.png|none|500px|caption]]
 +
'''Fig 4.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089]  digested by ''Pst'' I (about 5125 bp)
  
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Protocol:
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'''Note：'''E0420 is equal to B0034_E0020_B0015
 +
 
 +
'''Protocol:'''
  
 
1. Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution
 
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 OD600 increased to 0.6. 5.Induce for 6 hours and the condition is the same as before.
 
4.Add 100μL 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.
  
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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.
+
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.
 
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:
 
Here is the result:
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<table><tr><th>[[File:T--XMU-CHINA--figure_14.png|thumb|300px|Fig.3 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, pTrc2-derivative are used as the negative control group, the pTrc2-derivative-E0420(ECFP) are used as the positive control group while the pLtetO-1-LacI-pTrc2-E0420 (ECFP) and pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) are both experimental group.
 
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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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<table><tr><th>[[File:T--XMU-CHINA-yingguang.png|thumb|300px|Fig.4 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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[[File:T--XMU-CHINA--figure_14.png|none|500px|caption]]
 +
'''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.
 +
 
 +
The strength of pTrc-2-derivative and pTrc-2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, the pTrc2-derivative-E0420(ECFP) are used as the positive control group while the pLtetO-1_LacI_pTrc-2_E0420 (ECFP) and pLtetO-1_LacI_pTrc-2_derivative_E0420(ECFP) are both experimental group.
 +
 
 +
We can see, after adding IPTG to induce the two promoters, the fluorescence intensity/OD600 are both improved. The change of fluorescence intensity/OD600 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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 +
<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/5/51/T--XMU-China--XMU-China_2020-Ptrc2-de_pLtetO.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 6.''' In each group，the EP tubes on the left is non-induction group while the one on the right is induction group.
  
 
From this figure, the induction effect can be seen more intuitively.
 
From this figure, the induction effect can be seen more intuitively.

Revision as of 16:45, 27 October 2020


LacI-ssrAtag(mf-lon)

The LacI protein tagged with ssrAtag(mf-lon) is able to repress pTrc-2 promoter and pTrc-2 derivative promoter.We use it to repress pTrc-2 promoter and pTrc-2 derivative promoter.

Usage and Biology

LacI is a protein that can repress pTrc-2 and pTrc-2 derivative promoter in absence of IPTG. Especially, the part has ssrAtag(mf-lon), which means it can be degraded by mf-lon.

caption

Fig 1. Kill switch of the detection part

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 combines with tetR, so that pLtetO-1 promoter can’t be repressed. Then LacI which is controlled by pLtetO-1 can repress the 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 lead to the death of bacteria. 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 E.coli escape from our detection instrument, the effect can be reversed. That is to say, the E.coli will be killed by MazF. In the same way, we can see that in the presence of IPTG, MazF can be expressed and kill the E.coli.

In addition, LacI with ssrAtag(mf-lon) that can be degraded by mf-lon can accelerate the death.

Characterization:

caption

Fig 2. pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by EcoR I and Pst I (about 1018 bp)

caption

Fig 3. pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by Pst I (about 5086 bp)

caption

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 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 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 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:

caption

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.

The strength of pTrc-2-derivative and pTrc-2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, the pTrc2-derivative-E0420(ECFP) are used as the positive control group while the pLtetO-1_LacI_pTrc-2_E0420 (ECFP) and pLtetO-1_LacI_pTrc-2_derivative_E0420(ECFP) are both experimental group.

We can see, after adding IPTG to induce the two promoters, the fluorescence intensity/OD600 are both improved. The change of fluorescence intensity/OD600 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.

Fig 6. In each group，the EP tubes on the left is non-induction group while the one on the right is induction group.

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

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

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]