Difference between revisions of "Part:BBa K3332089"
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===Usage and Biology===
 
===Usage and Biology===
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This part can be used to test that if the LacI protein can repress the Ptrc-2 derivative promoter after the Ptrc-2 derivative promoter is characterized. With the expression of LacI, the bacteria can’t have fluorescence intensity.  
+
This part can be used to test that if the LacI protein can repress the pTrc-2 derivative promoter. With the expression of LacI, the bacteria have little fluorescence.  
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<table><tr><th>[[File:T--XMU-CHINA--circuit4.png|thumb|600px|Fig.1 Circuit.]]</th><th></table>
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/6/66/T--XMU-China--XMU-China_2020-pLtetO-1_RBS1_LacI-ssrAtag%28mf-lon%29_B0015_pTrc-2_derivative_B0034_ecfp_B0015.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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'''Fig1.''' pLtetO-1_RBS1_LacI_terminator_pTrc-2-derivative_RBS(B0034)_ecfp_terminator
 
===Characterization===
 
===Characterization===
−
The agarose gel electrophoresis images are below:  
+
The agarose gel electrophoresis images are shown as 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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[[File:T--XMU-CHINA-BBa K3332087.png|none|500px|caption]]
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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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'''Fig 2.''' pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by ''Eco''R I and ''Pst'' I (about 1018 bp)
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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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[[File:T--XMU-CHINA--BBa K3332088.png|none|500px|caption]]
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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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'''Fig 3.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by ''Pst'' I (about 5086 bp)
−
===Protocol===
+
[[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)
 +
 
 +
'''Note：'''E0420 is equal to B0034_E0020_B0015
 +
 
 +
'''Protocol'''
 
1. Preparation of stock solution
 
1. Preparation of stock solution
 +
 
Dissolve IPTG in absolute alcohol to make 1000× 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.
 
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.
+
3.Add 4mL of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
−
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.
+
 
 +
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.
 
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:
−
<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.
+
[[File:T--XMU-CHINA--figure 14.png|none|500px|caption]]
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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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'''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 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.  
 +
 
 +
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 the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.  
 +
 
 +
[[File:T--XMU-CHINA--pLtetO-1-LacI-pTrc2-E0420.png|none|500px|caption]]
 +
'''Fig 6.''' In each group，the EP tube on the left is non-induction while the one on the right is induction.
 +
 
 
From this figure, the induction effect can be seen more intuitively.
 
From this figure, the induction effect can be seen more intuitively.
 
===Sequence and Features===
 
===Sequence and Features===

Revision as of 18:29, 27 October 2020


pLtetO-1-RBS1-LacI-ssrAtag(mf-lon)-terminator-pTrc-2 derivative-RBS-ECFP-terminator

With this part, the LacI protein can be tested by observing the fluorescence intensity.

Usage and Biology

This part can be used to test that if the LacI protein can repress the pTrc-2 derivative promoter. With the expression of LacI, the bacteria have little fluorescence.

Fig1. pLtetO-1_RBS1_LacI_terminator_pTrc-2-derivative_RBS(B0034)_ecfp_terminator

Characterization

The agarose gel electrophoresis images are shown as below:

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 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:

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 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.

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 the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.

caption

Fig 6. In each group，the EP tube on the left is non-induction while the one on the right is 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 1489
  • 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