Difference between revisions of "Part:BBa K3332088"
 
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'''Fig 1.''' pLtetO-1_RBS1_LacI_terminator_pTrc-2_RBS(B0034)—ecfp-terminator
 
'''Fig 1.''' pLtetO-1_RBS1_LacI_terminator_pTrc-2_RBS(B0034)—ecfp-terminator
 
===Characterization===
 
===Characterization===
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The agarose gel electrophoresis images are shown as below:  
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We use pTrc-2 derivative_E0420_pUC57（<partinfo>BBa_K3332087</partinfo>）,pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57 and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57（<partinfo>BBa_K3332089</partinfo>） to characterize pTrc-2 promoter and pTrc-2 derivative promoter.
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The agarose gel electrophoresis images are below:  
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<table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|500px|'''Fig 2.''' pTrc-2 derivative_E0420_pUC57（<partinfo>BBa_K3332087</partinfo>） 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|500px|'''Fig 3.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57（<partinfo>BBa_K3332088</partinfo>） digested by ''Pst'' I (about 5086 bp).]]</th><th></table>
 +
<table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|500px|'''Fig 4.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57（<partinfo>BBa_K3332089</partinfo>）  digested by ''Pst'' I (about 5125 bp).]]</th><th></table>
  
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[[File:T--XMU-CHINA--BBa K3332087.png|none|500px|caption]]
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'''note:''' E0420 is equal to B0034_E0020_B0015.
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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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[[File:T--XMU-CHINA--BBa K3332088.png|none|500px|caption]]
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'''Fig 3.''' pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by ''Pst'' I (about 5086 bp)
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[[File:T--XMU-CHINA--BBa K3332089.png|none|500px|caption]]
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'''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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'''Note：'''E0420 is equal to B0034_E0020_B0015
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'''Protocol:'''
  
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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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1. Preparation of stock solution
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2.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.
  
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Dissolve IPTG in absolute alcohol to make 1000× stock solution
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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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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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4.Add 100 μL IPTG stock solution into the induction group when OD<sub>600</sub> increased to 0.6.  
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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.
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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 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 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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<html>
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    <figure>
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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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[[File:T--XMU-CHINA--figure 14.png|none|500px|caption]]
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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)（<partinfo>BBa_K3332087</partinfo>） 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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'''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.
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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 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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[[File:T--XMU-CHINA--pLtetO-1-LacI-pTrc2-E0420.png|none|500px|caption]]
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'''Fig 6.''' In each group，the EP tube on the left is non-induction while the one on the right is induction.
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From this figure, the induction effect can be seen more intuitively.
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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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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3332088 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332088 SequenceAndFeatures</partinfo>

Latest revision as of 22:25, 27 October 2020


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

With this part, the pTrc-2 promoter 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 promoter. With the expression of LacI, the bacteria have little fluorescence.

Fig 1. pLtetO-1_RBS1_LacI_terminator_pTrc-2_RBS(B0034)—ecfp-terminator

Characterization

We use pTrc-2 derivative_E0420_pUC57（BBa_K3332087）,pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57 and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57（BBa_K3332089） to characterize pTrc-2 promoter and pTrc-2 derivative promoter. 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 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 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)（BBa_K3332087） 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