Difference between revisions of "Part:BBa K3332088"
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<partinfo>BBa_K3332088 short</partinfo>
 
<partinfo>BBa_K3332088 short</partinfo>
  
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pLtetO-1 promoter_RBS1_Histag-LacI-ssrAtag(mf-lon)_terminator(B0015)_pTrc-2 promoter_RBS(B0034)_ECFP CDS_terminator(B0015).We used it to check the function of pTrc-2 promoter.
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With this part, the LacI protein can be tested by observing the fluorescence
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intensity.
  
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<!-- Add more about the biology of this part here
 
 
===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 promoter after the Ptrc-2 promoter is characterized. With the expression of LacI, the bacteria can’t have fluorescence intensity.
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<table><tr><th>[[File:T--XMU-CHINA--circuit3.png|thumb|600px|Fig.1 Circuit.]]</th><th></table>
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<span class='h3bb'>Sequence and Features</span>
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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_K3332088 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332088 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3332088 parameters</partinfo>
 
<partinfo>BBa_K3332088 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 04:17, 25 October 2020


pLtetO-1-RBS1-LacI-ssrAtag(mf-lon)-terminator-pTrc-2-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 promoter after the Ptrc-2 promoter is characterized. With the expression of LacI, the bacteria can’t have fluorescence intensity.

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
    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