Difference between revisions of "Part:BBa K3332086"
Line 3: Line 3:
 
<partinfo>BBa_K3332086 short</partinfo>
 
<partinfo>BBa_K3332086 short</partinfo>
  
−
With this part, the pTrc-2 promoter can be tested by observing the fluorescence intensity.
+
With this part, the pTrc-2 promoter can be tested by observing the fluorescence intensity/OD.
 
===Usage and Biology===
 
===Usage and Biology===
−
This part can be used to test that if the pTrc-2 promoter can work. The fluorescence intensity can reflect the strength of the pTrc-2 promoter.
+
This part can be used to test that if the pTrc-2 promoter can work. The fluorescence intensity can reflect the strength of the pTrc-2 promoter.  
  
−
<table><tr><th>[[File:T--XMU-CHINA--circuit2.png|thumb|600px|Fig.1 Circuit.]]</th><th></table>
+
<html>
 +
    <figure>
 +
        <img src="https://2020.igem.org/wiki/images/d/d1/T--XMU-China--XMU-China_2020-pTrc-2_B0034_ecfp_B0015.png" width="60%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
''Fig 1.'' pTrc-2_RBS(B0034)_ECFP(E0020)_terminator(B0015)
  
 
===Sequence and Features===
 
===Sequence and Features===
Line 14: Line 23:
 
===Characterization===
 
===Characterization===
 
The agarose gel electrophoresis images are below:  
 
The agarose gel electrophoresis images are below:  
−
<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.(about 1018 bp)]]</th><th></table>
+
[[File:T--XMU-CHINA-BBa K3332086.png|none|500px|caption]]
 +
'''Fig 2. '''pTrc-2_E0420_pSB1C3 digested by ''Xba ''I and ''Pst'' I(about 1003bp)
  
−
<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>
+
===Reference===
−
 
+
[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
−
<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.(about 5086 bp)]]</th><th></table>
+
−
 
+
−
<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(about 5125 bp).]]</th><th></table>
+
−
 
+
−
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:
+
−
 
+
−
<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>
+
−
 
+
−
The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure,  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 (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 and the pLtetO-1-LacI-pTrc2-E0420 (ECFP) group the difference of fluorescence intensity 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. That’s why after adding IPTG, the fluorescence intensity of pLtetO-1-LacI-pTrc2-E0420 (ECFP) group increases faster.
+
−
<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>
+
−
From this figure, the induction effect can be seen more intuitively.
+
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
Line 55: Line 33:
 
<partinfo>BBa_K3332086 parameters</partinfo>
 
<partinfo>BBa_K3332086 parameters</partinfo>
 
<!-- -->
 
<!-- -->
−
===Reference===
 
−
[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 19:06, 27 October 2020


pTrc-2-RBS-ECFP-terminator

With this part, the pTrc-2 promoter can be tested by observing the fluorescence intensity/OD.

Usage and Biology

This part can be used to test that if the pTrc-2 promoter can work. The fluorescence intensity can reflect the strength of the pTrc-2 promoter.

Fig 1. pTrc-2_RBS(B0034)_ECFP(E0020)_terminator(B0015)

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]

Characterization

The agarose gel electrophoresis images are below:

caption

Fig 2. pTrc-2_E0420_pSB1C3 digested by Xba I and Pst I(about 1003bp)

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