Difference between revisions of "Part:BBa K1819008"
 
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<partinfo>BBa_K1819008 short</partinfo>
 
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BBa_K1819008 construction indicates lactose promoter (pLac) efficiency to control a TetR promoter inhibited by TetR protein. This circuit was used to verify a kill switch mechanism (See more details in [http://2015.igem.org/Team:Brasil-USP]<a href=http://2015.igem.org/Team:Brasil-USP>Brasil-USP</a> team wiki)  
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BBa_K1819008 construction indicates lactose promoter (pLac) efficiency to control a TetR promoter inhibited by TetR protein. This circuit was used to verify a kill switch mechanism (See more details in <html><a href=http://2015.igem.org/Team:Brasil-USP>Brasil-USP</a></html> team wiki)  
  
 
In the absence of pLac inductor (Lactose or IPTG), RFP will be constitutively expressed under TetR promoter control. If inducer is added, TetR promoter is repressed and, consequently, RFP expression ceases and GFP expression starts.
 
In the absence of pLac inductor (Lactose or IPTG), RFP will be constitutively expressed under TetR promoter control. If inducer is added, TetR promoter is repressed and, consequently, RFP expression ceases and GFP expression starts.
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==Characterization==
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<html>
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<h3>DH5-alpha</h3>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We first started with DH5α. For protocols, we refer to our <a href="http://2015.igem.org/Team:Brasil-USP/Notebook/protocols">Protocols section</a>. We evaluated the fluorescence per cell by measuring Optical Density (OD) of our colonies and their fluorescences. We show in figures 1 and 2 our results.</p>
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<div class="fig" style="width: 600px;">
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  <img style="width:380px; display: block; margin: 20px auto 20px auto;" src="https://static.igem.org/mediawiki/2015/2/27/BrasilUS_IPTGODonlyregistry.png" />
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  <p><center>Figure 1 - Optical Density (OD) measurements over time, showing that all colonies were in their log phase.</center></p>
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</div>
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<div class="fig" style="width: 750px;">
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  <center><img style="width:350px; display: block; margin: 20px auto 20px auto;" src="https://static.igem.org/mediawiki/2015/b/b2/BrasilUSP_iptgpromotertestregistry.png" />
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  <p>Figure 2 - Fluorescence per cell, normalized by the fluorescence with no induction. In the x-axis, the higher the concentration label, the higher the concentration, with zero means no induction at all. All errors may be considered about 0.05 a.u.</center></p><br>
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</div>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Fluorescence was very weak and basically at the level of our negative control (E. Coli without plasmid), confirmed by a Mann-Whitney U test. Finally, no considerable changes were detected in the RFP measures. Since there results are not good indicatives, we performed this experiment three different times (to reduce possible experimental artifacts with assembling or other steps in our protocol). Yet we have always observed the same behavior consistently.</p>
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<h3>BL21 DE3</h3>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We performed the same tests in BL21 strain. For protocols, we refer to our <a href="http://2015.igem.org/Team:Brasil-USP/Notebook/protocols">Protocols section</a>. Again, our colonies were all in log phase (omitted) when we measured their fluorescence over time.</p>
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<div class="fig" style="width: 750px;">
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  <img style="width:750px; display: block; margin: 20px auto 20px auto;" src="https://static.igem.org/mediawiki/2015/0/09/Team-Brasil-USP_PTest_Lac_GFP_RFP.png" />
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  <p><center>Figure 3 - Fluorescence per cell for pLac, normalized by the fluorescence with no induction. We see that with IPTG, GFP activity incresases and RFP activity decreases, as it should happen.</center></p>
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<p>For more information visit our <a href="http://2015.igem.org/Team:Brasil-USP/Project/Results"> Results page</a> where we compared Lactose promoter with other promoters.</p>
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</div>
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</html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 23:26, 18 September 2015

Promoter test circuit to analyze the kill switch efficiency

BBa_K1819008 construction indicates lactose promoter (pLac) efficiency to control a TetR promoter inhibited by TetR protein. This circuit was used to verify a kill switch mechanism (See more details in Brasil-USP team wiki)

In the absence of pLac inductor (Lactose or IPTG), RFP will be constitutively expressed under TetR promoter control. If inducer is added, TetR promoter is repressed and, consequently, RFP expression ceases and GFP expression starts.


Characterization

DH5-alpha

    We first started with DH5α. For protocols, we refer to our Protocols section. We evaluated the fluorescence per cell by measuring Optical Density (OD) of our colonies and their fluorescences. We show in figures 1 and 2 our results.

Figure 1 - Optical Density (OD) measurements over time, showing that all colonies were in their log phase.

Figure 2 - Fluorescence per cell, normalized by the fluorescence with no induction. In the x-axis, the higher the concentration label, the higher the concentration, with zero means no induction at all. All errors may be considered about 0.05 a.u.


    Fluorescence was very weak and basically at the level of our negative control (E. Coli without plasmid), confirmed by a Mann-Whitney U test. Finally, no considerable changes were detected in the RFP measures. Since there results are not good indicatives, we performed this experiment three different times (to reduce possible experimental artifacts with assembling or other steps in our protocol). Yet we have always observed the same behavior consistently.

BL21 DE3

    We performed the same tests in BL21 strain. For protocols, we refer to our Protocols section. Again, our colonies were all in log phase (omitted) when we measured their fluorescence over time.

Figure 3 - Fluorescence per cell for pLac, normalized by the fluorescence with no induction. We see that with IPTG, GFP activity incresases and RFP activity decreases, as it should happen.

For more information visit our Results page where we compared Lactose promoter with other promoters.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2437
    Illegal AgeI site found at 2549
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 870