Difference between revisions of "Team:NAU-CHINA/Composite Part"

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             <h1>Our Best Composite Part:BBa_K2557010</h1>
 
             <h1>Our Best Composite Part:BBa_K2557010</h1>
 
             <figure>
 
             <figure>
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                     <img src="">
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                     <img src="https://static.igem.org/mediawiki/2018/c/c6/T--NAU-China--teto_recombinase.gif">
 
                 </figure>
 
                 </figure>
 
             <p>We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.</p>
 
             <p>We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.</p>
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                 <h2>Function In Our Genetic Circuit</h2>
 
                 <h2>Function In Our Genetic Circuit</h2>
 
                 <figure>
 
                 <figure>
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                     <img src="">
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                     <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--part_teto_anime.gif">
 
                 </figure>
 
                 </figure>
 
                 <p>This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.</p>
 
                 <p>This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.</p>
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                 <h2>Characterization</h2>
 
                 <h2>Characterization</h2>
 
                  
 
                  
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                 <p>TetR can effectively repress Tet operator in HEK 293T</p>
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                 <p><b>TetR can effectively repress Tet operator in HEK 293T</b></p>
 
                 <figure>
 
                 <figure>
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                     <img src="">
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                     <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--enhance_gfp.gif">
 
                 </figure>
 
                 </figure>
 
                 <figure>
 
                 <figure>
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                     <img src="">
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                     <img src="https://static.igem.org/mediawiki/2018/d/d7/T--NAU-China--newpart1.jpg">
 
                     <figcaption class="_table">Fig. 1 Inhibition of TetR on promoter with TetO sequence<br>
 
                     <figcaption class="_table">Fig. 1 Inhibition of TetR on promoter with TetO sequence<br>
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               (a)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.<br>
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               (A)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.<br>
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               (b)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.<br>
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               (B)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.<br>
 
             </figcaption> </figure>
 
             </figcaption> </figure>
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             <p>Bxb1 works well in HEK 293T</p>
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             <p><b>Bxb1 works well in HEK 293T</b></p>
 
             <figure>
 
             <figure>
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                     <img src="">
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                     <img src="https://static.igem.org/mediawiki/2018/f/f4/T--NAU-China--newpart7.jpg">
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                     <figcaption class="_table">Fig. 1a Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration.<br>
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                     <figcaption class="_table">Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells.<br>(A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments.<br>(B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.<br>(C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.</figcaption>
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Fig. 1b The reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration.
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                </figure>
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<br>
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            </figcaption></figure>
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                <p>The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.</p>
 
             <p>Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.</p>
 
             <p>Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.</p>
  
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  <figure>
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                    <img src="https://static.igem.org/mediawiki/2018/9/95/T--NAU-China--bxb1png.png">
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                    <figcaption class="_table">Fig. 1 Bxb1 structure homology-model provided by SWISS-MODEL.</figcaption>
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                </figure>
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                <div class="divvideo">
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                <video src="https://static.igem.org/mediawiki/2018/a/ab/T--NAU-China--bxb1mp4.mp4"  controls="controls">
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                </video>       
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                 </div>
 
                 </div>
  

Revision as of 15:45, 17 October 2018

Template:2018_NAU-CHINA

header
Safety

Our Best Composite Part:BBa_K2557010

We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.

Function In Our Genetic Circuit

This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.

Characterization

TetR can effectively repress Tet operator in HEK 293T

Fig. 1 Inhibition of TetR on promoter with TetO sequence
(A)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.
(B)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.

Bxb1 works well in HEK 293T

Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells.
(A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments.
(B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.
(C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.

The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.

Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.

Composite Part List

Name Type Description Length
BBa_K2557010 Composite TetO-miniCMV promoter-Bxb1 1890
BBa_K2557002 Composite Bxb1 attB-RFP-Bxb1 attP 935
BBa_K2557005 Composite PhiC31 attB-mCherry-PhiC31 attP 907
BBa_K2557008 Composite TP901 attB-mCherry-TP901 attP 949
BBa_K2557011 Composite TetO-EF1α promoter-Bxb1 2070
BBa_K2557012 Composite TetO-Ubc promoter-Bxb1 2258
BBa_K2557013 Composite TetO-miniCMV-PhiC31 2212
BBa_K2557014 Composite TetO-EF1α-PhiC31 2386
BBa_K2557015 Composite TetO-Ubc promoter-PhiC31 2574
BBa_K2557016 Composite TetO-miniCMV promoter-TP901 1935
BBa_K2557017 Composite TetO-EF1α promoter-TP901 2109
BBa_K2557018 Composite TetO-Ubc promoter-TP901 2297
BBa_K2557019 Composite miniCMV promoter-Bxb1-RDF 2391
BBa_K2557020 Composite EF1α promoter-Bxb1-RDF 2565
BBa_K2557021 Composite Ubc promoter-Bxb1-RDF 2753
BBa_K2557022 Composite miniCMV promoter-PhiC31-RDF 2676
BBa_K2557023 Composite EF1α promoter-PhiC31-RDF 2850
BBa_K2557024 Composite Ubc promoter--PhiC31-RDF 3038
BBa_K2557025 Composite miniCMV promoter-TP901-RDF 1842
BBa_K2557026 Composite EF1α promoter--TP901-RDF 2016
BBa_K2557027 Composite Ubc promoter--TP901-RDF 2204
BBa_K2557028 Composite TetO-miniCMV promoter- EGFP 1063
BBa_K2557029 Composite TetO-EF1α promoter -EGFP 1237
BBa_K2557030 Composite TetO-Ubc promoter- EGFP 1425
BBa_K2557031 Composite miniCMV promoter-Anti-GFP-mnotch-TEV protease-NLS 2455
BBa_K2557051 Composite EF1α promoter-TetR 1871