Difference between revisions of "Part:BBa K1758106"
 
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<partinfo>BBa_K1758106 short</partinfo>
 
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mRFP ([https://parts.igem.org/Part:BBa_K283035 BBa_K283035]) under control of the T7 promoter ([https://parts.igem.org/Part:BBa_I719005 BBa_I719005]). The part includes a translation enhancing sequence, 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]). The 5'-UTR sequence contains a spacer of 10 adenine bases which has been shown to further enhance translation efficiency ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).  
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mRFP ([https://parts.igem.org/Part:BBa_K283035 BBa_K283035]) under control of the T7 promoter ([https://parts.igem.org/Part:BBa_I719005 BBa_I719005]). The part includes a translation enhancing sequence, 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]). The 5'-UTR sequence contains a spacer of 10 adenine bases which has been shown to further enhance translation efficiency ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). For further details see [https://parts.igem.org/Part:BBa_K1758100 BBa_K1758100].
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1758106 SequenceAndFeatures</partinfo>
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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<html>
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<p>mRFP is synthesized when T7-polymerase is present. This part is a mRFP generator superior to <a href="https://parts.igem.org/Part:BBa_K1758105">BBa_K1758105</a>.</p>
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<h2> Results </h2>
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<p> This part was used to charactize the effect of our designed, translation enhancing 5'-untranslated region (5'-UTR; <a href="https://parts.igem.org/Part:BBa_K1758100">BBa_K1758100</a>), on protein production. </p>
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<p> We employed this mRFP generator, <a href="https://parts.igem.org/Part:Part:BBa_K1758105" target="_blank">BBa_K1758105</a>,  and two sfGFP generators (<a href="https://parts.igem.org/Part:Part:BBa_K1758102" target="_blank">BBa_K1758102</a> and <a href="https://parts.igem.org/Part:BBa_I746909" target="_blank">BBa_I746909</a>. This was done to check if the our designed, translation enhancing 5'-untranslated region (5'-UTR; <a href="https://parts.igem.org/Part:BBa_K1758100">BBa_K1758100</a>) was useful for translation in general. We constructed P<sub>T7</sub>-UTR-mRFP (this part) after assembling P<sub>T7</sub>-mRFP (<a href="https://parts.igem.org/Part:Part:BBa_K1758105" target="_blank">BBa_K1758105</a>). For mRFP fluorescence signal normalization on OD<sub>600</sub> we faced the problem that mRFP emits fluorescence at 607 nm (<a href= "http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Lentini2013">Lentini et al. 2013</a>). We also tried to express mRFP <i>in vitro</i> to check if it might be a reporter protein equally or better suited for our purposes. mRFP was excited at 580 nm, its emission was measured at 610 nm. </p>
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<h3> <i>In vivo</i> characterization </h3>
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<figure>
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<a href="https://static.igem.org/mediawiki/parts/7/7e/Bielefeld-CeBiTec_UTR_characterization_in_vivo_mrfp.jpg"><img src="https://static.igem.org/mediawiki/parts/7/7e/Bielefeld-CeBiTec_UTR_characterization_in_vivo_mrfp.jpg" alt="cultures of mRFP producting cells" style="width:300px"></a>
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<figcaption> <i>Image of mRFP producing <i>E. coli</i> cultures. Left: This part, right: <a href="https://parts.igem.org/Part:BBa_K1758106">BBa_K1758106</a>. Both cultures were cultivated for the same time span. You can see that our designed, translation enhancing 5'-untranslated region (5'-UTR; <a href="https://parts.igem.org/Part:BBa_K1758100">BBa_K1758100</a>), improves mRFP production <i>in vivo</i></i>. </figcaption>
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</figure>
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</br>
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<figure>
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    <a href="https://static.igem.org/mediawiki/2015/8/8d/Bielefeld-CeBiTec_CFPS_UTR_bar_red_big.png" data-lightbox="UTRcharact" data-title="<i>In vivo</i> characterization of our designed, translation enhancing 5'-untranslated region (5'-UTR) with mRFP. Relative fluorescence units were normalized on OD<sub>600</sub>. One might consider biases due to mRFP fluorescence emission at 607 nm like discussed previously."><img src="https://static.igem.org/mediawiki/2015/8/8d/Bielefeld-CeBiTec_CFPS_UTR_bar_red_big.png" alt="UTR test in vivo with mRFP" style="width:500px"></a>
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    <figcaption> <i><i>In vivo</i> characterization of our designed, translation enhancing 5'-untranslated region (5'-UTR; <a href="https://parts.igem.org/Part:BBa_K1758100">BBa_K1758100</a>) with mRFP. Relative fluorescence units were normalized on OD<sub>600</sub>. One might consider biases due to mRFP fluorescence emission at 607 nm like discussed previously. Error bars represent standard deviation of triplicates. PT7-mRFP: <a href="https://parts.igem.org/Part:BBa_K1758105">BBa_K1758105</a>; PT7-UTR-mRFP: This part</i> </figcaption>
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</figure>
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<h3> <i>In vitro</i> characterization </h3>
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<div>
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<p> <i>In vitro</i> mRFP expression with this construct (P<sub>T7</sub>-UTR-mRFP) showed us that mRFP was not nearly as well performing as sfGFP in <i>in vitro</i> experiments (see <a href="https://parts.igem.org/Part:Part:BBa_K1758102" target="_blank">BBa_K1758102</a>). Although we observed a fluorescence signal differing from the negative control, this signal was very low and raising very slowly, as depicted in the figure below. </p>
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        </div>
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        <div>
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<figure style="margin:auto">
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    <a href="https://static.igem.org/mediawiki/2015/0/08/Bielefeld-CeBiTec_CFPS_mRFP_in_vitro_performance.png" data-lightbox="in vitro UTR" data-title="Performance of mRFP in our CFPS reaction. mRFP was produced but the fluorescence signal was very low, even after several hours. Error bars represent standard deviation of triplicates."><img src="https://static.igem.org/mediawiki/2015/0/08/Bielefeld-CeBiTec_CFPS_mRFP_in_vitro_performance.png" alt="mRFP in vitro performance" style="width:600px"></a>
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  <figcaption><i> Performance of mRFP in our CFPS reaction. mRFP was produced but the fluorescence signal was very low, even after several hours. Error bars represent standard deviation of triplicates.</i> </figcaption>
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    </figure>
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        </div>
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</html>
  
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1758106 SequenceAndFeatures</partinfo>
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Latest revision as of 19:12, 19 September 2015

Translation enhancing 5-UTR + mRFP under control of T7 promoter

mRFP (BBa_K283035) under control of the T7 promoter (BBa_I719005). The part includes a translation enhancing sequence, 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]). The 5'-UTR sequence contains a spacer of 10 adenine bases which has been shown to further enhance translation efficiency ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). For further details see BBa_K1758100.

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 624
    Illegal AgeI site found at 736
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

mRFP is synthesized when T7-polymerase is present. This part is a mRFP generator superior to BBa_K1758105.

Results

This part was used to charactize the effect of our designed, translation enhancing 5'-untranslated region (5'-UTR; BBa_K1758100), on protein production.

We employed this mRFP generator, BBa_K1758105, and two sfGFP generators (BBa_K1758102 and BBa_I746909. This was done to check if the our designed, translation enhancing 5'-untranslated region (5'-UTR; BBa_K1758100) was useful for translation in general. We constructed PT7-UTR-mRFP (this part) after assembling PT7-mRFP (BBa_K1758105). For mRFP fluorescence signal normalization on OD600 we faced the problem that mRFP emits fluorescence at 607 nm (Lentini et al. 2013). We also tried to express mRFP in vitro to check if it might be a reporter protein equally or better suited for our purposes. mRFP was excited at 580 nm, its emission was measured at 610 nm.

In vivo characterization

cultures of mRFP producting cells
Image of mRFP producing E. coli cultures. Left: This part, right: BBa_K1758106. Both cultures were cultivated for the same time span. You can see that our designed, translation enhancing 5'-untranslated region (5'-UTR; BBa_K1758100), improves mRFP production in vivo.

UTR test in vivo with mRFP
In vivo characterization of our designed, translation enhancing 5'-untranslated region (5'-UTR; BBa_K1758100) with mRFP. Relative fluorescence units were normalized on OD600. One might consider biases due to mRFP fluorescence emission at 607 nm like discussed previously. Error bars represent standard deviation of triplicates. PT7-mRFP: BBa_K1758105; PT7-UTR-mRFP: This part

In vitro characterization

In vitro mRFP expression with this construct (PT7-UTR-mRFP) showed us that mRFP was not nearly as well performing as sfGFP in in vitro experiments (see BBa_K1758102). Although we observed a fluorescence signal differing from the negative control, this signal was very low and raising very slowly, as depicted in the figure below.

mRFP in vitro performance
Performance of mRFP in our CFPS reaction. mRFP was produced but the fluorescence signal was very low, even after several hours. Error bars represent standard deviation of triplicates.