Difference between revisions of "Part:BBa K1758102"
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<partinfo>BBa_K1758102 short</partinfo> | <partinfo>BBa_K1758102 short</partinfo> | ||
− | + | This part is based on [https://parts.igem.org/Part:BBa_I746909 BBa_I746909] but contains an optimized untranslated region (UTR, [https://parts.igem.org/Part:BBa_K1758100 BBa_K1758100]), which improves the sfGFP expression <i>in vivo</i> as well as <i>in vitro</i>. This 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), is especially helpful in ''in vitro'' cell free protein synthesis. 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]). The part also contains a double terminator made of [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0010 B0010] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0012 B0012]. | |
===Usage and Biology=== | ===Usage and Biology=== | ||
− | + | This part has been used in <i>E. coli</i>. When T7 polymerase is present, a fast production of sfGFP is observed. This part acts as a reliable reporter protein generator. | |
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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− | This part | + | == Performance == |
+ | <html> | ||
+ | <p> In our project, we developed cell-free biosensors with the help of <i>in vitro</i> protein synthesis. This part became our positive control due to superior performance compared to <a href="https://parts.igem.org/Part:BBa_I746909">I746909</a> </p> | ||
+ | <p> We were sure that a further optimization of sfGFP production was possible. Based on literature screening (for details see <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS">background page</a>), we designed a <b>translation enhancing sequence (5'-untranslated region, 5'-UTR, BBa_K1758100)</b> and inserted it into P<sub>T7</sub>-sfGFP, thereby improving it. The insertion led to the creation of this part. </p> | ||
+ | <p> We designed the 5'-UTR on assumption that if translation was a bottleneck in our cell-free protein synthesis system, this sequence would improve sfGFP production. As verification for the beneficial effect of the 5'-UTR, we performed <i>in vivo</i> experiments, normalizing the fluorescence signal to culture OD<sub>600</sub>. | ||
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+ | |||
+ | |||
+ | |||
+ | </html> | ||
+ | |||
+ | ==Characterization <i>in vivo</i>== | ||
+ | |||
+ | |||
+ | [[File:Bielefeld-CeBiTec_CFPS_UTR.png|thumb|600px|center|In vivo characterization of sfGFP with and without our designed, translation enhancing 5'-untranslated region (5'-UTR; BBa_K1758100). Relative fluorescence units were normalized on OD600. Error bars represent standard deviation of triplicates. PT7-sfGFP: BBa_I746909; PT7-UTR-sfGFP: BBa_K1758102 (This part)]] | ||
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As can be seen in the picture, the difference was observable with the naked eye as well. | As can be seen in the picture, the difference was observable with the naked eye as well. | ||
− | [[File:Bielefeld-CeBiTec_CFPS_UTR_cultures.png|thumb|400px|center|Comparision of cultures expressing sfGFP with the optimized UTR and without. Left: BBa_K1758102, right: BBa_I746909]] | + | |
+ | [[File:Bielefeld-CeBiTec_CFPS_UTR_cultures.png|thumb|400px|center|Comparision of cultures expressing sfGFP with the optimized UTR and without. Left: BBa_K1758102 (This part), right: BBa_I746909]] | ||
+ | |||
+ | ==Characterization <i>in vitro</i>== | ||
+ | |||
+ | [[File:Bielefeld-CeBiTec_CFPS_UTR_invitro.png|thumb|400px|center|Importance of 5'-untranslated region (UTR) for in vitro protein synthesis. T7 refers to T7 promoter. sfGFP lysate refers to cell lysate won by sonication; cells were induced to produce sfGFP in vivo. Decreasing sfGFP lysate signal probably relied on evaporation. Mean values from two technical replicates are shown]] | ||
+ | |||
+ | |||
+ | <html> | ||
+ | |||
+ | <p>As can be seen in the figure below, we improved <a href="https://parts.igem.org/Part:BBa_I746909">BBa_I746909</a> (PT7-sfGFP in the figure) by employing of the translation enhancing 5'-UTR (PT7-UTR-sfGFP in the figure) we designed. High yields for <i>in vitro</i> sfGFP production were only observed when 5'-UTR was present. </p> | ||
+ | |||
+ | |||
+ | <figure> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/7/7e/Bielefeld-CeBiTec_150729_CFPS_T7-UTR-sfGFP.png" data-lightbox="group_150729" data-title="CFPS kinetics of sfGFP in cell extract"><img src="https://static.igem.org/mediawiki/2015/7/7e/Bielefeld-CeBiTec_150729_CFPS_T7-UTR-sfGFP.png" target="_blank" alt="CFPS in Tecan plate reader"></a> | ||
+ | <figcaption> Tracing CFPS kinetics in Tecan platereader. RFU signals were normalized to first signal of sfGFP lysate. Purified GFP refers to the signal of a His-tagged GFP solution at a concentration of about 320 µg/mL we kindly received from our advisor Lukas. Error bars represent standard deviation of triplicates. </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | </html> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display |
Latest revision as of 19:16, 19 September 2015
Translation enhancing 5-UTR + sfGFP under control of T7 promoter
This part is based on BBa_I746909 but contains an optimized untranslated region (UTR, BBa_K1758100), which improves the sfGFP expression in vivo as well as in vitro. This 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), is especially helpful in in vitro cell free protein synthesis. 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]). The part also contains a double terminator made of B0010 and B0012.
Usage and Biology
This part has been used in E. coli. When T7 polymerase is present, a fast production of sfGFP is observed. This part acts as a reliable reporter protein generator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 82
Performance
In our project, we developed cell-free biosensors with the help of in vitro protein synthesis. This part became our positive control due to superior performance compared to I746909
We were sure that a further optimization of sfGFP production was possible. Based on literature screening (for details see background page), we designed a translation enhancing sequence (5'-untranslated region, 5'-UTR, BBa_K1758100) and inserted it into PT7-sfGFP, thereby improving it. The insertion led to the creation of this part.
We designed the 5'-UTR on assumption that if translation was a bottleneck in our cell-free protein synthesis system, this sequence would improve sfGFP production. As verification for the beneficial effect of the 5'-UTR, we performed in vivo experiments, normalizing the fluorescence signal to culture OD600.
Characterization in vivo
![](/wiki/images/0/0e/Bielefeld-CeBiTec_CFPS_UTR.png)
As can be seen in the picture, the difference was observable with the naked eye as well.
Characterization in vitro
![](/wiki/images/5/5c/Bielefeld-CeBiTec_CFPS_UTR_invitro.png)
As can be seen in the figure below, we improved BBa_I746909 (PT7-sfGFP in the figure) by employing of the translation enhancing 5'-UTR (PT7-UTR-sfGFP in the figure) we designed. High yields for in vitro sfGFP production were only observed when 5'-UTR was present.
![CFPS in Tecan plate reader](https://static.igem.org/mediawiki/2015/7/7e/Bielefeld-CeBiTec_150729_CFPS_T7-UTR-sfGFP.png)