Difference between revisions of "Part:BBa K1758102"
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<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> 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>
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<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)</b> and inserted it into P<sub>T7</sub>-sfGFP, thereby improving it. The insertion led to the creation of this part. </p>
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<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>.
 
<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>.
  

Revision as of 05:37, 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


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
    INCOMPATIBLE 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

In vivo characterization of sfGFP with and withour a translation enhancing 5'-UTR. Relative fluorescence units were normalized on OD600. Error bars represent standard deviation of triplicates.


As can be seen in the picture, the difference was observable with the naked eye as well.


Comparision of cultures expressing sfGFP with the optimized UTR and without. Left: BBa_K1758102, right: BBa_I746909


Characterization in vitro

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


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
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.