Difference between revisions of "Part:BBa K1758102"

Revision as of 00:24, 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), 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

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

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) and inserted it into P_T7-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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1758102 short</partinfo>
-sfGFP ([https://parts.igem.org/wiki/index.php?title=Part:BBa_I746916 BBa_I746916]) 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]) and 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].
+This part is based on [https://parts.igem.org/Part:BBa_I746909 BBa_I746909] but contains an optimized untranslated region (UTR), 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].
@@ Line 11: / Line 11: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1758102 SequenceAndFeatures</partinfo>
+== 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 </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)</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>.
+</html>
 ==Characterization <i>in vivo</i>==
-This part is based on [https://parts.igem.org/Part:BBa_I746909 BBa_I746909] but contains an optimized untranslated region (UTR), which improves the sfGFP expression <i>in vivo</i> as well as <i>in vitro</i>.
 [[File:Bielefeld-CeBiTec_CFPS_UTR.png|thumb|600px|center|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.]]