Difference between revisions of "Part:BBa K1758102"
Line 12: Line 12:
 
<partinfo>BBa_K1758102 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1758102 SequenceAndFeatures</partinfo>
  
−
 
+
==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>.
 
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>.
  
Line 23: Line 23:
 
[[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, 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]]
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1758102 parameters</partinfo>
 
<partinfo>BBa_K1758102 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 21:12, 18 September 2015

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

sfGFP (BBa_I746916) 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]) 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 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

Characterization in vivo

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.

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