Difference between revisions of "Part:BBa K2014004"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2014004 short</partinfo>
 
<partinfo>BBa_K2014004 short</partinfo>
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===Usage and Biology===
 
===Usage and Biology===
  
The promoter pxylS-M5'UTR (with a lab name: XylS-UTR ), tested in a biobrick pxylS-M5'UTR->sfGFP <b>(BBa_K2014004)</b> is a modified xylose-induced promoter/5’UTR controlling sfGFP protein expression. To make it we exchanged the 5’UTR sequence of xylA-proD5'UTR in a biobrick [https://parts.igem.org/Part:BBa_K2014009 <b>(BBa_K1741009)</b>] to a synthetic, unstructured M5’UTR (picture B) derived from the Mel2 promoter [https://parts.igem.org/Part:BBa_K1741004 <b>(BBa_K1741004)</b>], which we provided to iGEM Registry in 2015.  
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The promoter pxylS-M5'UTR (with a lab name: XylS-UTR ), tested in a biobrick pxylS-M5'UTR->sfGFP <b>(BBa_K2014004)</b> is a modified xylose-induced promoter/5’UTR controlling sfGFP protein expression. To make it we exchanged the 5’UTR sequence of xylA-proD5'UTR in a biobrick [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>] to a synthetic, unstructured M5’UTR (picture B) derived from the Mel2 promoter [https://parts.igem.org/Part:BBa_K1741004 <b>(BBa_K1741004)</b>], which we provided to iGEM Registry in 2015.  
  
  
If you are interested in the pathway of our modifications please click here-> [[https://static.igem.org/mediawiki/parts/c/c4/BBa_K2014004.pdf MODIFICATIONS OF XYLOSE INDUCED PROMOTERS]]
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If you are interested in the pathway of our modifications please click here-> [https://static.igem.org/mediawiki/parts/c/c4/BBa_K2014004.pdf MODIFICATIONS OF XYLOSE INDUCED PROMOTERS]
  
  
  
 
Biobricks used in description: <br><br>
 
Biobricks used in description: <br><br>
xylF-xylA - briefly called XylWT  <b>(BBa_K1741007)</b><br>
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xylF-xylA - briefly called XylWT  [https://parts.igem.org/Part:BBa_K1741007 <b>(BBa_K1741007)</b>]<br>
xylF-xylA-proD5'UTR - briefly called XylA1 <b>(BBa_K1741008)</b><br>
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xylF-xylA-proD5'UTR - briefly called XylA1 [https://parts.igem.org/Part:BBa_K1741008 <b>(BBa_K1741008)</b>]<br>
xylA-proD5'UTR - briefly called XylS  <b>(BBa_K1741009)</b><br>
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xylA-proD5'UTR - briefly called XylS  [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>]<br>
xylS-M5'UTR - briefly called XylS-UTR <b>(BBa_K2014004)</b><br>
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xylS-M5'UTR - briefly called XylS-UTR [https://parts.igem.org/Part:BBa_K2014004 <b>(BBa_K2014004)</b>]<br>
  
  
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{|align="center"
 
{|align="center"
 
  |-valign="top"
 
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  | colspan = 2 | [[Image:BBa K2014004-2.png|thumb|500px|center|<font size="2"><b>Figure 2</b> Comparison of xylose promoters tightness during overnight time course cultures of <i>E.coli</i> DH5α in 1xLB medium without xylose. Bacteria were transformed with constructs: XylWT (xylF-xylA->sfGFP; <b>BBa_K1741007</b>), XylS (xylA-proD5'UTR; <b>BBa_K1741009</b>), XylS-UTR (xylS-M5'UTR; <b>BBa_K2014004</b>) and XylS-lysozyme (the construct containing non fluorescent protein- lysozyme, under XylS protein), which was used as our background. </font>]]
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  | colspan = 2 | [[Image:BBa K2014004-2.png|thumb|500px|center|<font size="2"><b>Figure 2</b> Comparison of xylose promoters tightness during overnight time course cultures of <i>E.coli</i> DH5α in 1xLB medium without xylose. Bacteria were transformed with constructs: XylWT (xylF-xylA->sfGFP; [https://parts.igem.org/Part:BBa_K1741007 <b>BBa_K1741007</b>]), XylS (xylA-proD5'UTR; [https://parts.igem.org/Part:BBa_K1741009 <b>BBa_K1741009</b>]), XylS-UTR (xylS-M5'UTR; [https://parts.igem.org/Part:BBa_K2014004 <b>BBa_K2014004</b>]) and XylS-lysozyme (the construct containing non fluorescent protein- lysozyme, under XylS protein), which was used as our background. </font>]]
 
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{|align="center"
 
{|align="center"
 
  |-valign="top"
 
  |-valign="top"
  | colspan = 2 | [[Image:BBa K2014004-3.png|thumb|400px|center|<font size="2"><b>Figure 3 <br>A)</b> The most likely secondary structure that can be folded from the 5’UTR of XylWT promoter. <b>B)</b> The most likely secondary structures of M5’UTR from Mel2 and XylS promoters <b>(BBa_K1741004)</b>. The sequence of M5’UTR is designed to minimize the likelihood of secondary structure formation. Structures shown above, were generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi)  under the same parameters. RBS sequences are underlined with a green line. </font>]]
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  | colspan = 2 | [[Image:BBa K2014004-3.png|thumb|400px|center|<font size="2"><b>Figure 3 <br>A)</b> The most likely secondary structure that can be folded from the 5’UTR of XylWT promoter. <b>B)</b> The most likely secondary structures of M5’UTR from Mel2 and XylS promoters [https://parts.igem.org/Part:BBa_K1741004 <b>(BBa_K1741004)</b>]. The sequence of M5’UTR is designed to minimize the likelihood of secondary structure formation. Structures shown above, were generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi)  under the same parameters. RBS sequences are underlined with a green line. </font>]]
 
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{|align="center"
 
{|align="center"
 
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  | colspan = 2 | [[Image:BBa K2014004-5.png|thumb|700px|center|<font size="2"><b>Figure 4 <br>A)</b> Xylose inducibility of four xyl promoters/UTRs during 6h time course cultures of <i>E.coli</i> DH5α in LB medium containing 0.4% xylose. The efficiency of the improved promoter pxylS-M5'UTR (BBa_K2014004) is slightly higher, than all other versions of xylose responsive promoters we tested, and its induction seems to be faster (see 2h time-point). <b>B)</b> The growth rate of all constructs compared: Xyl-WT <b>(BBa_K1741007)</b>, XylA1 <b>(BBa_K1741008)</b>, XylS <b>(BBa_K1741009)</b> or XylS-UTR <b>(BBa_K2014004)</b> is very similar. </font>]]
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  | colspan = 2 | [[Image:BBa K2014004-5.png|thumb|700px|center|<font size="2"><b>Figure 4 <br>A)</b> Xylose inducibility of four xyl promoters/UTRs during 6h time course cultures of <i>E.coli</i> DH5α in LB medium containing 0.4% xylose. The efficiency of the improved promoter pxylS-M5'UTR ([https://parts.igem.org/Part:BBa_K1741004 <b>BBa_K2014004</b>]) is slightly higher, than all other versions of xylose responsive promoters we tested, and its induction seems to be faster (see 2h time-point). <b>B)</b> The growth rate of all constructs compared: Xyl-WT [https://parts.igem.org/Part:BBa_K1741007 <b>(BBa_K1741007)</b>], XylA1 [https://parts.igem.org/Part:BBa_K1741008 <b>(BBa_K1741008)</b>], XylS [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>] or XylS-UTR [https://parts.igem.org/Part:BBa_K2014004 <b>(BBa_K2014004)</b>] is very similar. </font>]]
 
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Latest revision as of 00:45, 22 October 2016

pxylS-M5'UTR->sfGFP


Usage and Biology

The promoter pxylS-M5'UTR (with a lab name: XylS-UTR ), tested in a biobrick pxylS-M5'UTR->sfGFP (BBa_K2014004) is a modified xylose-induced promoter/5’UTR controlling sfGFP protein expression. To make it we exchanged the 5’UTR sequence of xylA-proD5'UTR in a biobrick (BBa_K1741009) to a synthetic, unstructured M5’UTR (picture B) derived from the Mel2 promoter (BBa_K1741004), which we provided to iGEM Registry in 2015.


If you are interested in the pathway of our modifications please click here-> MODIFICATIONS OF XYLOSE INDUCED PROMOTERS


Biobricks used in description:

xylF-xylA - briefly called XylWT (BBa_K1741007)
xylF-xylA-proD5'UTR - briefly called XylA1 (BBa_K1741008)
xylA-proD5'UTR - briefly called XylS (BBa_K1741009)
xylS-M5'UTR - briefly called XylS-UTR (BBa_K2014004)


Figure 1 Synthetic evolution of E. coli xylose induced promoters in our lab up to xylS-UTR. The promoter pxylS-M5'UTR (XylS-UTR) contains only xylA part of E. coli double sided xylose operon promoter, since xylF part appeared to be very weak. The synthetic, unstructured M5’UTR containing a strong, well-positioned RBS slightly enhances the responsiveness of xylA promoter to xylose. Despite that the promoters are stronger, they are still tight (Figure 2.).


Figure 2 Comparison of xylose promoters tightness during overnight time course cultures of E.coli DH5α in 1xLB medium without xylose. Bacteria were transformed with constructs: XylWT (xylF-xylA->sfGFP; BBa_K1741007), XylS (xylA-proD5'UTR; BBa_K1741009), XylS-UTR (xylS-M5'UTR; BBa_K2014004) and XylS-lysozyme (the construct containing non fluorescent protein- lysozyme, under XylS protein), which was used as our background.


Figure 3
A)
The most likely secondary structure that can be folded from the 5’UTR of XylWT promoter. B) The most likely secondary structures of M5’UTR from Mel2 and XylS promoters (BBa_K1741004). The sequence of M5’UTR is designed to minimize the likelihood of secondary structure formation. Structures shown above, were generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) under the same parameters. RBS sequences are underlined with a green line.


Figure 4
A)
Xylose inducibility of four xyl promoters/UTRs during 6h time course cultures of E.coli DH5α in LB medium containing 0.4% xylose. The efficiency of the improved promoter pxylS-M5'UTR (BBa_K2014004) is slightly higher, than all other versions of xylose responsive promoters we tested, and its induction seems to be faster (see 2h time-point). B) The growth rate of all constructs compared: Xyl-WT (BBa_K1741007), XylA1 (BBa_K1741008), XylS (BBa_K1741009) or XylS-UTR (BBa_K2014004) is very similar.



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 162