Difference between revisions of "Part:BBa K2014004"
(13 intermediate revisions by 2 users not shown) | |||
Line 3: | Line 3: | ||
− | |||
+ | ===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_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] | ||
+ | Biobricks used in description: <br><br> | ||
+ | xylF-xylA - briefly called XylWT [https://parts.igem.org/Part:BBa_K1741007 <b>(BBa_K1741007)</b>]<br> | ||
+ | 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 [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>]<br> | ||
+ | xylS-M5'UTR - briefly called XylS-UTR [https://parts.igem.org/Part:BBa_K2014004 <b>(BBa_K2014004)</b>]<br> | ||
+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014004-1.png|thumb|500px|center|<font size="2"><b>Figure 1 Synthetic evolution of <i>E. coli</i> xylose induced promoters in our lab up to xylS-UTR.</b> The promoter pxylS-M5'UTR (XylS-UTR) contains only xylA part of <i>E. coli</i> 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.). </font>]] | ||
+ | |} | ||
+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | 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>]] | ||
+ | |} | ||
+ | {|align="center" | ||
+ | |-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 [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>]] | ||
+ | |} | ||
+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | 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>]] | ||
+ | |} | ||
− | |||
− | |||
− | |||
− | |||
− | |||
<!-- --> | <!-- --> |
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)
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 162