Generator

Part:BBa_K2014004

Designed by: Daria Niewiadomska   Group: iGEM16_UAM_Poznan   (2016-10-12)

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


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