Difference between revisions of "Part:BBa K2014002"
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− | <u>Xylose promoters- legend:</u> | + | <u>Xylose promoters- legend:</u><br> |
− | xylF-xylA - formerly called XylWT <b>(BBa_K1741007)</b> | + | xylF-xylA - formerly called XylWT <b>(BBa_K1741007)</b><br> |
− | xylF-xylA-proD5'UTR - formerly called XylA1 <b>(BBa_K1741008)</b> | + | xylF-xylA-proD5'UTR - formerly called XylA1 <b>(BBa_K1741008)</b><br> |
− | xylA-proD5'UTR - formerly called XylS <b>(BBa_K1741009)</b> | + | xylA-proD5'UTR - formerly called XylS <b>(BBa_K1741009)</b><br> |
− | xylA-M5'UTR – formerly called XylS-UTR <b>(BBa_K2014004)</b> | + | xylA-M5'UTR – formerly called XylS-UTR <b>(BBa_K2014004)</b><br><br> |
Revision as of 00:14, 20 October 2016
pxylS-E1_5'UTR->sfGFP
The construct pxylS-E1_5’UTR->sfGFP is composed of XylA part of Eschericha coli K-12 double sided xylose promoter in which we exchanged the native downstream 5’UTR with a E1_5’UTR (Fig. 1). E1_5’UTR contains an additional ribosome binding site from gene 10 of bacteriophage T7. The new synthetic promoter controls sfGFP expression. The fluorescent protein sfGFP is a marker of gene expression and protein synthesis/accumulation. Protein expression from all compared xylose responsive promoters was induced in rich media with 0.4% D-xylose.
pxylS-E1_5’UTR is most likely the strongest available version of a xylose induced promoter in E. coli.
pxylS-E1_5’UTR (XylS-E1) is the strongest among all xylose promoters provided so far to the iGEM community by our team UAM_Poznan (Fig. 2). XylS-E1 ensures aproximately 2-3-fold higher expression than its wild-type version (Bba_K1741007) and slightly lower than T7 promoter from pET systems (Fig. 3).
pxylS-E1_5’UTR activity was tested in E. coli DH5α grown in three different media (1xLB, 2xLB, SB-PKB) containing 0.4% xylose (Fig. 4). pxylS-E1_5’UTR was compared to previous xylose promoter versions: XylWT (Bba_K1741007), XylA1 (BBa_K1741008), XylS (BBa_K1741009) and to negative control – XylS promoter driving lysozyme gene expression to have identical growth conditions with no fluorescent protein production. The promoter pxylS-E1_5’UTR provided the highest protein expression in all media, at least 2-times higher than its wild-type version (Fig.4, Fig. 5).
Xylose promoters- legend:
xylF-xylA - formerly called XylWT (BBa_K1741007)
xylF-xylA-proD5'UTR - formerly called XylA1 (BBa_K1741008)
xylA-proD5'UTR - formerly called XylS (BBa_K1741009)
xylA-M5'UTR – formerly called XylS-UTR (BBa_K2014004)
References:
1. Olins PO, Rangwala SH.; A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):16973-6.
2. Davis J.H., Rubin A.J., Sauer R.T.; Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research, 2011, Vol. 39, No. 3 1131–1141
3. Song S., Park C.; Organization and Regulation of the D-Xylose Operons in Escherichia coli K-12: XylR Acts as a Transcriptional Activator. Journal of Bacteriology, Nov. 1997, p. 7025–7032
Long Description goes here 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 174