Difference between revisions of "Part:BBa K2014002"

Revision as of 23:52, 21 October 2016

pxylS-E1_5'UTR->sfGFP

Usage and Biology

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.

Fig. 1 Synthetic evolution of E. coli xylose induced promoters in our lab. pxylS-E1_5’UTR (XylS-E1) promoter contains only XylA part of E. coli double sided xylose operon promoter, since XylF part appeared to be very weak, and E1_5’UTR with the additional ribosome binding site from gene 10 of bacteriophage T7.

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).

Fig. 2 Comparison between pxylS-E1_5’UTR (XylS-E1) and our previous xylose responsive promoters. E.coli DH5α transformed with appropriate constructs containing sfGFP were cultured for 6h in LB medium containing 0.4% xylose.

Fig. 3 Comparison between pxylS-E1_5’UTR (XylS-E1) and its wild-type version (XylWT)- left panel. Comparison between pxylS-E_15’UTR (XylS-E1) and T7 promoter- right panel. E.coli DH5α (transformed with XylWT, XylSE1) or BL21-DE3 (transformed with T7-sfGFP) cells were cultured for 6h in LB medium supplemented with 0.4% xylose or lactose respectively.

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).

Fig. 4 Comparison between pxylS-E1_5’UTR (XylS-E1) and our previous xylose promoter versions in E. coli cultured in 1xLB, 2xLB, SB-PKB. E.coli DH5α with appropriate constructs containing sfGFP were cultured for 6h, after induction with D-xylose (0.4% final conc.). The efficiency of promoters was compared based on relative fluorescence intensity of produced sfGFP. OD600 shows that the growth rate of E. coli in all compared cultures is very similar (bacterial cultures were diluted 10-times).

Fig. 5 Photograph illustrating sfGFP fluorescence, which expression was controlled by different versions of xylose inducible promoters; E.coli DH5α were cultured for 6h in SB-PKB medium, with 0.4% xylose. Our newest promoter pxylS-E1_5’UTR (XylS-E1) is the strongest one.

Xylose promoters- legend:
xylF-xylA - formerly called XylWT (BBa_K1741007)
xylF-xylA-proD5'UTR - formerly called XylA1 (https://parts.igem.org/Part:BBa_K1741008 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

Sequence and Features