Generator

Part:BBa_K1741009

Designed by: Marta Żardecka   Group: iGEM15_UAM_Poznan   (2015-09-17)

sfGFP under shortened promoter xylA1 with improved 5'UTR

Design

Legend:

XylWT (XylA) [BBa_K1741007]

XylA1 [BBa_K1741008]

XylS [BBa_K1741009]

XylF [BBa_K1741010]


Genes involved in catabolism of xylose in E.coli are organized into two transcription units which are driven by two promoters - XylA and XylF. In order to increase the expression of a protein under control of our XylA1 promoter we added the 5'UTR of the proD promoter[BBa_K1741014] to the XylA of XylWT. In some experiments it seems to be slightly stronger than the original XylA promoter from part BBa_K1741007. The next step was to shorten the promoter by removing its XylF part while keeping the modified 5'UTR (from proD) behind XylA. Finally we tested the reversed XylF part of the XylWT promoter with sfGFP as the reporter gene. XylS is the strongest xylose-induced promoter. Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.

Results

Legend:

XylWT (XylA) [BBa_K1741007]

XylA1 [BBa_K1741008]

XylS [BBa_K1741009]

XylF [BBa_K1741010]


Teamuampoznanxyloselbgraph.png

Teamuampoznanxylosem9graph.png

Teamuampoznanxyloseod600graph.png


All our xylose promoters are induced by xylose and repressed by glucose. We also observed slight induction by arabinose. Short version of XylA1 promoter with known functional elements is stronger than two other versions (BBa_K1741007, BBa_K1741008). We observed that XylA1 and XylF had a lower expression than XylWT. XylS is the strongest xylose-induced promoter.

The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.


We also checked the tightness of our promoters.

All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose. Teamuampoznanpromoterscomparisongrpah.png


Contribution

Group: UAM_Poznan 2016
Author: Daria Niewiadomska


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 185


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