Generator

Part:BBa_K1741010

Designed by: Marta Żardecka   Group: iGEM15_UAM_Poznan   (2015-09-17)
Revision as of 07:25, 27 September 2015 by MartaZ (Talk | contribs) (→‎Design)

sfGFP under xylose promoter xylF (very weak)

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 our XylA1 promoter we have modified the 5'UTR of the XylWT promoter so it was similar to that of proD [BBa_K1741014], a strong constitutive promoter. In some experiments it seems to be slightly stronger than original XylA promoter from part BBa_K1741007. The next step was to shorten the promoter by removing its XylF part while keeping the modified UTR. Finally we tested the XylF promoter upstream of sfGFP. 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. We also tested the XylF promoter from the same double sided regulatory element (cf. BBa_K1741007), and XylF appears to be a very weak promoter slightly induced by xylose.

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


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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 23
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 223


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