Generator

Part:BBa_K1741007

Designed by: Marta Żardecka   Group: iGEM15_UAM_Poznan   (2015-09-17)
Revision as of 18:47, 27 September 2015 by Michal.michalak (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

sfGFP under xylose promoter xylA (xylWT)

SfGFP under xylose promoter XylA (wild type).

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 part of XylWT. In some experiments it seems to be slightly stronger than the original XylWT 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 resulting in the XylS construct. 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.

UAMpoznanXylWTmod.jpg

UAMpoznanXylA1mod.jpg

Results

Legend:

XylWT (XylA) [BBa_K1741007]

XylA1 [BBa_K1741008]

XylS [BBa_K1741009]

XylF [BBa_K1741010]


Teamuampoznanxyloselbgraph.png

Teamuampoznanxylosem9graph.png

Teamuampoznanxyloseod600graph.png

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

All our xylose promoters are induced by xylose and repressed by glucose. We also observed slight induction by arabinose.

We observed that XylA1 and XylF had a lower expression than XylWT. XylS is the strongest xylose-induced promoter.


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 153
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 408


[edit]
Categories
Parameters
None