Difference between revisions of "Part:BBa K1741008"
 
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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.
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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.  
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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.
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XylS is the strongest xylose-induced promoter. Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.
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Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.  
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===Results===
 
===Results===

Latest revision as of 08:52, 27 September 2015

sfGFP under 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.

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

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 419