Generator

Part:BBa_K1741000

Designed by: Marta Żardecka   Group: iGEM15_UAM_Poznan   (2015-09-17)
Revision as of 06:42, 27 September 2015 by Michal.michalak (Talk | contribs) (→‎Design)

sfGFP under arabinose promoter s1 without AraC (Arashort1)

In commercially available expression vectors arabinose promoter araBAD is usually used together with a gene for AraC transcription activator / repressor to make the system independent of chromosomal copy of AraC gene. We fused the araBAD promoter without AraC reading frame, as other sugar induced promoters to sfGFP. The shorter version of the popular promoter is still functional i.e. induced by arabinose. Arabinose induced (0,4%) in a a short time (1-8h) expression is a bit lower than driven by AraC-araBAD but after a longer time like 18h the GFP level is higher than from AraC-araBAD promoter. New shorter versions of arabinose induced promoters, all originating from E. coli genome were compared to the biobrick BBa_K1481002, provided last year by Poznan_Bioinf team.

Design

Legend

AraWT - [BBa_K1481002]

Arashort1 - [BBa_1471000]

Arashort2 - [BBa_1471002]

Link=https://static.igem.org/mediawiki/parts/d/d7/UAMpoznanaraall.jpg

For the design of our modified arabinose promoters we used the BioBrick created by our fellow iGEM team - PoznanBioInf. Their promoter was used as a control for out other arabinose promoters. We shortened the sequence of the original promoter by removing lengthy AraC part (we called that construct Arashort1) or removing AraC together with operator regions O1 and O2 (we called that construct Arashort2).

Characteristics and results

Legend

AraWT - [BBa_K1481002]

Arashort1 - [BBa_1471000]

Arashort2 - [BBa_1471002]

Teamuampoznanarabinose2lbgraph.png Teamuampoznanaram9.png

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

All of our arabinose promoters are induced by the arabinose and repressed by the glucose. Arashort1 works better than AraWT. On the other hand, Arashort2 is slightly weaker when compared to Arashort1, but still stronger than AraWT. Probably O1 and O2 regions that were removed in Arashort2 are necessary for the best promoter's performance.


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 BamHI site found at 236
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 71
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 53
    Illegal SapI.rc site found at 352



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