Composite

Part:BBa_K1649003

Designed by: Yang Yang   Group: iGEM15_NJAU_China   (2015-09-07)
Revision as of 16:49, 20 September 2015 by Yangshulantian (Talk | contribs)

YF1 blue light sensor with GFP reporter

GFP is used to report the function of blue light sensor. The LOV photo-switch YF1 was created by fusing the light perception domain of YtvA to the FixL kinase domain. YtvA acts as a blue-light photoreceptor and carries a blue-light-sensitive flavin-binding LOV domain. The response regulator FixJ forms a two-component system together with FixL.The sensor is inactive under blue light and active in dark.

Measurement

In order to test whether this part works,we cloned this part into pET28a using the NotI and XbaI sites. This plasmid contains a Kanamycin resistance gene and a pBR322 origin. BL21 strains are used to harbor blue light sensor plasmid.We measured OD600 and fluorescence of the strains at intervals. Plasmid Construction(gene circuit) BBa_K1649003 was cloned into pET28a using the NotI and XbaI sites. This plasmid contains a Kanamycin resistance gene and a pBR322 origin. Strains. BL21 strains are used to harbor blue light sensor plasmid. The control is BL21 strain. Culture BL21 strains transformed with blue light sensor plasmid (marked as pCNGG) and BL21 strains ( negative control) were each picked 3 colonies and grown overnight (~12 hours) at 37℃ and shaken at 200 rpm in 3 ml LB containing antibiotic. The overnights are then diluted 1:200 into 2 mL fresh media containing antibiotics in 2 Costar 24-well plates. One plate was exposed with blue LED which we call p1 and the other were wrapped in aluminum foil and kept in darkness which we call p2. All plates were maintained at 37℃. Each strain was taken 100μl into a new, identical set of 96-well plates and then measured OD600 and fluorescence at intervals. Result fpu: fluorescence per OD600 units

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15-NJAU-BBa K1649003.JPG BBa K1649003.png [[File:]] Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 605
    Illegal NgoMIV site found at 677
    Illegal NgoMIV site found at 767
    Illegal NgoMIV site found at 785
    Illegal NgoMIV site found at 1297
    Illegal NgoMIV site found at 1590
    Illegal NgoMIV site found at 1684
    Illegal AgeI site found at 319
    Illegal AgeI site found at 1465
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1354
    Illegal BsaI.rc site found at 218
    Illegal BsaI.rc site found at 2904


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