Composite

Part:BBa_K1084502

Designed by: Shinya Kawata   Group: iGEM13_HokkaidoU_Japan   (2013-09-26)
Revision as of 13:38, 30 September 2013 by Kenta (Talk | contribs)

Promoter Selector-aeBlue

This part is promoter 2 (BBa_K1084402) constructed as POK vector expresses aeBlue (Blue).

iGEM HokkaidoU Japan 2013 original promoter family is constructed from consensus promoter (BBa_K1084001) by adding mutation at -35 region. Other region is same among family members, and similar with promoter consensus sequence.

HokkaidoU2013_promoter_Result-fig1.png

Fig. 1 Randomized promoter family sequences

Correspondence of sequence and parts number is below. 

  -35        BBa_         t. e. rank
  TTGACA     K1084001     1   
  TAGGTC     K1084002     2
  CTGAAG     K1084003     6
  GGGGTG     K1084004     3
  GAGGAT     K1084005     5
  GCAATA     K1084006     7
  GGGGGG     K1084007     8
  TGTGTG     K1084008     4
  AGTGGG     K1084009     9
  TCTCGG     K1084010     10

t. e. = theoretical transcription efficyency

Promoter transcription efficiency was measured with mRFP1 (BBa_K1084401-K1084410), LacZ and Kanamycine (Km) resistance (BBa_K1084501-K1084505). HokkaidoU2013_promoter_Result-fig2.png

Fig. 2 mRFP1 assay result

HokkaidoU2013_promoter_Result-fig4.png

Fig. 3 β-Galactosidase assay result

HokkaidoU_2013_Result-f6_1_1.png

Fig. 4 Km resistance assay result

HokkaidoU_2013_Result-f7.png

Fig. 5 Comparison of assay results and modeling data

Theoretical promoter modeling also has done (see promoter Background page in HokkaidoU 2013 wiki). According to protein, these promoters show relatively another protein activity revel. Choose your best promoter by promoter optimization kit (POK). Plate image below is actual worked POK (Km resistance).

HokkaidoU_2013_POK_DEMO_48h_1.png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal suffix found in sequence at 213
    Illegal SpeI site found at 455
    Illegal SpeI site found at 673
    Illegal PstI site found at 687
    Illegal PstI site found at 1746
    Illegal PstI site found at 1880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 214
    Illegal SpeI site found at 455
    Illegal SpeI site found at 673
    Illegal PstI site found at 228
    Illegal PstI site found at 687
    Illegal PstI site found at 1746
    Illegal PstI site found at 1880
    Illegal NotI site found at 221
    Illegal NotI site found at 680
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal suffix found in sequence at 214
    Illegal SpeI site found at 455
    Illegal SpeI site found at 673
    Illegal PstI site found at 687
    Illegal PstI site found at 1746
    Illegal PstI site found at 1880
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 214
    Illegal SpeI site found at 455
    Illegal SpeI site found at 673
    Illegal PstI site found at 228
    Illegal PstI site found at 687
    Illegal PstI site found at 1746
    Illegal PstI site found at 1880
    Illegal AgeI site found at 70
    Illegal AgeI site found at 529
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 73
    Illegal BsaI site found at 532
    Illegal BsaI.rc site found at 67
    Illegal BsaI.rc site found at 526


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