Composite

Part:BBa_K1084504

Designed by: Shinya Kawata   Group: iGEM13_HokkaidoU_Japan   (2013-09-26)

Promoter Selector-mRFP

This part is promoter 9 (BBa_K1084009) constructed as Promoter Selector vector expresses mRFP1 (Red).

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 (Promoter Selector). Plate image below is actual worked Promoter Selector (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 PstI site found at 1294
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 214
    Illegal PstI site found at 228
    Illegal PstI site found at 1294
    Illegal NotI site found at 221
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal suffix found in sequence at 214
    Illegal PstI site found at 1294
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 214
    Illegal PstI site found at 228
    Illegal PstI site found at 1294
    Illegal AgeI site found at 70
    Illegal AgeI site found at 1014
    Illegal AgeI site found at 1126
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 73
    Illegal BsaI.rc site found at 67


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