Composite

Part:BBa_K3278006

Designed by: Mingxuan Chi   Group: iGEM19_ShanghaiTech_China   (2019-10-13)
Revision as of 04:45, 20 October 2019 by AliceZhou (Talk | contribs)


pDawn with YF2

pDawn is a blue light control system which can start the expression of target gene under the inducement of blue light. More information in BBa_K1075044 added by iGEM13_Bonn.

However, expression leakage and delay are the two main problems of pdawn system. Leakage refers to the expression of target protein without light. According to our measurement, samples strictly kept away from light still had significant expression of GFP after being cultured for one night. Therefore, we carefully studied the gene circuits of pdawn system [2] [3] and hypothesized that enhancing the strength of the promoter of YF1 may enhance the repression of protein expression in the dark, thus solving the leakage problem.

In this part, we tried to change YF1 into YF2. YF2 is reported in Andreas Möglich’s paper[2]. By changing 24 bps in the original sequence, YF2 was reported to have higher promotion effect on FixJ expression, which means stronger inhibitory effect on target gene’s expression.

pDawn with T7 promoter is abbreviated as T7c below. pDawn with tac promoter is abbreviated as tac below. pDawn with modified tac promoter is abbreviated as mtac below. pDawn with YF2 is abbreviated as YF2 below.

[Go for the protocol of characterization:https://2019.igem.org/Team:ShanghaiTech_China/LightControl]

pDawn-YF2-0 hour
Fig1. pDawn-YF2-0 hour


pDawn-YF2-6 hour
Fig2. pDawn-YF2-6 hour


pDawn-YF2-12 hour
Fig3. pDawn-YF2-12 hour


Dark to light-12 hour
Fig4. Dark to light-12 hour


pDawn-YF2-24 hour
Fig5. pDawn-YF2-24 hour


Dark to light-24 hour
Fig6. Dark to light-24 hour


pDawn-mEGFP-30 hour
Fig7. pDawn-mEGFP-30 hour


Dark to light-30 hour
Fig8. Dark to light-30 hour


pDawn-YF2-36 hour
Fig9. pDawn-YF2-36 hour


Dark to light-36 hour
Fig10. Dark to light-36 hour



To see more changes on pDawn, go to the websites below.
pDawn-mEGFP: https://parts.igem.org/Part:BBa_K3278000
pDawn with T7 promoter: https://parts.igem.org/Part:BBa_K3278001
pDawn with mtac promoter: https://parts.igem.org/Part:BBa_K3278002
pdawn with tac promoter: https://parts.igem.org/Part:BBa_K3278005
pDawn with YF2: https://parts.igem.org/Part:BBa_K3278006


References:
[1] From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Robert Ohlendorf1, Roee R. Vidavski, Avigdor Eldar, Keith Moffat, and Andreas Möglich. Journal of Molecular Biology, 2012, Vol.416, pp. 534-542.
[2] Design and Signaling Mechanism of Light-Regulated Histidine Kinases. Andreas Möglich, Rebecca A. Ayers and Keith Moffat. Biophysical Journal, 2009, Vol.96 (3), pp. 1433-1444.
[3] Oxygen-Regulated In Vitro Transcription of Rhizobium meliloti nifA and fixK Genes. JEAN-MARC REYRAT,' MICHEL DAVID,' CASIMIR BLONSKI,2 PIERRE BOISTARD,' AND JACQUES BATUT'*. Journal of Bacteriology, 1993, pp. 6867-6872.

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 2148
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 46
    Illegal NgoMIV site found at 178
    Illegal NgoMIV site found at 272
    Illegal NgoMIV site found at 565
    Illegal NgoMIV site found at 1059
    Illegal NgoMIV site found at 1077
    Illegal NgoMIV site found at 1167
    Illegal AgeI site found at 397
    Illegal AgeI site found at 1525
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1620
    Illegal BsaI.rc site found at 508


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