Difference between revisions of "Part:BBa K3278005"

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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.
 
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.
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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 use the tac promoter to substitute the lacIq promoter in front of YF1 and Fixk2 and compare its performance with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.
 
In this part, we use the tac promoter to substitute the lacIq promoter in front of YF1 and Fixk2 and compare its performance with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.
  
pDawn with T7 promoter is abbreviated as <b>T7c</b> below. pDawn with tac promoter is abbreviated as <b>tac</b> below. pDawn with modified tac promoter is abbreviated as <b>mtac</b> below. pDawn with YF2 is abbreviated as <b>YF2</b> below.
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pDawn with tac promoter is abbreviated as <b>tac</b> below.
  
 
[Go for the protocol of characterization:https://2019.igem.org/Team:ShanghaiTech_China/LightControl]
 
[Go for the protocol of characterization:https://2019.igem.org/Team:ShanghaiTech_China/LightControl]

Revision as of 13:14, 20 October 2019


pDawn with tac promoter

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 use the tac promoter to substitute the lacIq promoter in front of YF1 and Fixk2 and compare its performance with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.

pDawn with tac promoter is abbreviated as tac below.

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


pDawn-tac-0 hour
Fig1(a). pDawn-tac-0 hour
pDawn-tac-6 hour
Fig1(b). pDawn-tac-6 hour
pDawn-tac-12 hour
Fig1(c). pDawn-tac-12 hour
pDawn-tac-24 hour
Fig1(d). pDawn-tac-24 hour
pDawn-tac-30 hour
Fig1(e). pDawn-tac-30 hour
pDawn-tac-36 hour
Fig1(f). pDawn-tac-36 hour
Figure 1: Representative images of tac under different light illumination-durations. The groups labeled ‘light-on’ were under light induction, while controls labeled ‘light-off’ were kept in the dark wrapped with aluminum foil. pictures were taken with the same parameters as the Alexa Fluor 488 dye, with the same 1000-ms exposure time for all images. The Brightfield images were captured with 300-ms exposure time.


To see more changes on pDawn, go to the websites below.
origin pDawn: https://parts.igem.org/Part:BBa_K1075044
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 2161
  • 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