Composite

Part:BBa_K3278002

Designed by: Ailin Zhou   Group: iGEM19_ShanghaiTech_China   (2019-10-13)
Revision as of 14:02, 20 October 2019 by AliceZhou (Talk | contribs)


pDawn with modified 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 used the modified tac promoter(abbreviated as mtac promoter) to substitute the lacIq promoter in front of YF1 and Fixk2 and compared 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 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-mtac-0 hour
Fig1(a). pDawn-mtac-0 hour
pDawn-mtac-6 hour
Fig1(b). pDawn-mtac-6 hour
pDawn-mtac-12 hour
Fig1(c). pDawn-mtac-12 hour
pDawn-mtac-24 hour
Fig1(d). pDawn-mtac-24 hour
pDawn-mtac-30 hour
Fig1(e). pDawn-mtac-30 hour
pDawn-mtac-36 hour
Fig1(f). pDawn-mtac-36 hour
Figure 1: Representative images of mtac 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.


Result: Comparing with the original pDawn-mEGFP, both mtac and YF2 featured an ideal expression capacity and a minimum leakage level.


After that, we compared YF2, mtac with pDawn-mEGFP as below.

Figure 3 zal.png
Figure 3: Average F.I. line graphs of pDawn-mEGFP, mtac, YF2, tac normalized based on duration = 0 h. The detailed methods of data analysis and normalizing are shown at the bottom of this part.

Result: Comparing with the original pDawn-mEGFP, a similar effect can be observed in YF2 and mtac. To quantify the expression efficiency and leakage level, further analysis was carried out as below.

The effect on reducing leakage level was proven with the following graphs.

Figure 4 zal.png
Figure 4: Average F.I. bar graphs of pDawn-mEGFP, mtac, YF2, tac in separate timepoints normalized based on duration = 0 h. Timepoints were chosen based on the tendency of the line graph with 12h as the rapid growth period, 24h as the development period and 30h, 36h as the stable period.
Figure 5 zal.png
Figure 5: Bar graphs of average F.I. in light-off group of pDawn-mEGFP, mtac, YF2, tac at the timepoints of 12h, 24h, 30h, 36h after normalization based on the original pDawn-mEGFP groups, characterizing the leakage level of the plasmids


Result: Figures above clearly show the decreased background expression level of mtac and YF2 compared with pDawn-mEGFP. Specifically, the YF2 maintained a relatively low level of leakage which also dipped over time, while mtac showed a downward trend after 12h before becoming stable after 30h. The lowest leakage of the mtac and YF2 occurred at 12h and 36h respectively, with mtac reducing the leakage by 78.74% and YF2 by 78.98% when compared with pDawn-mEGFP.

Expression efficiency was compared in the following graphs:

Figure 6 zal.png
Figure 6: Bar graphs of relative F.I. with the fluorescence level of the light-on group divided by dark group of pDawn-mEGFP, mtac, YF2, tac at the timepoints of 12h, 24h, 30h, 36h, characterizing the expression efficiency of the system.


Result: Comparing with the original pDawn-mEGFP, a similar expression efficiency can be observed in YF2 and mtac and the timepoint of 12h and 36h witnessed a substantial climb of the YF2 and mtac respectively, exceeding pDawn-mEGFP by approximately 50%.
As a result, mtac and YF2 can be characterized as the most ideal modified components among all of the candidates, with similar efficiency as the original one and better performance on leakage reducing. Though the expression level of the 2 modified components is not yet ideal enough, we can expect a potential more excellent result obtained under suitably higher light intensity or a more proper inducing environment, which will be our plan for the future.



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(better)
pDawn with tac promoter: https://parts.igem.org/Part:BBa_K3278005
pDawn with YF2: https://parts.igem.org/Part:BBa_K3278006(better)


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 2159
  • 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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