Difference between revisions of "Part:BBa K3278006"

Latest revision as of 14:06, 20 October 2019

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. Moreover, we characterized it 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]

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.

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.

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:

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