Characterization

To ensure that our system of the real part can work as we expect and we can achieve our goal, we did some verifications and tests.

the verification of oscillator

When the genetic oscillator was completed, we verified its function by using GFP as a reporter indicating the state of oscillation. We gained some pictures and videos by observing the GFP in E.coli through fluorescence microscope.

Fig 1:The E.coli with fluorescence through fluorescence microscope.

video:The state of E.coli cultured in microfluidic chip through fluorescence microscope.

Furthermore, due to the GFP protein as the reporter has delay and accumulation character in our experiment, we also tried to verify the oscillator in transcriptional level using a RNA aptamer as the reporter, which is sensitive and can real-time reflect the state of oscillation.

We adopted a new generation RNA aptamer named dBroccoli. (Filonov G S, et al. JACS, 2014) It can activate the fluorescence of DFHBI-1T when binding with DFHBI-1T and shows green fluorescence in cells. ex = 465nm,em = 535nm. When the related gene added the RNA aptamer sequence, we can observe the state of oscillator in E.coli through fluorescence microscope. Meanwhile the function of the RNA aptamer was verified in our experiment showing as the following pictures.

Fig 2: The fluorescence in E.coli through fluorescence microscope.

Ps:

1) the Molecular Formula of DFHBI-1T is

            (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1Himidazol-5(4H)-one

2) The Chemical synthesis process of DFHBI-1T

Fig 3

3) Excitation peak: 465 nm Emission peak: 535 nm

The effect of light on the expression of genes

We verified the effect of light on the expression of genes by controlling the light condition of E.coli transformed with the genetic circuit of light control system.

Two sample owning the same genetic circuit were set in our test experiment, the one in dark, the other in red light, and each sample had three biologic repeats. Besides we designed a simple and practical device to control the light condition of E.coli as the following picture shown.

Fig 4:A red LED powered by battery is set at the bottom of a cup to provide red light.

Fig 5: Two light conditions in our test device.

After some times gropes and tests, we gained related data and did correlation analysis as showing from the following table. As we can see from it, the presence of red light can inhibit the expression of target genes.(In the test, we used GFP as the target gene).

Fig 6:The effect of red light on the expression of genes

the test of the related promoter

In order to fully understand the features of the oscillator, we specifically ran some related tests on the hybrid promoter(Plac/ara-1). We put a mRFP behind the promoter as a reporter in our experiment. We had used two different chemical inducer(Arabinose/IPTG) in our test to run respective tests on the promoter. A series of different inducer's concentrations are set to test amples, and the expressions level of mRFP is linked with the influence that different concentration of inducer had on the promoter.

We got and analysed related datas showing as the following charts.

Fig 7:The effect on the promoter of Arabinose.

Fig 8:The effect on the promoter of IPTG.

Reference

1.Filonov G S, Moon J D, Nina S, et al. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.[J]. Journal of the American Chemical Society, 2014, 136.