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Measurement of LuxBrick’s Characteristic
In order to figure out the basic characteristic of k325909 submitted by Cambridge 2010, we measured growth curves of dH5a transformed k325909, which was induced by 0.3% arabinose (added from the very beginning of the incubation, and keep adding during the whole incubating process) under low temperature (25℃). As it shows in the chart, cells with and without induction grew to the logarithmic growth phase (about 5 hrs after inoculation) and stable phase (about 20 hrs after inoculation) were practically at the same time. But cells induced by arabinose displayed a significant decrease compared with those without induction.
Figure 1: growth curve of K325909 in a E.coli strain dH5a.
Detection of the Modified Protein
Detection of the Modified Protein’s Expression
Expression of mRNA for our modified protein was detected through reverse transcription PCR. The gel demonstrates that no problem exists in the transcription of the coding sequence of the modified protein lov-HTH.
The result of SDS-PAGE gel displays a satisfying expression of the modified protein lov-HTH.
Figure2: rupturing Top10 (invitrogen) cells transformed modified protein coding sequence promoted by araBAD. Extract supernatant ran the SDS-PAGE gel with PageRuler™ Prestained Protein Ladder. The electrophoretic band lies on the 24kb line confirms the expression of the modified protein.
Detection of the Modified Protein’s Function
The result of western-blot that we used to measure the expression of GFP in cells incubated under different conditions shows that the GFP expression of cells incubated under 450nm light (37℃) is about 30% lower than cells incubated in darkness.
Figure3: rupturing BL21(DE3) cells transformed modified protein coding sequence promoted by araBAD followed by GFP promoted by ptetO (pSB1A2) incubating with and without induction of arabinose. Extract supernatant to do the western-blot. The two bands corresponding to the two samples incubated under different condition show 30% distinction.
To reassure the result of the western-bolt, we measured the relative fluorescence intensity under different gradients of IPTG induction between 0- 0.5%. The 3D diagram presents a result that the GFP expressions of cells incubated under 450nm light and in darkness are of significant difference. Moreover, the 2D diagram indicates that 0.5% IPTG induction caused the largest distinction (28%) between the light and dark among the gradients.
Figure4: Top10 (invitrogen) transformed modified protein coding sequence promoted by T7 promoter followed by GFP promoted by ptetO (pSB1A2) incubating with induction of IPTG in gradient between 0~0.5% under 450nm light (a) and in dark (b). Relative fluorescence intensity of both samples shows a decrease go with the increase of induction.
Figure5: Top10 (invitrogen) transformed modified protein coding sequence promoted by T7 promoter followed by GFP promoted by ptetO (pSB1A2) incubating with induction of 0.5% IPTG under 450nm light and in dark. The curve of relative fluorescence intensity corresponding with the cells incubated under 450nm light shows 30% lower than in dark.
The Formation of Synchronized Oscillation
The analysis of a series of images that present variations of the culture disks incubating cells transformed luxbrick following the modified protein displays a well-performed synchrony. As it can be seen in analysis diagram, above 90% of the spectrum distribution of the 2000 sampling points randomly chosen reveals a significant concentration in the cycle. The analysis result of culture disks incubating luxbrick promoted by ptetO as control indicates that with the same number of sampling points, the spectrum distribution is rarely concentrated. The result is a perfect match for the simulation.
Figure6: Spectrum analyse by randomly choosing 2000 sampling points. (a).above 90% of the sampling points’ cycles concentrate between 800 s to 1000 s. (b). the cycles of all sampling points distribute between 600 s to 2600 s. Comparing with the simulation (c), Top10(Invitrogen) transformed LuxBrick followed by the modified protein lov-HTH (pSB1A2) displays a well-performed synchrony.
