Difference between revisions of "Part:BBa K3052030"

 
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==2019 Team XJTU-CHINA's improvement of BBa_K2598038==
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===Overview===
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This year team XJTU-CHINA aim to improve a well-characterized red-light-activated sensor which is part of the GRB system of 2018 team UCAS-China(Part:BBa_K2598038). This part contains red-light sensor cph8(BBa_I15010) and repressor CI (BBa_C0051) under its regulation.
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Base on this red-light-activated sensor, we add a CI regulated promoter - Pλ to this part and enable it to realize the function of two-phases switch (BBa_K3052030). Besides, we also added two fluorescent proteins - superfolder GFP (BBa_K3052041) and mRFP (BBa_K3052042) under the control of pomp (BBa_R0082) and Pλ(BBa_K1145005) respectively to report the expression level of each phase. Theoretically, RFP will expresses under red light and GFP expresses in the absence of red light.
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<div>[[File:T--XJTU-CHINA--CIRCUIT.png |700px|thumb|center|<b>Figure 1:</b> bidirectional light-responsive switch]]</div>
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===Characterization===
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The light-fluorescent circuits (see circuits) were constructed by two-step Golden Gate Assembly and were confirmed by colony PCR. The length of this fragment is 1467 bp and the following results showed the successful constructions.
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<div>[[File:T--XJTU-CHINA--CPCR.png |700px|thumb|center|<b>Figure 1:</b> PCR confirmation of light-fluorescent circuit]]</div>
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In order to validate the effectiveness of this switch, we qualitatively measure the fluorescent of this circuit at different time. Sample of DH5α E. coli transformed with BBa_K3052030 is incubated for 24 hours without red light and then continue incubating 24 hours with red light. We photographed both the sample of control group (DH5α E.coli wildtype) and experimental group(E.coli with light-fluorescent circuit ) by a fluorescence microscope at 24 hours (24 hours without light treatment) and 48 hours (24 h without light treatment and 24 h with red light treatment). The figure indicated that the bidirectional switch was functional to switch from GFP to RFP.
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<div>[[File:T--XJTU-CHINA--FLU.png |700px|thumb|center|<b>Figure 2:(A) The images of control group. (B)The images of experimental group which has been incubated for 24 hours without light treatment. (C)The images of experimental group which has been continually incubated with red light for 24 hours after incubating for 24 hours without light treatment. (The pictures from top to bottom represent: GFP, RFP and bright)]]</div>
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Furthermore, we used COFOCAL to sensitively detect GFP and RFP while switch is shifting. After the sample above had been incubated for 24 hours without light treatment, it was treated with intense red light (wavelength=640nm). After incubating for another 3 hours with red light treatment, we took out some of the sample and perform CONFOCAL imaging. The result shows that the RFP was appearing with red light treatment, which indicates this switch can be effectively triggered by red light.
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<div>[[File:T--XJTU-CHINA--COFOCAL.png |700px|thumb|center|<b>Figure 3:</b> Results from COFOCAL: (A)Images of the sample. (B)Zoomed view of a specific area. (The pictures in A and B respectively represent: (a)GFP, (b)RFP, (c)Bright, (d)Merge) ]]</div>
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To quantitatively test the efficiency of this light switch, we decided to cultivate the cells in dark environment first and then trigger the switch with intense red light. When they had been incubated for 30 hours without red light, we start to put intense red light around the medium. During the experiment, we measured the GFP and RFP intensity of the sample every 2 hours.
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<div>[[File:T--XJTU-CHINA--QUA.png |700px|thumb|center|<b>Figure 4:</b> Quantitative result of GFP and RFP fluorescent intensity of the light-controlling switch.(The blue dotted line represents the time point of starting red light treatment)]]</div>
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From the figure 4 we can see that the GFP intensity dropped sharply to a low and stable value in 4 hours when the sample was treated with red light, additionally the increase of RFP intensity also indicates the effectiveness of this bidirection switch.

Revision as of 18:27, 21 October 2019


Light-fluoresce circuit (GFP&RFP)

This circuit uses a light control system wherein the central protein cph8 is a photosensitive protein that can response to red light and block the expression of downstream genes. The variables of this two-phases can be calculated by measuring the fluoresce intensity of sfGFP and mRFP.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 5032
    Illegal NheI site found at 5055
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2298
    Illegal BglII site found at 6592
    Illegal XhoI site found at 405
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6145
    Illegal AgeI site found at 4801
    Illegal AgeI site found at 4913
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2504


2019 Team XJTU-CHINA's improvement of BBa_K2598038

Overview

This year team XJTU-CHINA aim to improve a well-characterized red-light-activated sensor which is part of the GRB system of 2018 team UCAS-China(Part:BBa_K2598038). This part contains red-light sensor cph8(BBa_I15010) and repressor CI (BBa_C0051) under its regulation. Base on this red-light-activated sensor, we add a CI regulated promoter - Pλ to this part and enable it to realize the function of two-phases switch (BBa_K3052030). Besides, we also added two fluorescent proteins - superfolder GFP (BBa_K3052041) and mRFP (BBa_K3052042) under the control of pomp (BBa_R0082) and Pλ(BBa_K1145005) respectively to report the expression level of each phase. Theoretically, RFP will expresses under red light and GFP expresses in the absence of red light.

Figure 1: bidirectional light-responsive switch

Characterization

The light-fluorescent circuits (see circuits) were constructed by two-step Golden Gate Assembly and were confirmed by colony PCR. The length of this fragment is 1467 bp and the following results showed the successful constructions.

Figure 1: PCR confirmation of light-fluorescent circuit

In order to validate the effectiveness of this switch, we qualitatively measure the fluorescent of this circuit at different time. Sample of DH5α E. coli transformed with BBa_K3052030 is incubated for 24 hours without red light and then continue incubating 24 hours with red light. We photographed both the sample of control group (DH5α E.coli wildtype) and experimental group(E.coli with light-fluorescent circuit ) by a fluorescence microscope at 24 hours (24 hours without light treatment) and 48 hours (24 h without light treatment and 24 h with red light treatment). The figure indicated that the bidirectional switch was functional to switch from GFP to RFP.

Figure 2:(A) The images of control group. (B)The images of experimental group which has been incubated for 24 hours without light treatment. (C)The images of experimental group which has been continually incubated with red light for 24 hours after incubating for 24 hours without light treatment. (The pictures from top to bottom represent: GFP, RFP and bright)
</div>


Furthermore, we used COFOCAL to sensitively detect GFP and RFP while switch is shifting. After the sample above had been incubated for 24 hours without light treatment, it was treated with intense red light (wavelength=640nm). After incubating for another 3 hours with red light treatment, we took out some of the sample and perform CONFOCAL imaging. The result shows that the RFP was appearing with red light treatment, which indicates this switch can be effectively triggered by red light.

<b>Figure 3:</b> Results from COFOCAL: (A)Images of the sample. (B)Zoomed view of a specific area. (The pictures in A and B respectively represent: (a)GFP, (b)RFP, (c)Bright, (d)Merge)

To quantitatively test the efficiency of this light switch, we decided to cultivate the cells in dark environment first and then trigger the switch with intense red light. When they had been incubated for 30 hours without red light, we start to put intense red light around the medium. During the experiment, we measured the GFP and RFP intensity of the sample every 2 hours.

<b>Figure 4:</b> Quantitative result of GFP and RFP fluorescent intensity of the light-controlling switch.(The blue dotted line represents the time point of starting red light treatment)

From the figure 4 we can see that the GFP intensity dropped sharply to a low and stable value in 4 hours when the sample was treated with red light, additionally the increase of RFP intensity also indicates the effectiveness of this bidirection switch.