Difference between revisions of "Part:BBa K3927020"

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===Design===
 
===Design===
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[[File:T--NUS Singapore--pGH3CF.png|700px|thumb|center|pGH3CF construct schematic]]
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[[File:T--NUS Singapore--pGH3CF.png|700px|thumb|center|Figure 1: pGH3CF construct schematic]]
  
 
To test out the functionality of the driver circuit, a construct with 3C120-CYC-LacO controlling the FLO1 gene was designed (figure 1). Anticipating the need to test integration, this plasmid was also designed to include the HBD2 gene under the control of the GAL1 promoter.
 
To test out the functionality of the driver circuit, a construct with 3C120-CYC-LacO controlling the FLO1 gene was designed (figure 1). Anticipating the need to test integration, this plasmid was also designed to include the HBD2 gene under the control of the GAL1 promoter.
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===Characterization===  
 
===Characterization===  
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Blue light induced flocculation protocol was replicated for pRL3CM(BY474D), and the reduction in OD600 after 24 hours of flocculation as well as the unflocculation index plotted.
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Blue light induced flocculation protocol was replicated for pRL3CM(BY474D), based off existing protocol used for characterization of blue light induced flocculation. The reduction in OD600 after 24 hours of flocculation as well as the unflocculation index plotted.
  
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[[File:T--NUS_Singapore--3C120-CYC-LacO-Flo1.png |800px|thumb|center|Figure 1: Blue light induced flocculation of pC120-Flo-EL-U (BY4741) (Top right), and uninduced culture (Top left), galactose induced flocculation of pGFT-H (BY4741) (Bottom right), and uninduced culture (Bottom left), after 6 hours (Left side) and 24 hours (Right side)]]
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Please refer to https://2021.igem.org/Team:NUS_Singapore/Results for more information on the data and work done to characterize this part.
  
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[[File:T--NUS_Singapore--3C120-CYC-LacO-Flo1_ODgraph.png |800px|thumb|center|Figure 2: Change in OD600 of supernatant of static pGH3CF-U(BY474D) culture after 24 hour in either dark or blue light, compared to pC120-Flo-EL222-U(BY4741).]]
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[[File:T--NUS_Singapore--3C120-CYC-LacO-Flo1.png |800px|thumb|center|Figure 2: Blue light induced flocculation of pC120-Flo-EL-U (BY4741) (Top right), and uninduced culture (Top left), galactose induced flocculation of pGFT-H (BY4741) (Bottom right), and uninduced culture (Bottom left), after 6 hours (Left side) and 24 hours (Right side)]]
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 +
[[File:T--NUS_Singapore--3C120-CYC-LacO-Flo1_ODgraph.png |800px|thumb|center|Figure 3: Change in OD600 of supernatant of static pGH3CF-U(BY474D) culture after 24 hour in either dark or blue light, compared to pC120-Flo-EL222-U(BY4741).]]
  
  

Latest revision as of 14:18, 18 October 2021


3C120-CYC-LacO-Flo1

3C120-CYC-LacO controlling of expression such that in the presence of blue light(dependent on EL222 expression) AND NOT LacI, yeast express the flocculation phenotype.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 203
    Illegal SpeI site found at 535
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 185
    Illegal SpeI site found at 535
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 203
    Illegal SpeI site found at 535
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 203
    Illegal SpeI site found at 535
    Illegal AgeI site found at 439
    Illegal AgeI site found at 481
    Illegal AgeI site found at 616
    Illegal AgeI site found at 886
  • 1000
    COMPATIBLE WITH RFC[1000]

Description

Design

Figure 1: pGH3CF construct schematic

To test out the functionality of the driver circuit, a construct with 3C120-CYC-LacO controlling the FLO1 gene was designed (figure 1). Anticipating the need to test integration, this plasmid was also designed to include the HBD2 gene under the control of the GAL1 promoter.

Usage

This part requires the host organism to be able to express BBa_K3570021 for it to work.

Characterization

Blue light induced flocculation protocol was replicated for pRL3CM(BY474D), based off existing protocol used for characterization of blue light induced flocculation. The reduction in OD600 after 24 hours of flocculation as well as the unflocculation index plotted.

Please refer to https://2021.igem.org/Team:NUS_Singapore/Results for more information on the data and work done to characterize this part.

Figure 2: Blue light induced flocculation of pC120-Flo-EL-U (BY4741) (Top right), and uninduced culture (Top left), galactose induced flocculation of pGFT-H (BY4741) (Bottom right), and uninduced culture (Bottom left), after 6 hours (Left side) and 24 hours (Right side)
Figure 3: Change in OD600 of supernatant of static pGH3CF-U(BY474D) culture after 24 hour in either dark or blue light, compared to pC120-Flo-EL222-U(BY4741).