Difference between revisions of "Part:BBa K4244015"

 
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This plasmid contains a system to control the amount of dCas9 with the amount of rhamnose. Here the amount of dSpyCas9 will decrease when more rhamnose is present. This was accomplished by placing the repressor gene CI under the rhamnose inducible promoter. Due to this, more CI will produced the more rhamnose is present. This CI then represses the pR promoter that controls the expression of dSpyCas9. To check this, a lucifirase protein was fused to dSpyCas9. For this system a titratable inducer was needed. Therefore the experiment must be done in an E. coli strain containing two knock-outs, one for rhamnose transport and one for rhamnose catabolism, these were obtained form papers [1]. This system was tested, by placing cells on different amounts of rhamnose and then measuring luciferase expression (Figure 1)
 
This plasmid contains a system to control the amount of dCas9 with the amount of rhamnose. Here the amount of dSpyCas9 will decrease when more rhamnose is present. This was accomplished by placing the repressor gene CI under the rhamnose inducible promoter. Due to this, more CI will produced the more rhamnose is present. This CI then represses the pR promoter that controls the expression of dSpyCas9. To check this, a lucifirase protein was fused to dSpyCas9. For this system a titratable inducer was needed. Therefore the experiment must be done in an E. coli strain containing two knock-outs, one for rhamnose transport and one for rhamnose catabolism, these were obtained form papers [1]. This system was tested, by placing cells on different amounts of rhamnose and then measuring luciferase expression (Figure 1)
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[[File:BBa K4244015 fig1.svg|thumb|500px|center|<font size="1">Figure 1: Rhamnose response curve on expression of luciferase. Increasing rhamnose concentrations lead to a lower amount of luciferaase expression, due to the CI inverter. A) A downward trend was observed 90 minutes after induction, experiment was done with concentrations until 500 µM. B) A downward trend was observed 120 minutes after induction, experiment done with concentrations until 125 µM.</font>]]
  
 
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<partinfo>BBa_K4244015 parameters</partinfo>
 
<partinfo>BBa_K4244015 parameters</partinfo>
 
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===References===
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1. Hjelm A, Karyolaimos A, Zhang Z, Rujas E, Vikström D, Slotboom DJ, et al. Tailoring Escherichia coli for the l -Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS Synth Biol. 2017;6(6):985–94.

Revision as of 16:33, 8 October 2022


dCas9 double repression system

This plasmid contains a system to control the amount of dCas9 with the amount of rhamnose. Here the amount of dSpyCas9 will decrease when more rhamnose is present. This was accomplished by placing the repressor gene CI under the rhamnose inducible promoter. Due to this, more CI will produced the more rhamnose is present. This CI then represses the pR promoter that controls the expression of dSpyCas9. To check this, a lucifirase protein was fused to dSpyCas9. For this system a titratable inducer was needed. Therefore the experiment must be done in an E. coli strain containing two knock-outs, one for rhamnose transport and one for rhamnose catabolism, these were obtained form papers [1]. This system was tested, by placing cells on different amounts of rhamnose and then measuring luciferase expression (Figure 1)


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Figure 1: Rhamnose response curve on expression of luciferase. Increasing rhamnose concentrations lead to a lower amount of luciferaase expression, due to the CI inverter. A) A downward trend was observed 90 minutes after induction, experiment was done with concentrations until 500 µM. B) A downward trend was observed 120 minutes after induction, experiment done with concentrations until 125 µM.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 33
    Illegal EcoRI site found at 1038
    Illegal EcoRI site found at 2410
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 33
    Illegal EcoRI site found at 1038
    Illegal EcoRI site found at 2410
    Illegal NheI site found at 2169
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 33
    Illegal EcoRI site found at 1038
    Illegal EcoRI site found at 2410
    Illegal BamHI site found at 4448
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 33
    Illegal EcoRI site found at 1038
    Illegal EcoRI site found at 2410
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 33
    Illegal EcoRI site found at 1038
    Illegal EcoRI site found at 2410
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Hjelm A, Karyolaimos A, Zhang Z, Rujas E, Vikström D, Slotboom DJ, et al. Tailoring Escherichia coli for the l -Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS Synth Biol. 2017;6(6):985–94.