Difference between revisions of "Part:BBa K4218003"
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<p>In order to test the ability of MAP3K7-LUC and ZNF91-LUC to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmids MAP3K7-LUC and ZNF91-LUC were transfected into 293T cells, respectively. PB (final concentration: 1 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensors, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced (more details about the principle were listed in the project description). After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1 and 2, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1 and 2, Fig 5). The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB, compared with normal cells (Table 1 and 2, Fig 5). These results suggested MAP3K7-LUC and ZNF91-LUC sensors can detect the alteration of RNA splicing in cells.
 
<p>In order to test the ability of MAP3K7-LUC and ZNF91-LUC to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmids MAP3K7-LUC and ZNF91-LUC were transfected into 293T cells, respectively. PB (final concentration: 1 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensors, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced (more details about the principle were listed in the project description). After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1 and 2, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1 and 2, Fig 5). The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB, compared with normal cells (Table 1 and 2, Fig 5). These results suggested MAP3K7-LUC and ZNF91-LUC sensors can detect the alteration of RNA splicing in cells.
 
<p class="blog-p mb-25" style="text-align:center; font-weight:bold; font-family:'Times New Roman', Times, serif;padding-top:15px;">Table 1. The value of fluorescence in cell transfected with MAP3K7-LUC</p>
 
<p class="blog-p mb-25" style="text-align:center; font-weight:bold; font-family:'Times New Roman', Times, serif;padding-top:15px;">Table 1. The value of fluorescence in cell transfected with MAP3K7-LUC</p>
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Revision as of 07:29, 8 October 2022


T7-double Toehold switch-mcherry

Toehold switch is a special RNA hairpin structure, which contains trigger strand, ribosome binding site, translation start codon and a report gene. Our double toehold switch sequence (BBa_BBa_K4218016) is based on an article called Toehold Switches: De-Novo-Designed Regulators of Gene Expression (Green A, et al., 2014) and the mCherry is added to the downstream of the RNA hairpin structure.

Result

Functional verification of MAP3K7-LUC and ZNF91-LUC plasmid sensors</h>

In order to test the ability of MAP3K7-LUC and ZNF91-LUC to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmids MAP3K7-LUC and ZNF91-LUC were transfected into 293T cells, respectively. PB (final concentration: 1 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensors, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced (more details about the principle were listed in the project description). After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1 and 2, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1 and 2, Fig 5). The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB, compared with normal cells (Table 1 and 2, Fig 5). These results suggested MAP3K7-LUC and ZNF91-LUC sensors can detect the alteration of RNA splicing in cells. <p class="blog-p mb-25" style="text-align:center; font-weight:bold; font-family:'Times New Roman', Times, serif;padding-top:15px;">Table 1. The value of fluorescence in cell transfected with MAP3K7-LUC

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]