Difference between revisions of "Part:BBa K4789009"

Revision as of 04:41, 30 September 2023

The MAP3K7-LUC improved plasmids

The introduction of our experiment Our improved experiment is based upon the YiYe-China iGEM project from 2022(https://2022.igem.wiki/yiye-china/, https://parts.igem.org/Part:BBa_K4218004). The project aims to provide a plasmid sensor to detect intracellular alternative splicing for early diagnosis of myelodysplastic syndromes(MDS). The project had established a dual luciferase reporter system to monitor the alteration of RNA splicing in cells These reporters incorporate segments of both the intronic and exonic sequences of MAP3K7 or ZNF91 within the luciferase gene (Fig 1). The activity of luciferase can be used to measure the changes of intracellular splicing, so as to play an auxiliary role in the diagnosis of MDS.

          
                    Fig .1 The dual luciferase reporter system

Sequence and Features

Assembly Compatibility:

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COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 2459
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INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 3661

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 ===Usage and Biology===
+The dual luciferase reporter system works as follow: fusion proteins (Fluc-Rluc) were expressed in cells, but when RNA splicing was disrupted, only Fluc proteins were induced. In normal cells, the expression of fusion proteins (Fluc-Rluc) was notably high. Treatment with PB (dysregulate RNA splicing) led to the downregulation of these fusion proteins. The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB compared to normal cells. These findings strongly indicate that the MAP3K7-LUC and ZNF91-LUC sensors can effectively detect alterations in RNA splicing in cells. Through these results, we found that the MAP3K7-LUC plasmid exhibited higher luciferase activity after transfection, which could potentially make the system more sensitive. Consequently, we focused on evaluating the performance of the MAP3K7-LUC sensor in detecting cellular RNA splicing abnormalities. Our goal is to improve the plasmid by improving the recognition ability of inserted introns, thereby increasing the sensitivity and splicing efficiency of this MAP3K7-LUC sensor and further improving its role in MDS diagnosis.
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