Difference between revisions of "Part:BBa K4218003"
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<p>In our project, the trigger sequence is linked to this part by DNA amplification and was added into the cell free system together with the plasmid contain toehold sequence, the report fluorescent protein will be expressed. The visible color and fluorescent signal can indicate the methylation degree of the TFPI2 gene in CRC patients.</p> | <p>In our project, the trigger sequence is linked to this part by DNA amplification and was added into the cell free system together with the plasmid contain toehold sequence, the report fluorescent protein will be expressed. The visible color and fluorescent signal can indicate the methylation degree of the TFPI2 gene in CRC patients.</p> | ||
==Results== | ==Results== | ||
| − | + | <partinfo>The introduction of our experiment:<partinfo> | |
<p>It has been established a toehold switch that combines with a fluorescent protein, mCherry, on its tail to detect methylated TFPI2 for early screening of non-invasive diagnosis of CRC (https://2021.igem.org/Team:YiYe-China). It is reported that SDC2 methylation and TFPI2 methylation are highly sensitive to CRC (DOI:10.2147/cmar.s375358) . In order to further improve the detection accuracy of CRC compared to last year, we combined the detection of methylated SDC2 with methylated TFPI2 this year. We constructed a toehold switch consisting of the plasmids of double-toehold pSB1C3 (more details were listed below) and double-trigger pCOLADuetTM-1, and then we have a functional verification for the plasmids in the BL21 cells. Finally, we detected methylation SDC2 gene and methylated TFPI2 through the expression of fluorescent protein in cell-free protein expression system.</p> | <p>It has been established a toehold switch that combines with a fluorescent protein, mCherry, on its tail to detect methylated TFPI2 for early screening of non-invasive diagnosis of CRC (https://2021.igem.org/Team:YiYe-China). It is reported that SDC2 methylation and TFPI2 methylation are highly sensitive to CRC (DOI:10.2147/cmar.s375358) . In order to further improve the detection accuracy of CRC compared to last year, we combined the detection of methylated SDC2 with methylated TFPI2 this year. We constructed a toehold switch consisting of the plasmids of double-toehold pSB1C3 (more details were listed below) and double-trigger pCOLADuetTM-1, and then we have a functional verification for the plasmids in the BL21 cells. Finally, we detected methylation SDC2 gene and methylated TFPI2 through the expression of fluorescent protein in cell-free protein expression system.</p> | ||
==Double-toehold switch== | ==Double-toehold switch== | ||
Revision as of 16:50, 11 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.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]