Designed by: Mathis LAVAUD   Group: iGEM21_UParis_BME   (2021-09-27)

Toehold_miR21-5p_2 (to Toehold_miR21_7)

This part enables expression of the variable part of the toehold switch (Fig.1) targeting human microRNA-21-5p (hsa-miRNA-21-5p) via T7 RNA polymerase activity. It is flanked by BsaI restriction sites with appropriate cutting sites for this type IIS restriction enzyme The sequence encoding for the variable part of the toehold switch is preceded by the T7 promoter. To produce a complete toehold switch, this part has to be assembled in a plasmid, directly followed by a repressed gene and the T7 terminator.

Figure 1: Scheme of toehold switch with the variable part composed of the toehold region and the hairpin. Created with BioRender.

Usage and Biology

This part is flanked by BsaI restriction sites at both ends. It allows rapid assembly in a plasmid using Golden Gate Assembly [1, 2]. To produce a fully working toehold switch, the part to be inserted in a plasmid and followed by the repressed gene and the T7 terminator, such as Ba_K3453101.

Figure 2. Principle of the insertion of the variable part of the toehold switch in a plasmid. Created with BioRender.

This part once cloned into a plasmid such as Ba_K3453101 and when expressed in a cell or cell free system containing T7 RNA polymerase, will produce a functional toehold switch targeting hsa-miR-21-5p. A random nucleotide sequence has been designed between the BsaI sites at the 5’ end and the T7 promoter to lengthen the size of the part. It enables easy visualisation on an agarose gel. E. coli BL21 DE3 strain can be used to express this part in vivo.

This part has not been characterized yet. It is one candidate of the bank of toehold switches targeting hsa-miR-21-5p ( Ba_K38780071, Ba_K3878013, Ba_K3878014, Ba_K3878015, Ba_K3878016, Ba_K3878017, Ba_K3878018, ) designed with the SwitchMi Designer. ==References== [1] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One (2008) 3, e3647. [2] Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One (2009) 4, e5553. Sequence and Features BBa_K3878014 SequenceAndFeatures