Latest revision as of 02:51, 9 October 2021

T7-Toehold Switch-mcherry

This part is composite of T7 promoter, our toehold switch part BBa_K3822000,FP mcherry and terminator.

Sequence and Features

Usage and Biology

In our project, the trigger sequence is linked to DNA fragment of biomarker by DNA amplification and then was constructed into a plasimd. After con-transformation into E.coli with the plasmid contain toehold sequence, the biomarker RNA will also be connected with trigger. The product with trigger can open the stem-loop structure and form a red fluorescent protein, which is the visible observation result.

result

Co-transformation verification

1. plasmid construction E.coli

In order to realize the co-transformation of two different plasmids to E.coli to run the functions, two plasmids need to have different origin of replication(ori). Therefore, we constructed the Toehold sequence and mcherry sequence onto PSB1C3 and constructed the trigger sequence containing the target gene onto pCOLADuet. We used SpeI / XbaI enzyme digestion PSB1C3 vector, ECOR I / Sal I enzyme digestion pCOLADuet vector, the synthesis of Toehold sequence and TFPI2-trigger sequence were linked to the enzyme digestion of PSB1C3 vector and pCOLADuet vector. Then the ligation products were transformed into DH5alpha bacteria, positive clones were selected on LB plate containing chloramphenicol and kana antibiotics respectively. After overnight incubator at 37 ℃ , the positive clones were picked out and amplified in LB tube for 6 hours, and then 100ul bacteria solution was extracted and sent to Tsingke biology company for sequencing of bacteria solution.

Figure1: Construction of Toehold switch-related plasmids. A: PSB1C3 plasmid vector map and pCOLADuet™-1 plasmid vector map ; B: PSB1C3 restriction enzyme digest with SpeI/XbaI (left) and pCOLADuet™-1 restriction enzyme digest with EcoR I/Sal I（right); C: Positive clone of PS1C3-Toehold recombinant (left) and positive clone of pCOLAduet-TFPI2-trigger recombinant (right); D：Sequencing results of PS1C3-Toehold recombiner and pCOLAduet-TFPI2-trigger recombiner.

2. pCOLAD-TFPI2-trigger andPB1C3-Toehold-mcherry were co-transferred into E.coli

The constructed plasimds PSB1C3-Toehold and pCOLADuet-TFPI2-trigger were cotransforred into E.coli DH5alpha. the constructed stain was then cultured overnight in 37 ℃ incubator in a double resistance LB plate containing chloramphenicol and kanamycin. Positive cloney was selected into 6ml LB. The bacteria was shaked at 160rpm in a 37-degree shaker until the OD600 reaches 0.4-0.6, add IPTG, a protein inducer with a final concentration of 1mM, and induce overnight at 160rpm in a 26-degree shaker. After induction, We can obersve that there is no fluorescence in the toehold alone group and there is bovious red light in the Toehold with TFPI2-trigger group and the positive control group (Part K3577001), which is in consistent with the fluorescence results observed under fluorescence microscope. 100ul bacteria solution was taken from each group and was used to detect the red fluorescence intensity on Microplate Reader at 587 nm excitation light 610nm receiving light. The results showed that TFPI2-trigger group had obvious red fluorescence intensity compared with thecontrol group. In general, tthe toehold system inserted with the target gene TFPI2 sequence can work as expected.

Figure2: Toehold switch function verification. A: PSB1C3-Toehold and pCOLADuet-TFPI2-trigger were co-transferred into DH5α; B: Expression of red Fluorescent induced by IPTG in different bacterias. C: Observation of bacterias under fluorescence microscope; D: Statistics of detection data and results of fluorescent bacteria on Microplate Reader.

3. Conclusion

By the co-transformation experiment, we can obviously see red light from the phenotype. At the same time through fluorescence detection, we can also see that the fluorescence signal is significantly different form that of the control group. In general, the Toehold switch system we designed works as expected.