Difference between revisions of "Part:BBa K4195155"

Revision as of 23:50, 13 October 2022

T7-B0034-sfG(R)FP-T7t

Biology

Ribozyme ENabled Detection of RNA (RENDR) RENDR is a high-performing, plug-and-play RNA-sensing platform (1). RENDR utilizes a split variant of the Tetrahymena thermophila ribozyme by synthetically splitting it into two non-functional fragments. Two fragments are each appended with designed RNA guide sequences, which can interact with the RNA input of interest. The split ribozyme is then inserted within a desired gene output. When binded with the RNA input, two transcribed split ribozyme fragments are triggered to self-splice and thus the intact transcript of the protein output will form.

Fig. 1. Schematic of RENDR that uses an RNA input to template a split ribozyme.

Usage and design

We replicate the circuit used in the literature as reference. The splicing ribozyme was inserted within the coding sequence (CDS) of a super folder green fluorescent protein (sfGFP) gene such that translation of the full-length protein is disrupted in the absence of splicing. Upon transcription and splicing, the ribozyme removes itself from the flanking exons and forms a functional sfGFP mRNA, which is then translated to produce a fluorescent protein output. The combined one (BBa_K4195155) was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. In Vivo Verification

1) Agarose Gel Electrophoresis

After constructing the part on the expression vector pSB1C3, the plasmid was transferred into E. coli BL21(DE3). The positive transformants were selected by chloramphenicol and confirmed by colony PCR.

Fig. 2 The result of colony PCR. Plasmid pSB1C3.

2) Bioluminescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of sfGFP is observed under blue-light gel imager.

Fig. 3 The result of induction.

2. In Vitro Verification

Plasmid was put into the cell-free system for expression. The expression behavior of sfGFP is observed by measuring the fluorescence by using microplate reader.

Fig. 4 Experiment result under 37 °C. BBa_K4195152 was set as positive control (PC), and the sterile deionized water was set as negative control (NC).

Reference

1. L. Gambill et al., https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).

Sequence and Features