Part:BBa_K4195076

Biology

This sequence is the first part of guide designed for detection of toxin gene pirA.
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 (Fig. 1). 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 bound 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 illustration of RENDR.

Usage and Design

The conserved region of pirA gene is set as the RNA input. The guide sequences were designed based on NUPACK prediction(2). Based on the model provided (Equation. 1), we calculate the free energy difference of candidate sequences at 37 °C, and select guide pair g1 and g2 with 244.16 kcal/mol and 232.86 kcal/mol. The optimized ribozyme split sites are selected from the literature, and named α (split site 15) and β (split site 402)(1). Equation. 1 ln(FL/OD) ~ΔG_{Guide 1} + ΔG_{Guide 2} + ΔG_{RNA input} − ΔG_SC. Two parts of the split ribozyme are separately transcribed with different transcription start sites. We separately designed two split ribozymes as different parts BBa_K4195057 and BBa_K4195076, then the combined one (BBa_K4195183) was assembled into the vector pSB3K3 by standard BioBrick assembly. The constructed plasmids were transformed into 'E. coli' BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. Plasmid No part name specified with partinfo tag. and plasmid No part name specified with partinfo tag. were transformed into E. coli BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.