View source for Part:BBa K4195076


__NOTOC__
='''Biology'''=
This sequence is the first part of guide designed for detection of toxin gene ''pirA''.<br/>
Ribozyme ENabled Detection of RNA (RENDR)<br/>
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.
[[File:T--XMU-China--RENDR.png|400px]]

'''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<sub>Guide 1</sub> + ΔG<sub>Guide 2</sub> + ΔG<sub>RNA input</sub> − ΔG<sub>SC</sub>.'''

Two parts of the split ribozyme are separately transcribed with different transcription start sites. We separately designed two split ribozymes as different parts <partinfo>BBa_K4195057</partinfo> and <partinfo>BBa_K4195076</partinfo>, then the combined one (<partinfo>BBa_K4195183</partinfo>) 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 <partinfo>BBa_K4195168</partinfo>_pSB3K3 and plasmid <partinfo>BBa_K4195179</partinfo>_pSB1C3 were transformed into ''E. coli'' BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.

='''Characterization'''=
==''In vivo'' Verification==
#Agarose Gel Electrophoresis
<partinfo>BBa_K4195179</partinfo> and <partinfo>BBa_K4195168</partinfo> were 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.
[[File:T--XMU-China--pirA g1α.png|400px]]

'''Fig. 2 The result of colony PCR. Plasmid pSB1C3.'''
#Double transformation
Plasmid <partinfo>BBa_K4195168</partinfo>_pSB3K3 and plasmid <partinfo>BBa_K4195179</partinfo>_pSB1C3 were transformed into ''E. coli'' BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.
#Fluorescence measurement
Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of GFP is observed by measuring the Fluorescence/OD600 as time progressed using microplate reader.
[[File:T--XMU-China--GFP detection.png|400px]]

''Fig. 3 '' In vivo'' behavior of detection systems.''' <br/>'''a''' pirA detection systems and ori detection system were assembled into the vector pSB1C3. <br/>'''b''' pirA/ ori detection system and the target input were assembled separately into the vector pSB1C3 and pSB3K3. <br/>'''c''' pirB detection systems and ori detection system were assembled into the vector pSB1C3. <br/>'''d''' pirB/ ori detection system and the target input were assembled separately into the vector pSB1C3 and pSB3K3.

='''Reference'''=
#L. Gambill ''et al''., https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).
#J. N. Zadeh ''et al''., NUPACK: Analysis and design of nucleic acid systems. ''J Comput Chem'' '''32''', 170-173 (2011)