Part:BBa_K4195060

pirA_g2αR_G

Biology

This sequence is the second 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.

GFP
The GFP derived from jellyfish Aequeora Victoria (BBa_E0040) is designed by team Antiquity in 2004. It’s a commonly used reporter.

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.

Fig. 2 The MFE structure of g2 guide-input complex at 37℃.ΔG_Guide1 and ΔG_Guide2 = The minimum free energy (MFE) of the two RNA guide sequences attached to each fragment of the RENDR ribozyme. ΔG_RNAinput = The MFE of the RNA input. ΔG_SC = The duplex binding energy of the complex.ΔG_Guide1 = -10.1 kcal/mol, ΔG_Guide2 = -28.4 kcal/mol, ΔG_RNAinput = -38.0 kcal/mol, ΔG_SC = -309.36 kcal/mol, ΔG_Guide1 + ΔG_Guide2 + ΔG_RNAinput - ΔG_SC = 232.86 kcal/mol.

GFP was chosen as the reporter, and the split ribozyme was inserted between the Ribosome-binding site and the coding sequence of reporter gene. Two parts of the split ribozyme are separately transcribed with different transcription start sites. We separately designed two split ribozymes as different parts BBa_K4195060 and BBa_K4195079, then the combined one (BBa_K4195169) was assembled into the vector pSB3K3 by standard BioBrick assembly. We assembled BBa_K4195179 and BBa_K4195169 into the vector pSB1C3 by standard BioBrick assembly. For optimizing this detection system, we also assembled BBa_K4195169 into the vector pSB3K3 by standard BioBrick assembly, and performed double transformation of plasmid BBa_K4195169_pSB3K3 and BBa_K4195179_pSB1C3. All the constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin or chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. In vivo Verification

(1) Agarose Gel Electrophoresis

BBa_K4195179 and BBa_K4195169 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.
Fig. 3 The result of colony PCR. Plasmid pSB1C3.

(2) Double transformation

Plasmid BBa_K4195169_pSB3K3 and plasmid BBa_K4195179_pSB1C3 were transformed into E. coli BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.

(3) 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.

Fig. 4 In vivo behavior of detection systems. a pirA detection systems and ori detection system were assembled into the vector pSB1C3. b pirA/ ori detection system and the target input were assembled separately into the vector pSB1C3 and pSB3K3. c pirB detection systems and ori detection system were assembled into the vector pSB1C3. d pirB/ ori detection system and the target input were assembled separately into the vector pSB1C3 and pSB3K3.

Reference

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

2. J. N. Zadeh et al., NUPACK: Analysis and design of nucleic acid systems. J Comput Chem 32, 170-173 (2011)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 435
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 337
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1162