Part:BBa_K4195142

T7-pirA_g2α_Nu-T7t

Biology

This sequence is the 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.

NanoLuc

NanoLuc is a novel engineered luciferase enzyme that relies on the substrate furimazine to produce high intensity, glow-type luminescence. With high stability, small size and bright luminescence, it is an attractive luminescent reporter (2).

Fig. 2 The bioluminescent reaction catalyzed by NanoLuc® luciferase (2).

Usage and Design

The conserved region of pirA gene is set as the RNA input. The guide sequences were designed based on NUPACK prediction[3].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 (Fig. 3). 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. 3 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_{Guide 1} + ΔG_{Guide 2} + ΔG_{RNA input} − ΔG_SC= 232.86 kcal/mol.

NanoLuc 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_K4195044 and BBa_K4195079, then the combined one (BBa_K4195142) 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.

Characterization

1. In vivo Verification

1) Agarose Gel Electrophoresis

BBa_K4195142 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.

Fig. 4 The result of colony PCR. Plasmid pSB3K3.

2) Double transformation

Plasmid BBa_K4195142_pSB3K3 and plasmid BBa_K4195179_pSB1C3 were transformed into E. coli BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.

3) Bioluminescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of NanoLuc is observed by measuring the bioluminescence as time progressed using microplate reader.

Fig. 5 In vivo behavior of pirA_g2α_Nu as time progressed.

Reference

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

2. C. G. England, E. B. Ehlerding, W. Cai, NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug Chem 27, 1175-1187 (2016).

3. 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 174
  Illegal NheI site found at 693
  Illegal NheI site found at 1300
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 595
  Illegal XhoI site found at 38
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 822
  
• 1000
  COMPATIBLE WITH RFC[1000]