Part:BBa_K4195158

T7-pirB_g1α_B-T7t

This sequence is the first part of guide designed for detection of toxin gene pirB.

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 (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.

AmpC

β-lactamase (AmpC) is a bacterial enzyme that facilitates resistance against β-lactam antibiotics by hydrolyzing the β-lactam ring, deactivating the antibiotic. It can also catalyze the hydrolysis reaction of nitrocefin, resulting in a distinct color change from yellow to red (2).

Fig. 2 The color transformation catalyzed by AmpC (2).

Usage and design

The conserved region of pirB 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 215.36 kcal/mol and 205.86 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 g1 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 = -13.2 kcal/mol, ΔG_Guide2 = -10.6 kcal/mol, ΔG_RNAinpu = -27.9 kcal/mol, ΔG_SC = -267.06 kcal/mol, ΔG_{Guide 1} + ΔG_{Guide 2} + ΔG_{RNA input} − ΔG_SC = 215.36 kcal/mol.

β-lactamase (AmpC) 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_K4195050 and BBa_K4195077, then the combined one (BBa_K4195158) 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_K4195158 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_K4195158_pSB3K3 and plasmid BBa_K4195180_pSB1C3 were transformed into E. coli BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.

3) Absorbance measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of AmpC is observed by measuring the absorbance in 490 nm as time progressed using microplate reader.

Fig. 5 In vivo behavior of pirB_g1α_B as time progressed.

2. In Vitro Verification

Plasmid was put into the cell-free system for expression. The expression behavior of AmpC is observed by measuring the absorbance in 490 nm as time progressed using microplate reader.

Fig. 6 In vitro behavior of pirB_g1α_B .

Reference

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

2. K. E. Boehle, C. S. Carrell, J. Caraway, C. S. Henry, Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics. ACS Sens 3, 1299-1307 (2018).

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 200
  Illegal NheI site found at 693
  Illegal NheI site found at 1918
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 595
  Illegal XhoI site found at 38
  Illegal XhoI site found at 1060
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]