Part:BBa_K4195176

T7-ori_i-T7t-T7-ori_g1α_G-T7t

Biology

This composite is the detection system taken from the literature (1).

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

We replicate the circuit used in the literature as reference for our design. 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 split ribozymes are designed separately as different parts BBa_K4195066 and BBa_K4195067, then the combined one (BBa_K4195177) is assembled into the vector pSB1C3 by standard BioBrick assembly. We assembled BBa_K4195177 and BBa_K4195178 into the vector pSB1C3 by standard BioBrick assembly. For optimizing this detection system, we also assembled BBa_K4195177 into the vector pSB3K3 by standard BioBrick assembly, and performed double transformation of plasmid BBa_K4195177_pSB3K3 and BBa_K4195178_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_K4195177 and BBa_K4195178 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.

(2) Double transformation

Plasmid BBa_K4195177_pSB3K3 and plasmid BBa_K4195178_pSB1C3 were transformed into E. coli BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol.

Fig. 2 The result of colony PCR. Plasmid pSB1C3.

(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. 3 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).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 205
  Illegal NheI site found at 461
  Illegal NheI site found at 954
  Illegal NheI site found at 1762
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 856
  Illegal XhoI site found at 299
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1681