Difference between revisions of "Part:BBa K4195041"

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===Biology===
 
===Biology===
  
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Ribozyme ENabled Detection of RNA (RENDR)
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'''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.
 
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.
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[[File:T--XMU-China--RENDR.png|500px]]
 
[[File:T--XMU-China--RENDR.png|500px]]
  
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'''Fig. 1 Schematic illustration of RENDR'''
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'''Fig. 1 Schematic illustration of RENDR.'''
  
  
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NanoLuc
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'''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'').
 
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'').
  
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[[File:T--XMU-China--Nu.png|500px]]
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[[File:T--XMU-China--NanoLuc.png|500px]]
  
 
'''Fig. 2 The bioluminescent reaction catalyzed by NanoLuc® luciferase (''2'').'''
 
'''Fig. 2 The bioluminescent reaction catalyzed by NanoLuc® luciferase (''2'').'''
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===Usage and design===
 
===Usage and design===
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[[File:T--XMU-China--A g1 Nupack.png|400px]]<br/>
 
[[File:T--XMU-China--A g1 Nupack.png|400px]]<br/>
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'''Fig. 2 The MFE structure of g1 guide-input complex  at 37℃. '''ΔG<sub>Guide1</sub> and ΔG<sub>Guide2</sub> = The minimum free energy (MFE) of the two RNA guide sequences attached to each fragment of the RENDR ribozyme. ΔG<sub>RNAinput</sub> = The MFE of the RNA input. ΔG<sub>SC</sub> = The duplex binding energy of the complex. ΔG<sub>Guide1</sub>= -11.5 kcal/mol, ΔG<sub>Guide2</sub>= -17.0 kcal/mol, ΔG<sub>RNAinput</sub>= -38.0 kcal/mol, ΔG<sub>SC</sub>= -310.66 kcal/mol, ΔG<sub>SC</sub>-ΔG<sub>Guide1</sub>+ΔG<sub>Guide2</sub>+ΔG<sub>RNAinput</sub>= 244.16 kcal/mol.
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'''Fig. 3 The MFE structure of g1 guide-input complex  at 37℃. '''ΔG<sub>Guide1</sub> and ΔG<sub>Guide2</sub> = The minimum free energy (MFE) of the two RNA guide sequences attached to each fragment of the RENDR ribozyme. ΔG<sub>RNAinput</sub> = The MFE of the RNA input. ΔG<sub>SC</sub> = The duplex binding energy of the complex. ΔG<sub>Guide1</sub>= -11.5 kcal/mol, ΔG<sub>Guide2</sub>= -17.0 kcal/mol, ΔG<sub>RNAinput</sub>= -38.0 kcal/mol, ΔG<sub>SC</sub>= -310.66 kcal/mol, ΔG<sub>Guide 1</sub> + ΔG<sub>Guide 2</sub> + ΔG<sub>RNA input</sub> − ΔG<sub>SC</sub>= 244.16 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 <partinfo>BBa_K4195041</partinfo> and <partinfo>BBa_K4195076</partinfo>, then the combined one (<partinfo>BBa_K4195141</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.
 
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 <partinfo>BBa_K4195041</partinfo> and <partinfo>BBa_K4195076</partinfo>, then the combined one (<partinfo>BBa_K4195141</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.
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===Characterization===
 
===Characterization===
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[[File:T--XMU-China--K4195141 (K4195141 pSB3K3, colony PCR).png|500px]]
 
[[File:T--XMU-China--K4195141 (K4195141 pSB3K3, colony PCR).png|500px]]
  
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'''Fig. 4 The result of colony PCR. Plasmid pSB3K3'''
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'''Fig. 4 The result of colony PCR. Plasmid pSB3K3.'''
  
  
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'''Fig. 5 ''In vivo'' behavior of pirA_g1α_Nu as time progressed.'''
 
'''Fig. 5 ''In vivo'' behavior of pirA_g1α_Nu as time progressed.'''
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===Reference===
 
===Reference===

Latest revision as of 09:34, 13 October 2022


pirA_g1αR_Nu


This sequence is the second part of guide designed for detection of toxin gene pirA.

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.

T--XMU-China--RENDR.png

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).

T--XMU-China--NanoLuc.png

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) ~ΔGGuide 1 + ΔGGuide 2 + ΔGRNA input − ΔGSC.

T--XMU-China--A g1 Nupack.png
Fig. 3 The MFE structure of g1 guide-input complex at 37℃. ΔGGuide1 and ΔGGuide2 = The minimum free energy (MFE) of the two RNA guide sequences attached to each fragment of the RENDR ribozyme. ΔGRNAinput = The MFE of the RNA input. ΔGSC = The duplex binding energy of the complex. ΔGGuide1= -11.5 kcal/mol, ΔGGuide2= -17.0 kcal/mol, ΔGRNAinput= -38.0 kcal/mol, ΔGSC= -310.66 kcal/mol, ΔGGuide 1 + ΔGGuide 2 + ΔGRNA input − ΔGSC= 244.16 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_K4195041 and BBa_K4195076, then the combined one (BBa_K4195141) 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_K4195141 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.

T--XMU-China--K4195141 (K4195141 pSB3K3, colony PCR).png

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


2) Double transformation

Plasmid BBa_K4195141_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.

A1a-Nu pSB3K3.png

Fig. 5 In vivo behavior of pirA_g1α_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 409
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 311
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 538
  • 1000
    COMPATIBLE WITH RFC[1000]