Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria
Abstract
:1. Introduction
2. Materials and Methods
2.1. ALP* and NLuc Constructs
2.2. Expression and Purification of ALP* and Nluc
2.3. Enzyme Activity of Purified ALP* and NLuc
2.4. Construction of Reporter Phages ALP* and Nluc
2.5. Quantitation of Reporter Protein through the Phage Assay
2.6. Limit of Detection of Reporter Phage for E. coli
3. Results
3.1. Expression and Purification of ALP* and NLuc
3.2. Enzyme Activity of Purified ALP* and NLuc
3.3. Construction of Reporter Phages ALP* and NLuc
3.4. Quantitation of Reporter Protein through the Phage Assay
3.5. Limit of Detection of Reporter Phage for E. coli
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Appendix A
References
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NLuc | Restriction Site | RBS | STOP | START | Primer |
---|---|---|---|---|---|
Forward Primer | EcoRI GAATTC | AGGAGG | TGATGA | ATG | 5′CGGGAATTCATGATGAGCGAGGAGGGCGATGATGGTCTTCACACTC 3′ |
Reverse Primer | HindIII AAGCTT | TGA | 5′CGGAAGCTTCTAGTGGTGGTGGTGGTGGTGCGCCAG 3′ | ||
ALP* | Restriction Site | RBS | STOP | START | Primer |
Forward Primer | EcoRI GAATTC | AGGAGG | TGATGA | ATG | 5′CGGCGGAATTCATGATGAGCGAGGAGGCGCATGCGTACACCGGAAATGCCG 3′ |
Reverse Primer | HindIII AAGCTT | TGA | 5′CGGAAGCTTTCAGTGGTGGTGGTGGTGGTGCTCGAGTTTCAG 3′ |
Ref. | Type of Phage | E. coli Strain | Reporter Modification | Phage Available for Infection | Pre/Enrich-ment of E. coli | Incubation Time-Volume | Reporter Concentration | Reaction Volume | Detection Matrix | Detection Method | Definition of the Detection Limit- Total Assay Time |
---|---|---|---|---|---|---|---|---|---|---|---|
[4] | T7_ALP* | BL21 derivative | CBM(Cex) | 1:10 CFU:PFU | 4 h | 2 h- 100 mL | Magnetic cellulose particles | 60 µL | LB | Abs | 3LOQ: <10 CFU/100 mL-8 h |
[7] | T7_ALP* | BL21 | His-tag | 107 PFU/mL | - | 16 h- | - | 50 µL | LB | Abs | 7LOD: ∼1 × 105 CFU/mL |
[10] | T7_NLuc | BL21 | CBM2a/pelB | 2 × 109 PFU | 8–12 h | 1.5 h- 2 mL | Cellulose filter | ~300 µL | Drinking water | Abs/LU | Matched EPA results |
[19] | T7_NLuc | BL21 | CBM/pelB | 106 PFU | 1 h | 2 h- 1 mL | - | 200 µL | LB | LU | 6Detectable signal: 5 × 102 CFU/mL-2 h |
[43] | T7_NLuc | BL21 | CBM/pelB | 109 PFU | 1 h (pre-) | 1.5 h-126 mL | Micro-crystalline cellulose | 100 µL | Drinking water | LU | 4LOD: <10 CFU/mL-3 h |
[44] | T7_NLuc | BL21 | CBM | 1.25 × 108 PFU | - | 3 h- 0.25 mL | Nitro-cellulose membrane | 75 µL | Drinking water | LU | 1LOD: 4.1 CFU-5.5 h |
[45] | T7_NLuc | ECOR13 | CBM | 107 PFU/mL | 3 h (pre-) | 1.5 h- | Cellulose filter | - | Drinking water | LU | 2LOD: <20 CFU-5 h |
[46] | T4_NLuc | ECOR13 | CBM | 107-6 PFU | 3 h (pre-) | 3 h- 0.5 mL | Cellulose filter | 50 µL | Drinking water | LU | 5LOD: <10 CFU/100 mL-7 h |
[47] | K1E_NLuc | K1 | - | 106 PFU | - | 1 h- 0.1 mL | - | - | LB | LU | 8LLOD: 8 CFU/well-1 h |
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Wijeratne, S.; Bakshi, A.; Talbert, J. Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria. Bioengineering 2022, 9, 479. https://doi.org/10.3390/bioengineering9090479
Wijeratne S, Bakshi A, Talbert J. Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria. Bioengineering. 2022; 9(9):479. https://doi.org/10.3390/bioengineering9090479
Chicago/Turabian StyleWijeratne, Shalini, Arindam Bakshi, and Joey Talbert. 2022. "Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria" Bioengineering 9, no. 9: 479. https://doi.org/10.3390/bioengineering9090479