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The MphR sensing module consists of the TetR-family allosteric transcription factor (aTF) MphR in the ROSALIND system. The ROSALIND system is based on the regulation of an in vitro transcription of a fluorescence activating RNA known as the 3WJdB (three-way junction dimeric Broccoli) aptamer. In the presence of a suitable binding dye (DFHBI-1T), transcription of the 3WJdB aptamer can be measured in real-time. Transcription can be regulated by including the MphR protein in the in vitro transcription reaction with a suitably encoded DNA transcription template that includes the MphR binding site (known as the operator sequence, mphO). Repression can be relieved through induction of MphR and resulting transcription can be monitored fluorescently (see References and Resources below for a more thorough description).
MphR induces transcription in the presence of macrolides - a class of antibiotics (e.g., azithromycin, erythromycin) that are widely used in human and animal health and are included on WHO’s list of essential medicines. The availability of biological sensors for macrolides will help the discovery and development of new macrolides and macrolide analogues, and enable the ability to monitor the presence of antibiotics in the environment, which give rise to antibiotic resistance. These compounds represent candidates for antifungal, antimicrobial, and antibacterial medicines that are important for human, animal, and planetary health. Such biological sensors can also enable scalable screening and process optimization in biomanufacturing.
Protein: MphR-6xHis
Sequence: MPRPKLKSDDEVLEAATVVLKRCGPIEFTLSGVAKEVGLSRAALIQRFTNRDTLLVRMMERGVEQVRHYLNAIPIGAGPQGLWEFLQVLVRSMNTRNDFSVNYLISWYELQVPELRTLAIQRNRAVVEGIRKRLPPGAPAAAELLLHSVIAGATMQWAVDPDGELADHVLAQIAAILCLMFPEHDDFQLLQPHAKLAAALEHHHHHH
Plasmid: pJBL715 (Backbone: pET28c, Addgene #140385 - UBMTA)
DNA-reporter: T7-mphO-3WJdB-T
Sequence:
gcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgactctagataatacgactcactataggagggaatataaccgacgtgactgttacatttaggtggcccacatactctgatgatccgagacggtcgggtccagatattcgtatctgtcgagtagagtgtgggctcggatcattcatggcaagagacggtcgggtccagatattcgtatctgtcgagtagagtgtgggctcttgccatgtgtatgtgggtagcataaccccttggggcctctaaacgggtcttgaggggttttttg
Plasmid: pUC19-T7-mphO-3WJdB-T ****(Backbone: pUC19, Addgene #140386- UBMTA)
<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment
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| Name | Product | Manufacturer | Part # | Price | Link | Storage Conditions |
|---|---|---|---|---|---|---|
| Spermidine | Spermidine, ≥99% (GC) | Sigma-Aldrich | S2626-5G | $176 | [link] | -25C to -15C |
| Tris-HCL | Trizma® hydrochloride | |||||
| anhydrous, free-flowing, Redi-Dri™, ≥99.0% | Sigma-Aldrich | RDD009-1KG | $268.60 | [link] | 4C to 30C | |
| MgCl2 | Magnesium chloride hexahydrate, BioXtra ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | [link] | 4C to 30C |
| DTT | ||||||
| NaCl | Sodium chloride, anhydrous, Redi-Dri™, free-flowing, ACS reagent, ≥99% | Sigma-Aldrich | 746398-500G | $79.70 | [link] | 4C to 30C |
| rATP | Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial | Sigma-Aldrich | A7699-1G | $125.00 | [link] | -25C to -15C |
| rGTP | Guanosine 5′-triphosphate sodium salt hydrate, ≥ 95% (HPLC), powder | Sigma-Aldrich | G8877-100MG | $155 | [link] | -25C to -15C |
| rCTP | Cytidine 5′-triphosphate disodium salt, ≥95% | Sigma-Aldrich | C1506-100MG | $102.00 | [link] | -25C to -15C |
| rUTP | Uridine 5′-triphosphate trisodium salt hydrate, Type IV, ≥93.0% (HPLC) | Sigma-Aldrich | U6750-100MG | $58.20 | [link] | -25C to -15C |
| 0.3 U TIPP | Thermostable Inorganic Pyrophosphatase | New England Biolabas | M0296S | $82.00 | [link] | -25C to -15C |
| DFHBI-1T (dye) | DFHBI 1T | Tocris Bioscience | 5610 | $346.00 | [link] | -25C to -15C |
| MphR (plasmid) | pJBL715 | AddGene | 140385 | $85.00 | [link] | -25C to -15C |
| T7-mphO-3WJdb-T (plasmid) | pJBL716 | AddGene | 140386 | $85.00 | [link] | -25C to -15C |
| Forward primer | GCGGATAACAATTTCACACAGGAAACAGC | -25C to -15C | ||||
| Reverse primer | CAAAAAACCCCTCAAGACCCG | -25C to -15C | ||||
| PCR Kit | Phusion® High-Fidelity PCR Kit | New England Biolabs | E0553S | $84.00 | [link] | -25C to -15C |
| T7 RNAP* | T7 RNA Polymerase | New England Biolabs | M0251S | $74.00 | [link] | -25C to -15C |
pT7-lacO-UTR1-T7RNAP-tT7hyb6Purified MphR from the plasmid pJBL715 is described in Jung et al (DOI: 10.1038/s41587-020-0571-7) and is likely compatible with the following guides: