MphR Sensing Module (Stemloop)

Overview

Description of Module

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.

Component specifications

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)

Materials and Equipment

* Alternatively, the T7 RNAP is available within the DNA Distribution as the component pT7-lacO-UTR1-T7RNAP-tT7hyb6

Usage

Preparation of purified MphR

Purified 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:

• Grow and Induce Expression Strains
• Purify Proteins by Ni-His Gravity Column

Preparation of linear DNA templates

The DNA templates used in the ROSALIND system are purified linear PCR amplicons from the plasmid pJBL716 using the forward and reverse primers as described in Jung et al (DOI: 10.1038/s41587-020-0571-7).

It is recommended to use the Phusion PCR kit with the following thermocycle parameters: 98 C for 3 min—(98 C for 30 s, 71 C for 30 s, 72 C for 15 s) 25 cycles—72 C for 10 min—hold at 12 C. Purify the product via spin column into MQ water.

Characterize the size and purity of the product by running ~50–100 ng of the purified DNA template on a 2% TAE agarose gel to confirm the size and purity of the product.

Stocks solutions and buffers

Stock solutions

1) Input Template, Dye, aTF and ligand stock concentrations. Add desired amounts per reaction for template, Dye and aTF. (Ligand is added below in step 6).

10x ROSALIND buffer

TRIS buffered nucleotides

100 mM rNTPs are buffered in a 11.4 mM solution of TRIS-HCL (pH 7.5) and store at -25C to -15C.

Transcription Mastermix

2) Prepare a mastermix by mixing the following components at room temperature.

3) For each desired reaction, aliquot 15 µL of incubated mastermix in each reaction tube.

4) Incubate the aliquoted mastermix at 37° C for 15 min to allow MphR:DNA equillibration.

5) Meanwhile prepare 1:10 dilution of T7 RNAP stock (stock concentration: 1 µg/µL).

6) For each desired reaction, add ligand, diluted T7 RNAP, and remaining water according to the table below. (Modify the table according to your experiment design.) Add these components immediately before transferring to a plate reader for measurement.

Performance Data

The performance of the MphR macrolides sensor is characterized using erythromycin - a common antibiotic used to treat a variety of bacterial infections. Here, 12.5 uM of erythromycin (Sigma Aldrich, cat no. E5389) dissolved in ethanol is added to a ROSALIND reaction containing 208 uM MphR. The fluorescence response is expressed in units of micromolar equivalent fluorescein (MEF) using a NIST traceable fluorescein standard (Invitrogen, cat no. F36915)

MphR represses transcription until 2.5 uM erythromycin is introduced with a response saturation occurring at approximately 10 uM erythromycin. The kinetic time series data displayed for 12.5 uM erythromycin shows a response of 0.5 MEF within 1 hour.

The complete study wherein the behavior of MphR is characterized using the ROSALIND system can be found Jung et al (DOI: 10.1038/s41587-020-0571-7). There, additional macrolides - azithromycin, clarithromycin, and roxithromycin - were also characterized with slightly higher dose response thresholds to obtain saturation. Despite these different saturation thresholds, visible amounts of fluorescence could be observed within similar time frames. The author’s of that study note that the “broad range of MphR specificity is expected based on its known promiscuity, and is probably a common feature of xenobiotic-sensing aTFs.”

Resources and References

• Other Protocols
• Jung, J. K., Alam, K. K., & Lucks, J. B. (2022). ROSALIND: Rapid Detection of Chemical Contaminants with In Vitro Transcription Factor-Based Biosensors. In Cell-Free Gene Expression (pp. 325–342) [link]
• Papers
• Alam, K. K., Tawiah, K. D., Lichte, M. F., Porciani, D. & Burke, D. H. A Fluorescent Split Aptamer for Visualizing RNA–RNA Assembly In Vivo. ACS Synthetic Biology vol. 6 1710–1721 (2017) [link]
• Jung, J. K. et al. Cell-free biosensors for rapid detection of water contaminants. Nature Biotechnology vol. 38 1451–1459 (2020) [link]
• Kasey, C. M., Zerrad, M., Li, Y., Cropp, T. A. & Williams, G. J. Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology. ACS Synthetic Biology vol. 7 227–239 (2017) [link]
• Resources
• N/A

Credits

• Developers
• Stemloop
• Testers
• Untested