Homebrew reagents for low cost RT-LAMP

Matute, Tamara; Núñez, Isaac; Rivera, Maira; Reyes, Juan G.; Blázquez‐Sánchez, Paula; Arce, Aníbal; Brown, Andrew J.; Gandini, Chiara; Molloy, Jenny; Ca, Ramirez-Sarmiento; Federici, Fernán

doi:10.1101/2021.05.08.21256891

Cited by 5 publications

(3 citation statements)

References 42 publications

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“…While the RNA amplification step still requires commercial inputs, such reactions can also be made inhouse (Compton, 1991;Aufdembrink et al, 2020). Future work will focus on the in-house preparation of NASBA from locally purified, and IP-free RNAseH, T7 RNAP, pyrophosphatase, and reverse transcriptase enzymes (Aufdembrink et al, 2020;Reclone, 2020;Matute et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

Arce

Chavez

Gandini

et al. 2021

Front. Bioeng. Biotechnol.

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Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

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Section: Discussionmentioning

confidence: 99%

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

Arce

Chavez

Gandini

et al. 2021

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Compton, 1991;Lauren M. Aufdembrink et al, 2020). Future work will focus on the in-house preparation of NASBA from locally purified, and IP-free RNAseH, T7 RNAP, pyrophosphatase, and reverse transcriptase enzymes (Lauren M. Aufdembrink et al, 2020;Reclone, 2020;Matute et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

Arce

Chavez

Gandini

et al. 2021

Preprint

Self Cite

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Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toeholds sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

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“…In earlier work, we have reported the local production of a self-indicating mCherry fused BST LF with a silica binding tag for immobilization, that was produced locally in Ghana for a loop-mediated isothermal amplification (LAMP) for malaria diagnosis of clinical samples (Seevaratnam et al, 2022). The COVID-19 pandemic has also shown that the enzymes for RT-LAMP can be successfully produced in-house locally without the need for import of these vital reagents (Matute et al, 2021) and Mote et al have produced a PCR master mix with reagents produced in-house (Mote et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic synthesis of 2′‐deoxynucleotide 5′‐triphosphates from bacterial genomic DNA: Proof of principle

Bird

Molloy

Hall

2023

Biotech & Bioengineering

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2′-deoxynucleoside 5′-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR).These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in lowand middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solidstate bioreactor for on-demand dNTP production.

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Homebrew reagents for low cost RT-LAMP

Cited by 5 publications

References 42 publications

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

Biocatalytic synthesis of 2′‐deoxynucleotide 5′‐triphosphates from bacterial genomic DNA: Proof of principle

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