Producing molecular biology reagents without purification

We recently developed ‘cellular’ reagents–lyophilized bacteria overexpressing proteins of interest–that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Supplementary Information
Materials and methods S1          Phusion DNA polymerase (NEB) was used for PCR amplification of sequences for subsequent cloning.
Standard Gibson assembly techniques were used for cloning unless otherwise noted. Plasmid constructs for expressing Taq DNA polymerase, KlenTaq DNA polymerase, Bst-LF DNA polymerase, RTX DNA polymerase, MMLV RT, and Br512 DNA polymerase were described previously [1,2]. Briefly, Taq, KlenTaq, Bst-LF, and MMLV-RT are expressed from an anhydrotetracycline inducible pAtetO promoter in the ampicillin resistant pATetO 6xHis plasmid. RTX expression is driven from a T7 promoter-containing pET vector (Addgene; Plasmid #102787). Br512 is also expressed from a T7 promoter in an in-house E.
Construction of T4 DNA ligase, T7 DNA ligase, BsaI, and BsmAIM plasmids E. coli codon optimized T4 (UniProtKB -P00970) or T7 (UniProtKB -P00969) DNA ligase encoding gene blocks were inserted immediately downstream of the Factor X cleavage site in the pATetO 6xHis plasmid [1,3]. This is an in-house designed vector based on the pASK-IBA37plus vector (IBA GmbH) from which the multiple cloning site and Rop gene have been removed to improve plasmid copy number [3]. The plasmid also features a modified anhydrotetracycline controlled pATetO promoter with a single point mutation to make it unidirectional. Assembled plasmids were transformed into chemically competent Top10 E. coli and verified by Sanger sequencing at the Institute of Cellular and Molecular Biology Core DNA Sequencing Facility.
A constitutive expression plasmid for Geobacillus stearothermophilus methyltransferase, BsmAIM (UniProtKB -Q6UQ63), was built by inserting two E. coli codon optimized gBlocks comprising the BsmAIM coding sequence downstream of a constitutive pLac promoter in a plasmid bearing pBR322 origin of replication and a kanamycin resistance gene (S16 Fig). Assembled plasmid was transformed into chemically competent Top10 E. coli and verified by Sanger sequencing at the Institute of Cellular and Molecular Biology Core DNA Sequencing Facility. An inducible expression system for the Geobacillus stearothermophilus restriction endonuclease (BsaIR; UniProtKB -Q6SPF4) was constructed by inserting an E. coli codon optimized gBlock comprising the enzyme coding sequence beginning with a TTG start codon in an ampicillin resistant plasmid with a p15a origin of replication and a LacI expression cassette (S16 Fig). The BsaIR gene was flanked by an upstream LacO-regulated T7 promoter and two downstream T7 terminators to control gene expression.
Assembled plasmid was co-transformed along with the BsmAIM expression plasmid into chemically competent Top10 E. coli. Transformants were allowed to recover for 1 h in a 37 °C shaker prior to adding kanamycin at a concentration of 50 µg/mL and continuing recovery for another 1 h in the 37 °C shaker.
Subsequently, the transformants were plated on Luria Bertani agar plates containing 100 µg/mL ampicillin and 50 µg/mL kanamycin and allowed to grow overnight at 30 °C. Plasmids were isolated from individual transformants resistant to both ampicillin and kanamycin and verified by Sanger sequencing.
Construction of pOBL1 expression vector for the expression of OpenVent pOBL1 carries the gene encoding for an off-patent DNA polymerase from Pyrococcus sp. The corresponding protein is sold by NEB under the name DeepVent™ [4,5]. We have renamed the gene product OpenVent. The protein sequence was retrieved from the patent [4] and back translated. The OpenVent gene sequence was then optimized for expression in E. coli using OPTIMIZER [6] (guided random optimization) and domesticated for type II restriction enzymes. The optimized and domesticated sequence was then sent to be chemically synthesized (Twist Bioscience). The gene was cloned in a custom built backbone named pOBL, based on the open source minimal vector built by Staal and coworkers [7] and containing a Kanamycin resistance cassette. pOBL and the OpenVent gene were assembled to give the final pOBL1 using Klenow assembly [8]. pOBL1 map, gb and FASTA files are available in Supporting information (S11 Fig, and supplementary Genbank and FASTA files).

McFarland standards preparation
McFarland turbidity standards [9] were prepared as reported in [10,11]. Prepare a 1.0% (wt/vol) solution of anhydrous barium chloride (BaCl2, 0.048 M) and a 1.0% (vol/vol) solution of sulfuric acid (H2SO4, 0.18 M). The solutions are mixed according to the Table S2. Turbidity standards should be aliquoted in clear tubes of the same size and shape, the volume of the standard should be filling the tube to a height of around 2.5 cm. Place the tubes in a rack that allows the tubes to be viewed from the side (S1 Fig). Print or draw on paper a black and white pattern to place behind the standards. To compare your culture with the standards, you should aliquot some culture in the same tube used for the standards and compare the turbidity against the black and white pattern, diluting your culture if the turbidity is equal to 1 OD600. The sample tube should be placed next to the turbidity standards to be compared side by side. The standard that best represents the turbidity of the sample will be the one that obscures to the same extent the black and white pattern.

Protein Expression Analysis
For protein analysis, cells were harvested before induction and at the time of final collection. Cell samples equivalent to 1 mL of 1 OD600 were harvested in microcentrifuge tubes, centrifuged at 5000 x g for 5 minutes in a table top centrifuge and the supernatant was discarded. Cell pellets were resuspended in 1x Laemli buffer (0.33 M TRIS pH 6.8, 7% SDS, 10% glycerol, 5% 2-Mercaptoethanol), boiled at 95°C for 5 minutes and centrifuged at 13 000 x g for 2 minutes. The supernatant was transferred in a new microcentrifuge tube and the pellet discarded. 10 µL of each sample were loaded on a 12% Bis-Tris gel (SurePAGE, Genscript) and run as suggested by the manufacturer. The protein gel was stained using

Assembly instructions for a DIY incubator
Documentation including laser cutting design files and the Arduino firmware program can be found here https://github.com/FOSH-following-demand/Incubator. The incubator is made up of three main parts: 1. Temperature control unit made of an Arduino Uno that thermostatically controls the temperature using a DHT22 temperature sensor and a 75W incandescent light bulb. The temperature can be visualized by an LCD screen and adjusted through push-buttons. Materials: Arduino UNO, LCD screen, potentiometer, DHT22 temperature sensor, laser cut acrylic box, push buttons, wires as needed.

LAMP-OSD
All assay mixes were assembled and kept on ice prior to initiation of amplification. Human glyceraldehyde- In some reactions, reverse transcription was performed by directly adding 3 mm paper discs containing 2 x 10 7 evaporated MMLV RT cellular reagents. Following 1 h of reverse transcription at 42 °C, 5 µL of the resulting cDNA-containing solution was analyzed by TaqMan qPCR using Taq DNA polymerase. Briefly, Taq DNA polymerase TaqMan qPCR reactions were prepared in 25 µL volume containing a final concentration of 0.32 μM each of forward (Zika-4481_F) and reverse (Zika-4552c) primers [12].
Amplification was performed in 1X ThermoPol buffer (NEB) containing 0.4 mM dNTPs, 2.5 units of Taq DNA polymerase, and 80 nM TaqMan probe (Zika-4507c-FAM) [12]. For real-time signal measurement, the TaqMan qPCR reactions were placed in a LightCycler 96 real-time PCR machine and subjected to 10 min at 95 °C followed by 45 cycles of 15 sec at 95 °C (denaturation) and 30 sec at 55 °C (annealing and extension). Fluorescence signals were recorded in the FAM channel during the annealing/extension step in each cycle. All data were analyzed using the LightCycler 96 software.