Construction of recombinant plasmids
The pET17b-GST was constructed by using Glutathione S-Transferase encoding gene (Genbank accession number M97937.1). This gene was inserted into NdeI and SacI restriction sites of pET17b plasmid. The pET28a-MrNV was synthesized by General Biosystems. To construct the plasmid, a gene encoding an MrNV capsid protein (Genbank accession number EU150129.1) with a C-terminus six-histidine was inserted between NcoI and XhoI restriction sites of an empty pET28a plasmid.
Preparation of donor bacteriophage P1
To prepare P1 lysate from a donor strain, HT115(DE3) was grown overnight with shaking at 37 °C in 5 ml lysogeny broth (LB) with 12.5 μg/ml tetracycline. The overnight culture was diluted 100-fold with 4 ml antibiotic-free LB containing 5 mM CaCl2, 10 mM MgCl2 and 5 mM glucose. The culture was incubated at 37 °C for 1 h with shaking at 250 rpm. Then, 100 μl of E. coli bacteriophage P1 (ATCC® 25404B1™) was added to the donor bacteria culture and incubated at 37 °C with aeration overnight. Afterwards, 50 μl of chloroform was added and mixed by vortex for 30 s. The mixture was centrifuged at 13,200×g for 1 min, before collecting the P1 phage-containing supernatant into a fresh tube.
P1 phage transduction
To infect the recipient strain BL21(DE3) with the P1 lysate, BL21(DE3) was grown overnight at 37 °C in 5 ml antibiotic-free LB broth. Then, 2 ml of the culture was centrifuged at 4500×g. The BL21(DE3)-containing pellet was resuspended in 250 μl of LB broth containing 10 mM MgSO4 and 5 mM CaCl2. Next, 100 μl of the P1 lysate from the previous step was mixed with 100 μl of the resuspended recipient cells and incubated at 37 °C for 30 min on a bench. To chelate calcium and minimize secondary infection of P1 phage , 1 ml of LB media containing 0.2 M citrate was added. The mixture was further incubated with shaking at 250 rpm for 1 h at 37 °C, before being centrifuged at 4500×g for 1 min. The resulting sediment was resuspended in 100 μl of a fresh LB media containing 0.2 M citrate. The suspension was equally divided and plated on LB agar plates with and without 12.5 μg/ml tetracycline.
Selection and verification of desirable mutants
Colonies grown on the tetracycline-supplemented plates were selected for further chromosomal DNA extraction by Qiagen genomic DNA extraction kit. The resulting DNA was used as a template DNA to amplify the rnc14 gene by polymerase chain reaction (PCR) using an rnc-KO forward primer (5′-AAA CTG CAG CGA AGC AGT TA-3′) and an rnc-KO reverse primer (5′-TCA TTC CAG CTC CAG TTT TT-3′). The primer sequences were designed based on the E. coli str. K-12 substr. MG1655 genome (GenBank accession number NC_000913.3). The forward primer annealed upstream from the start codon between nucleotide positions 2,704,131 and 2,704,150. The reverse primer annealed adjacent to the stop codon between the nucleotide positions 2,703,383 and 2,703,402. A 50 μl PCR reaction mixture contained a final concentration of 1x reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM of each forward and reverse primers, 100 ng extracted DNA, and 1 unit of Taq DNA polymerase (Invitrogen). Thermocycling conditions were at 94 °C for 3 min; followed by 30 cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 1 min; then a final extension at 72 °C for 10 min. To identify a mutation, PCR amplicons from wild type and mutant bacteria were purified and sent for sequencing by using the rnc-KO forward and reverse primers (Macrogen).
Determination of the gene knockout effect on bacterial growth
To test the effect of rnc14 knockout on the growth of the novel E. coli strain, the pET28a-GW182 plasmid  was transformed into BL21(DE3), HT115(DE3) and DualX-B15(DE3) for simulation of the actual expression condition of double-stranded RNA (dsRNA). The transformants were grown in LB media containing 50 μg/ml kanamycin (for all strains) and 12.5 μg/ml tetracycline (for HT115(DE3) and DualX-B15(DE3)). The overnight cultures were diluted in 15 ml of fresh LB media supplemented with the corresponding antibiotics and OD600 was adjusted to 0.05. Then, they were incubated at 37 °C with shaking at 250 rpm. The growth of bacterial strains was observed by measuring OD600 every 20 min for 160 min by NanoDrop One Spectrophotometer (Thermo Fisher Scientific). Experiments were performed in triplicate to obtain average OD600 at each time point.
Expression of dsRNA
To express dsRNA, the pET28a-GW182 plasmid  was transformed into BL21(DE3), HT115(DE3) and DualX-B15(DE3). The transformants were grown in LB media containing 50 μg/ml kanamycin (for all strains) and 12.5 μg/ml tetracycline (for the HT115(DE3) and DualX-B15(DE3)) overnight. The cultures were diluted 100-fold in 25 ml of antibiotic-supplemented fresh LB media and OD600 was adjusted to 0.1. Then, they were incubated at 37 °C with shaking at 250 rpm for 2 h until OD600 reached 0.4. To induce the expression of dsRNA, 1 mM IPTG was added to the cultures for 3 h at 37 °C before the final OD600 was measured by a spectrophotometer. The cultures were harvested by centrifugation at 4500×g for 5 min.
Purification of dsRNA
To extract dsRNA by the ethanol extraction method , 50 mg of the resulting wet bacterial pellet was resuspended with 1 ml of 75% ethanol in 1x PBS (Sigma-Aldrich) and incubated at − 20 °C overnight. The mixture was centrifuged at 6000×g for 5 min at 4 °C, resuspended with 0.3 ml of 150 mM NaCl, and incubated at room temperature for 1 h. To isolate the soluble fraction, the suspension was centrifuged at 8000×g for 10 min at 4 °C and dsRNA-containing supernatant was collected.
To compare the yields of dsRNA-GW182 among different E. coli strains, the bacteria culture was done in triplicate. To visualize the extracted RNA, RNA isolates were analyzed by 1.5% agarose gel electrophoresis. Analysis of RNA band intensity was performed using ImageJ. The concentration of dsRNA was calculated by comparing the band intensity with that of the corresponding band in a 2-log DNA marker (New England Biolabs).
RNase treatment of the purified RNA
To verify that the RNA expressed in the engineered strain was in the form of dsRNA, the resulting RNA was divided into three equal parts. One fraction was mixed with 1x RNase A buffer and distilled water for the untreated condition. The other fraction was treated with 0.01 μg/μl RNase A enzyme (New England Biolabs) in 10 μl reaction mixture containing 1x RNase A buffer. The remaining fraction was treated with 1 unit of RNase III enzyme (New England Biolabs) in 10 μl reaction mixture containing 1x RNase III buffer and 1x MnCl2. The reactions were incubated at 37 °C for 5 min before being analyzed by 1.5% agarose gel electrophoresis alongside a 2-log DNA ladder staining with ethidium bromide and then visualized under UV lamp of Gel Documentation (Bio-Rad).
Expression of GST protein
To compare protein expression capacity of the three E. coli strains, the pET17b-GST plasmid was transformed into HT115(DE3), BL21(DE3) and DualX-B15(DE3). The bacteria were grown in LB medium containing 100 μg/ml ampicillin (for all strains) and 12.5 μg/ml tetracycline (for HT115(DE3) and DualX-B15(DE3)) at 37 °C with shaking at 250 rpm until OD600 reached 0.6. Protein expression was induced with IPTG at a final concentration of 1 mM and cells were then incubated at 25, 30 and 37 °C overnight. After centrifugation at 4500×g for 5 min, 50 mg of wet bacterial pellet was resuspended with 1 ml of lysis buffer pH 8 (50 mM NaH2PO4, 150 mM NaCl and 10 mM imidazole). Sonication of the cell suspension was performed at 40% amplitude with 2 cycles of 10 short bursts of 5 s followed by 5 s intervals for cooling (Sonics VCX 750). The lysates were then fractionated by centrifugation at 12,000×g. The total protein concentration was measured by using the Bradford reagent (Bio-Rad). The fractions were analyzed on 12.5% SDS-PAGE and Western blot using a mouse anti-GST antibody with a pre-stained protein ladder (Thermo Scientific).
To compare the GST yields among three E. coli strains, cell lysates were analyzed by SDS-PAGE to detect protein band. Analysis of protein band intensity was performed using ImageJ. The concentration of GST was calculated by comparing the band intensity with that of the corresponding band in 4 μl pre-stained protein ladder (Thermo Scientific).
Co-expression of dsRNA and CP
To co-express dsRNA-VP28 and MrNV-CP in the three bacterial strains, HT115(DE3), BL21(DE3) and DualX-B15(DE3) were co-transformed with the pGEM-VP28  and pET28a-MrNV plasmids for dsRNA and CP expression, respectively. The expression temperatures were varied at 25, 30 and 37 °C. The optimized condition of co-expression was found to be at 25 °C overnight because, while dsRNA-VP28 could be expressed at all of the above temperature (Fig. S1), the best expression temperature of MrNV-CP was 25 °C as previously reported in Jariyapong et. al, 2015 . The bacterial cells were grown in LB medium containing 50 μg/ml ampicillin, 25 μg/ml kanamycin (for all strains) and 6.25 μg/ml tetracycline (for HT115(DE3) and DualX-B15(DE3)) at 37 °C with shaking at 250 rpm until OD600 reached 0.6. Co-expression of dsRNA and CP was induced with IPTG at a final concentration of 1 mM and cells were then incubated at 25 °C overnight before harvest by centrifugation at 4500×g for 5 min.
The cell wet weight used for dsRNA and CP extraction was 100 mg. Half of the bacteria culture was used for protein analysis, while the other half of culture was subjected to dsRNA purification and RNase treatment (see above). To analyze protein expression, the total amount of 7.5 μg total protein determined by Bradford assay was loaded in each lane of gel electrophoresis. The MrNV-CP expression was confirmed by Western blot. For dsRNA-VP28 analysis, the total amount of 2 μg RNA, as determined by a spectrophotometer, was loaded in agarose gel electrophoresis.
After SDS-PAGE, proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane by Pierce Power Blotter (Thermo Scientific) for Western blot according to the manufacturer’s protocol. The membranes were immersed in 5% skim milk in PBS to block non-specific antibody binding at room temperature for 1 h with constant shaking. For GST protein detection, the membranes were immersed in a 1:1000 dilution of a mouse anti-GST antibody (Bio-Rad). For MrNV-CP expression, the membranes were immersed in a 1:100 dilution of an anti-MrNV-CP monoclonal antibody . The membranes were washed three times for 5 min each with PBS containing 0.05% Tween-20 at room temperature with shaking. A secondary antibody (horseradish peroxidase conjugated goat anti-mouse IgG) was added at 1:2500 dilution and incubated for 1 h at room temperature with agitation. The membranes were washed three times with PBS containing 0.05% Tween-20. The antigen-antibody reactivity was detected by chemiluminescent method using Clarity Western ECL substrate (Bio-Rad).