RNAi by bacterial feeding was performed as previously described (Timmons and Fire, 1998 ). The C. elegans strain TLG281 rrf-3(pk1426) II; texIs100 IV; dbl-1(nk3) V was used for this experiment (Beifuss and Gumienny, 2012 (link)). Bacteria from the Vidal and Ahringer cDNA libraries (Open Biosystems and Source BioScience, respectively) were used (Kamath and Ahringer, 2003 (link); Rual et al., 2004 (link)). C06C3.5, a predicted pseudogene, was used as the negative control. bli-4(RNAi) was used as the positive control (Thacker et al., 1995 (link)). Bacteria from single colonies were grown overnight in Luria–Bertani broth (Sigma, St. Louis, MO) containing 50 µg/ml carbenicillin (Gold Biotechnology, St. Louis, MO) and induced to express dsRNA using 1 µg/ml isopropyl β-d -1-thiogalactopyranoside (IPTG) for 4 h. After induction, bacterial broth was plated on nematode growth medium containing 50 µg/ml carbenicillin and 1 µg/ml IPTG and dried. Embryos staged by bleaching were transferred to these plates and grown to the young adult stage for observation as previously described (Kamath and Ahringer, 2003 (link); Rual et al., 2004 (link); Beifuss and Gumienny, 2012 (link)).
Carbenicillin
Manufactured by GoldBio
Sourced in United States
Carbenicillin is a semi-synthetic penicillin antibiotic used in laboratory settings. It inhibits the synthesis of bacterial cell walls, preventing the growth and replication of susceptible bacteria.
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Lab products found in correlation
Kanamycin, by GoldBio (7 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (7 mentions)
Chloramphenicol, by GoldBio (5 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
L arabinose, by GoldBio (2 mentions)
M toluic acid, by Merck Group (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Kanamycin, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Dextrose, by Thermo Fisher Scientific (1 mentions)
Ammonium sulfate, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Hygromycin b, by Santa Cruz Biotechnology (1 mentions)
Luria bertani media, by Thermo Fisher Scientific (1 mentions)
Naringenin, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
E coli dh5α, by Zymo Research (1 mentions)
Top10 competent cell, by Thermo Fisher Scientific (1 mentions)
24 protocols using carbenicillin
1
RNAi by Bacterial Feeding in C. elegans
2
Ahringer Library RNAi Protocol
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Feeding-RNAi constructs (expressed in HT115 bacteria) were obtained from the Ahringer library60 (link). RNAi-feeding bacteria were induced in 1 mM IPTG in liquid 2xYT media plus 100 µg ml−1 carbenicillin (Gold Biotechnology) for 5–7 h with the exception of lin-23, let-363, and atfs-1 RNAi bacteria, which was induced by plating overnight cultures of the lin-23 RNAi bacteria on 1 mM isopropyl β-D -1-thiogalactopyranoside (IPTG) plus 100 µg ml−1 carbenicillin plates. Double RNAi treatments were performed by combining RNAi bacteria at a 1:1 ratio (unless otherwise indicated) using OD600 optical densities to quantify the bacteria prior to seeding plates. Unless otherwise indicated, eggs were placed on the RNAi plates and adults from the next generation were analyzed.
3
Engineered E. coli and Yeast Strains for Metabolite Production
4
Cloning and Validation of Truncated CENP-A
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Genomic DNA from wild-type fission yeast cells was used as a template for the PCR amplification of N-terminally truncated CENP-A gene copies using Phusion polymerase (Thermo Fisher Scientific, Waltham, MA, USA), whereas plasmids pFA6a-6xGLY-3xFLAG-kanMX6 [49 (link)] and pFA6a-GFP(S65T)-kanMX6 [50 (link)] were used as templates [50 (link)] for FLAG and GFP protein tags, respectively. All PCR products were ethanol-precipitated and subjected to gel purification using a DNA gel extraction kit (Qiagen) according to manufacturer’s instructions. Both plasmid pREP41 and purified DNA (PCR products) were treated with restriction enzymes (New England Biolabs (NEB), Ipswich, MA, USA) and subjected to agarose gel purification, as described previously [28 (link)]. Digested gene inserts were ligated to pREP41 plasmids with T4 ligase (NEB), and transformed into TOP10 competent cells (Thermo-Fisher Scientific), before plating onto LB + Car agar plates (Luria–Bertani medium with 100 μg/mL carbenicillin (GoldBio, St. Louis, MO, USA). Positive transformants were then expanded by plasmid miniprep (Qiagen, Hilden, Germany) and extracted plasmids were sequenced using the Sanger method for confirmation of the correct gene sequences, frame, and orientation.
5
Plasmid Construction for VEGAS Evolution
6
Antibiotic and Induction Compound Preparation
7
Cloning and Transforming E. coli
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All constructs were designed with a CMV promoter using Gibson or restriction cloning (see list of constructs) and transformed into chemically competent DH5ɑ E. coli cells (Invitrogen). Transformed E. coli were plated on either 50 μg/mL kanamycin (Teknova, cat #K2151) or 100 μg/mL carbenicillin (Gold Biotechnology, cat #C-103–5) LB agar (Teknova, cat #L9115) plates overnight at 37 °C. Single colonies were picked and grown overnight in 5 mL LB Broth (Alfa Aesar, cat #AAJ75854A1) with kanamycin or carbenicillin at 37 °C with shaking at 220 rpm. The following day, shaking cultures were mini-prepped (Zymo, cat #D4212) and sent for Sanger Sequencing (Azenta). Sequence-verified plasmids were then midi-prepped (Zymo, cat #D4201) for use in transfection.
8
Engineered E. coli Strains for Recombineering
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This work used the following E. coli strains: NEB 5-alpha (NEB, C2987; not authenticated), BL21-AI (Thermo Fisher, C607003; not authenticated), bMS.346 and bSLS.114. bMS.346 (used previously20 (link)) was generated from E. coli MG1655 by inactivating the exoI and recJ genes with early stop codons. bSLS.114 (used previously29 (link)) was generated from BL21-AI by deleting the retron Eco1 locus by lambda Red recombinase-mediated insertion of an FRT-flanked chloramphenicol resistance cassette, which was subsequently excised using FLP recombinase42 (link). bCF.5 was generated from bSLS.114, also using the lambda Red system. A 12.1kb region was deleted that contains a partial lambda*B prophage that is native to BL21-AI cells within the attB site, where temperate lambda integrates into the bacterial genome43 (link).
Phage retron recombineering cultures were grown in LB, shaking at 37 °C with appropriate inducers and antibiotics. Inducers and antibiotics were used at the following working concentrations: 2 mg/ml L-arabinose (GoldBio, A-300), 1 mM IPTG (GoldBio, I2481C), 1mM m-toluic acid (Sigma-Aldrich, 202-723-9), 35 μg/ml kanamycin (GoldBio, K-120), 100 μg/ml carbenicillin (GoldBio, C-103) and 25 μg/ml chloramphenicol (GoldBio, C-105; used at 10 μg/ml for selection during bacterial recombineering for strain generation).
Phage retron recombineering cultures were grown in LB, shaking at 37 °C with appropriate inducers and antibiotics. Inducers and antibiotics were used at the following working concentrations: 2 mg/ml L-arabinose (GoldBio, A-300), 1 mM IPTG (GoldBio, I2481C), 1mM m-toluic acid (Sigma-Aldrich, 202-723-9), 35 μg/ml kanamycin (GoldBio, K-120), 100 μg/ml carbenicillin (GoldBio, C-103) and 25 μg/ml chloramphenicol (GoldBio, C-105; used at 10 μg/ml for selection during bacterial recombineering for strain generation).
9
Construction and Transformation of GmFT7 Overexpression
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For gene expression construct, the coding sequence of Glyma02g07650 (GmFT7) was amplified (with XhoI and BamHI restriction enzyme sites on 5′ and 3′ end, respectively) and ligated to pGEM-T Easy vector (Promega). The vector was digested, and gene fragment was ligated to pRT101 at Xho I and Bam HI sites, to obtain 35S promoter and polyA terminator. Then the 35S:GmFT7:polyA cassette was excised using Hind III and ligated to pUQC10255 expression vector (Supplementary Fig. S4 ). Transformation of Arabidopsis Col (WT) and ft-10 mutant was performed using the floral dip method53 (link).
Bragg cultivar was used for soybean transformation following the protocol by Li et al.40 (link) with modifications; selection on SIM medium containing 4 mg/L glufosinate (Sigma, USA) was started during the second subculture step SIM and continued till SEM medium. 100 mg/L Cefotaxime (GoldBio, USA), 50 mg/L Vancomycin (GoldBio, USA) and 50 mg/L Ticarcillin (GoldBio, USA) were used instead of carbenicillin on SIM, SEM and RM medium. Five independent experiments were conducted using an average of 110 seed explants per experiment and recovered five transgenic shoots. However, one-line produced seeds. The transgenic seeds were multiplied and selected for the Bar gene. Three T2 lines were used for analysis.
Bragg cultivar was used for soybean transformation following the protocol by Li et al.40 (link) with modifications; selection on SIM medium containing 4 mg/L glufosinate (Sigma, USA) was started during the second subculture step SIM and continued till SEM medium. 100 mg/L Cefotaxime (GoldBio, USA), 50 mg/L Vancomycin (GoldBio, USA) and 50 mg/L Ticarcillin (GoldBio, USA) were used instead of carbenicillin on SIM, SEM and RM medium. Five independent experiments were conducted using an average of 110 seed explants per experiment and recovered five transgenic shoots. However, one-line produced seeds. The transgenic seeds were multiplied and selected for the Bar gene. Three T2 lines were used for analysis.
10
Culturing HEK293T and E. coli Rosetta Cells
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