Experiments

Plasmid extraction

E. coli DH5α or DH5α -Z1 cells harbouring the pLO_SNAP, pET28, pSynSense2.5000 and pBAD plasmids were grown overnight in 5mL Luria-Bertani (LB) broth medium with 100 µg/mL ampicillin, 50 µg/mL kanamycin, 34 µg/mL and 50 µg/mL kanamycin respectively. The tubes were centrifuged for 10 minutes at 4000 g and 20°C, the supernatant was decanted. The plasmids are extracted from the cell pellets using the E.N.Z.A.® Plasmid DNA Mini Kit I (Omega Bio-tek, Georgia, USA) following the manufacturer’s instructions. The dsDNA concentration and quality were measured using the SPECTROstarNano (BGM Labtech, Ortenberg, Germany).

Primer designs and polymerase chain reaction (PCR)

Primers were designed using SnapGene 5.08 ® software (from Insightful Science; available at snapgene.com) and checked for secondary structures and/or primer dimers using OligoAnalyzer^TM Tool (IDT, Iowa, USA). The annealing sequence is 15-25 bp long and the melting temperature (T_m) of corresponding forward and reverse primers differed no more than 3°C. Overhangs are added to each primer depending on the intended cloning strategy. For type IIS assembly, the recognition site for BsaI-HF®-v2 was added. For Gibson assembly, the overhangs of the primer used to amplify the insert are homologous to the overhang of the primer used to amplify the vector. Primers were ordered from Integrated DNA Technologies (IDT, Iowa, USA). An overview of the PCR reaction mixtures, the thermocycler conditions and the extension time calculation can be found in tables 1, 2 and 3. 5X MyTaq reaction was obtained from Meridian Bioscience (Ohio, USA). Q5 reaction buffer, Q5® High-Fidelity polymerase, Taq polymerase and Q5 High GC enhancer were obtained from NEB (New England Biolabs, Massachusetts, USA). The Doppio thermocycler from VWR (Pennsylvania, USA) was used to perform the reactions.

Table 1: Primers designed for constructing the plasmids of the Eat-Shoot parts. gBlocks were ordered from IDT and TwistBioscience.

Table 2: Primers designed for constructing the plasmids of the Leave part. gBlocks were ordered from IDT and TwistBioscience.

Table 3: PCR protocols: reaction mixture (50 µL)

Table 4: PCR protocols: thermocycler conditions

Table 5: Calculation of the extension time: Q5 HF polymerase PCR reaction

Table 6: Calculation of the extension time: Taq polymerase PCR reaction

To analyze whether the desired fragment length was amplified, an agarose gel electrophoresis is carried out. Depending on the expected length, a 1.0% (400 bp – 10kb) or a 2.0% (100 bp – 2 kb) agarose gel is prepared according to the manufacturer’s instructions. 0.50 g (1.0% gel) or 1.00 g (2.0% gel) UltraPure™ Agarose (Invitrogen) was added to 50 mL LiAc (Lithium acetate) buffer and microwaved for 1-2 minutes. 4µL SYBR Safe DNA Gel Stain (Invitrogen) was added to the cleared agarose solution which was casted and left for 10 minutes to polymerize. The 1 kb DNA ladder (all obtained from NEB) was always used as a reference. Samples were prepared by adding 2 µL of 6X Gel Loading Dye Purple (NEB) to 10 µL of the PCR reaction mixture.

DNA extraction

PCR products were extracted from the PCR reaction mix with the MicroElute® Cycle – Pure Kit (Omega Bio-tek, Georgia, USA) according to the manufacturer’s instructions. When multiple fragments are visualised using agarose gel electrophoresis, the Monarch® DNA Gel Extraction Kit (New England Biolabs, Massachusetts, USA) was used to extract the desired fragments.

Type IIS cloning

The following digestion mix (50µL) was set up on ice: 1µg DNA, 5µL 10X NEB buffer r3.1, 1µL type IIS restriction enzyme, mQ water up to 50µL. The reaction was incubated at the at 37°C for 1 hour followed by a heat inactivation at 65°C for 20 minutes. All enzymes and reaction buffers were obtained from NEB. After digestion, DNA was extracted using the appropriate technique (NEBcloner, 2022). Alternatively, heat inactivation can be skipped to protect the sample when purification and ligation are done right after digestion. It is also possible to premix the digested products before purification. The enzymes used were BsaI-HF, HindIII-HF, NcoI-HF and SalI-HF. BsaI-HF was used during the cloning process, SalI-HF was used to remove the stopcodon from the toxin construct and finally HindIII-HF and NcoI-HF were used to linearise plasmids in order to visualize them with agarose gel electrophoresis. The following ligation reaction (20µL) was set up on ice: 1µL 10X T4 DNA ligase buffer, 50 ng vector DNA (4 kb), 37.5 ng insert DNA (1 kb), 1µL T4 DNA ligase, mQ water up to 20µL. The vector: insert ratios were optimized according to the lengths with the NEBioCalculator. For troubleshooting, different ratios of insert and vector can be tried. The reaction was incubated at room temperature for 10 minutes and the enzyme was inactivated at 65°C for 20 minutes (NEBcloner, 2022). Before transformation, the sample is stored at -20°C. All enzymes and reaction buffers were obtained from NEB.

Gibson assembly

To perform Gibson assembly, the following reaction mixture (20µL) was set up: 0.02 – 0.5 pmols of DNA (total amount of fragments), 10 µL 2X Gibson Assembly Master Mix, mQ water up to 20 µL. To optimize the cloning efficiency, 50-100 ng of vector DNA was added to a 2-3 fold excess of insert DNA. The sample was incubated at 50°C for 15 minutes and stored at -20°C before transformation (Gibson Assembly® | NEB, 2022).

Transformation

50 µL of chemically competent cells (stored at -80°C) were thawed on ice and 1 pg – 100 ng of plasmid DNA was added. The tube was then flicked 4-5 times and the mixture was put on ice for 5 to 30 minutes. Following this, the sample was heat shocked at 42°C for 30 to 60 seconds and the sample was put back on ice for 5 minutes. Then, 450 µL of sterile LB medium was added to the cell suspension and this was incubated at 37°C for 1 hour while shaking. Lastly, 25 to 100 µL of this sample was plated on the appropriate selection plate which was then incubated at 37°C overnight. DH5α cells were obtained from NEB or competent cells were prepared using the following protocol. Alternatively, when difficulties occur, the sample can be incubated without shaking and avoid pipetting up and down to mix. After plating 25 to 100 µL, centrifuge the remaining cells and discard the supernatant. Resuspend with 100 µL LB medium and plate this on another plate.

Competent cell preparation

500 µL of an overnight cell culture (3mL LB with kanamycin, 37°C, 180 rpm) was diluted in a sterile Erlenmeyer containing 50 mL LB. It was incubated at 37°C until the cells were grown to an OD₆₀₀ of 0.2 – 0.6. The cells were cooled down on ice and centrifuged (4°C, 5000 rpm, 10 min). The cell pellet was then resuspended in ice cold, sterile 0.1 M CaCl₂ and put on ice for at least 25 min. Following this, the cells were centrifuged again (4°C, 5000 rpm, 10 min) and the pellet resuspended in 500 µL ice cold, sterile 0.1 M CaCl₂. 100 µL ice cold, sterile 80% glycerol was added to this solution. Lastly, this cell suspension was divided into sterile Eppendorf tubes (50 µL each) and stored at -80°C.

Colony PCR and sequencing

After transformation, colonies were picked with a swab which was washed in the PCR reaction mixture. Under sterile conditions, it is streaked onto a new LB agar selection plate which is incubated at 37°C overnight. The PCR tubes were incubated for 2 minutes at 95°C to lyse the cells which was followed by the appropriate thermocycling conditions. Two primers were designed to amplify a region where the insert and the vector are ligated. The correct length can be verified with an agarose gel and if the amplicon had the expected length, the samples were sequenced. The sample preparation for Sanger sequencing was done according to the instructions of Eurofins.

Induction tests

TOXIN CONSTRUCTS - To test the toxin constructs, E. coli DH5αZ1 cells containing different plasmids are grown overnight (37°C, LB medium). The overnight cultures were diluted 50X in 96 well plates (TPP Tissue culture test plate 96F, Techno Plastic Products AP) and grown until they reached an OD₆₀₀ of 0.2 - 0.6. OD₆₀₀ and fluorescence measurements were done with the CLARIOstar (BMG Labtech, Ortenberg, Germany). All measurements were performed in triplicate. Four colonies were tested either with the plasmid pLO_toxin or pBAD_toxin. An overview of the different test conditions is provided in the table below.

Table 5: Different test conditions for the toxin constructs.

ANTITOXIN CONSTRUCTS - Two 96 well plates were prepared with 200 µL of the relevant selection medium (LB + 100 µg/mL ampicillin) and the plates were covered with a breathable film. The cultures were diluted 50 times in these well plates and incubated at different temperatures overnight at 180 rpm. Temperatures tested ranged from 20°C to 42°C. The next day, both the OD₆₀₀ as the fluorescence intensity were measured with the CLARIOstar (BMG Labtech, Ortenberg, Germany). Blanks (LB) and negative controls (culture with pLO_SNAP plasmid) are subjected to the same protocol. All measurements were performed in triplicate.