Electroporator

BL21(DE3) cells were co-transformed with a pET-GFP-TAG-66 plasmid (0.5 μL) and pEVOL-3FY plasmid (0.5 μL) using an Eppendorf electroporator (Eppendorf, Hauppague, NY, USA). Cells were then plated on LB-agar plates supplemented with ampicillin (50 mg/mL) and chloramphenicol (34 mg/mL) and grown at 37 °C. After 16 h, a single colony was selected and used to inoculate LB media (4 mL) supplemented with ampicillin and chloramphenicol. The culture was grown at 37 °C for 12 h. The culture was used to initiate an expression culture of LB media (10 mL) at OD₆₀₀ 0.1, then incubated at 37 °C, to an OD₆₀₀ of ~0.6, at which point cells were induced with 1 M IPTG (10 μL), 20% arabinose (10 μL) and 100 mM (100 μL) of respective fluorotyrosine (3-FY; 2,3-F₂Y; 3,5-F₂Y; 2,3,5-F₃Y; 2,3,6-F₃Y). Cultures were grown for an additional 16 h at 37 °C, then harvested by centrifugation (10 min at 10,000 rpm). The media was removed and the cell pellet placed in the −80 °C freezer for at 20 min. Purification was accomplished using commercially available Ni-NTA spin columns and according to manufacturer’s protocol. Protein yield and purity was assessed by SDS-PAGE, and with a Nanodrop spectrophotometer (ThermoFisher, Pittsburgh, PA, USA).

The Aa113 genomic DNA bacterial artificial chromosome (BAC) library was constructed as described by Brosch et al. (1998) (link) with minor modifications. After HindIII partial digestion, DNA fragments 75 to 120 kb in size were ligated into plasmid pBeloBAC11 and electroporated into E. coli TG1 using an electroporator (Eppendorf, Germany). After electroporation, the cells were resuspended in 600 µl of LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1000 ml deionized water), allowed to recover for 1 h at 37° with gentle shaking, and then plated on LB agar containing chloramphenicol (12.5 μg/ml). The plates were incubated overnight, and the recombinants were picked manually and transferred to 96-well plates that were incubated for 10 h with shaking at 220 rpm. The BAC library was stored at −80°. Screen of the BAC clone by pool method was first performed with the markers SC403 and SC254. Gaps present in the assembled sequence were further filled as described by Zhou et al. (2006) (link). The selected BAC clones were sequenced, and the obtained sequences were assembled using the Bioedit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html).

Trypsinized and washed donor cells were transferred into the perivitelline spaces of enucleated oocytes with a 25-μm micropipette. Cell couplets were briefly washed in fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, 0.05% fatty acid-free BSA) and fused by two DC pulses of 100V for 15 μs each using an Eppendorf electroporator. Couplets were removed from the fusion chamber and put back into Hepes-TCM-199 to score fusion success and detect detached or lysed donor cells. Reconstructs were activated 1.5 h post fusion with 5 μM ionomycine followed by exposure to 6-dimethylaminopurine (6-DMAP) for 4 h, as described previously [14 (link)]. The activated oocytes were then transferred to 500 μL of embryo culture medium I (modified potassium simplex optimization medium with essential and non-essential amino acids [KSOMaa] supplemented with 1% BSA and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. On Day 3 (Day 0 = day of activation) the cleaved embryos were transferred into 500 μL of embryo culture medium II (modified KSOMaa supplemented with 10% FCS) and cultured under the same conditions until Day 7. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture.

The plasmids were transformed into M. extorquens AM1 by electroporation as described in a previous study [53 (link)], with a few adjustments. Initially, M. extorquens AM1 cells were grown in a 50-mL flask until the culture reached OD₆₀₀ of 0.8. The cells were then collected, washed thrice with ice-cold water to prepare competent cells, and resuspended in 1 mL ddH₂O for distribution. Next, 100 µL of competent cells mixed with 2 µg of plasmid DNA were transferred into a 1 mm-gap cuvette placed on ice and operated at a voltage of 1800 V using the electroporator (Eppendorf, Germany). The mixture was transferred into 2 mL liquid medium in a 10 mL tube and incubated at 30℃ with a rotational speed of 200 rpm for 5 h. 100 µL of the culture was subsequently spread onto selective plates. The objective colonies appeared after 3–5 days via antibiotic resistance, which were confirmed by colony PCR.