Strains and culture conditions
The strains used in this study are listed in Table S1. E. coli was cultured at 37°C in lysogeny broth medium (LB, 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl). Streptomycin (100 μg/mL) or kanamycin (50 μg/mL) was added to the medium where appropriate. R. eutropha was cultured in LB at 30°C. Gentamycin (10 μg/mL) or kanamycin (200 μg/mL) was added where appropriate.
Primers and Plasmids
Primers (Table S2) used for the construction of plasmids (Table S3) in this study were designed using j5 Device Editor [17]. The other primers were designed using Clonemanager software. High fidelity DNA polymerase was purchased from Takara (Dalian, China) or New England Biolabs (USA). BsaI restriction endonuclease and T4 ligase were purchased from New England Biolabs (USA) and Thermo-Fisher Scientific (USA), respectively. Plasmids were constructed using Golden Gate [18], Gibson [19] or CPEC[20] methods, and used to transform E. coli or R. eutropha. Plasmids were extracted using the AxyPrep Plasmid Miniprep Kit (AXYGEN, China) according to the manufacturer’s instructions, with minor modifications.
Genome Integration of R. eutropha
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, the above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Integration of araC and T7 RNA polymerase genes into R. eutropha C5 (R. eutropha H16∆H16_A0006∆H16_A0008-9)[21] was performed with the pk18mobsacb suicide plasmid with similar procedure as our previous work [21]. For construction of the integration plasmid pj5_00015, the DNA cassettes of left homologous arm, AraC gene, PBAD promoter, T7 RNA polymerase gene and terminator were assembled into pk18mobsacb plasmid with the Golden gate method. Plasmid pj5_00015 was then transferred into R. eutropha C5 strain by bacterial transconjugation. And the strain with kanamycin resistance was screened on agar plates containing 200 μg/ml kanamycin. The strain with genomic integrated pj5_00015 was selected by colony PCR. To eliminate the DNA cassette containing antibiotic marker, The above selected strain was cultured in NaCl free LB liquid media containing 10% sucrose, and then plated agar plate containing the same content to screen for strain with no kanamycin resistance. The obtained strain was confirmed again with colony PCR and sequencing of the PCR products, which was designated as R. eutropha C5T7 strain.
Assessment of RFP expression levels
Red fluorescence intensity was measured using a microplate reader (Infinite M200 PRO, TECAN) after the strains were induced with L-arabinose for 48 hours. The excitation and emission wavelengths were 585 and 620 nm respectively[6]. Optical density at 600 nm (OD600) was also measured using the microplate reader. The RFP expression levels were determined as the ratio of the red fluorescence intensity to the OD600.
Identification of the region necessary for plasmid replication
Different primers for Golden Gate were designed using j5 DeviceEditor, and PCR was implemented using the plasmid pBBR1-PT7-rfp as template. The products of the PCR were purified using the SanPrep DNA Purification Kit (Sangon Biotech, China), and then cyclized via Golden Gate assembly. As a result, some regions of the pBBR1-PT7-rfp were deleted, and the minimized plasmids were used to transform E. coli. Transformed colonies would be obtained if the core region for plasmid replication was intact, while transformation would be unsuccessful if the core region were damaged.
Electroporation of R. eutropha and evaluation of transformation efficiency
Competent cells of R. eutropha were prepared according to the protocol described in our previous work[21]. Briefly, R. eutropha was cultured in 100 mL LB at 30°C and 200 rpm, then chilled on ice for 5-10 min after the OD600 reached 0.8. The cells were collected by centrifugation at 3000× g for 5 min and washed with ice-cold sterile 10% (V/V) glycerol three times. Finally, the cells were suspended in about 0.5 mL 10% glycerol and 0.1 mL portions transferred to sterile 1.5-mL tubes. The prepared R. eutropha competent cells were used immediately, or frozen in liquid nitrogen and preserved at -80°C for a few weeks.
Electroporation of R. eutropha was implemented according to the protocol described in our previous work[21]. Briefly, appropriate plasmids were added to the competent cells and mixed gently. The mixture was transferred into a pre-chilled 2-mm electroporation cuvette (Bio-Rad, USA), and incubated on ice for 5 min, after which electroporation was performed at a voltage of 2.3 kV. Immediately afterwards, 1 mL of LB preincubated at 30°C was added to the cells. All the cells were transferred to a sterile 1.5 mL tube and incubated at 30°C for 2 h. Then, a portion of the cells was spread on LB agar plates with appropriate antibiotics, and cultured at 30°C until colonies were visible. Electroporation efficiencies were calculated as the ratio of colony-forming units (CFU) to the amount of the used plasmid DNA.