Figure Legends
Figure 1 Construction and identification of the tet-pL-Eselection/counter-selection cassette. (A) Schematic of constructing plasmid pBBR1-E. (B) Schematic of constructing the tet-pL-Eselection/counter-selection cassette. DNA fragment in the dotted box indicates the goal region of the constructed tet-pL-E cassette. (C) PCR identification of strain CWE-1. The PCR products were analyzed on a 1% agarose gel.
Lane 1 shows a 851-bp amplicon in CWE-1, while no band is observed in the control strain MG-10 (Lane Con). M represents the DNA marker. (D) PCR identification of strain CWE-2. The PCR products were analyzed on a 1% agarose gel. Lane 1 shows a 1,430-bp amplicon in CWE-2, while no band is observed in the control strain MG1655 (Lane Con). M represents the DNA marker.
Figure 2 Potential and functional testing of lysis geneE as a counter-selection marker gene. (A) Cells were grown overnight at 30 °C or 42 °C after streak cultivation. (B) Serial spot dilution of cells was grown in LB agar plates at 30 °C or 42 °C. (C) Colony formation of CWE-2 at 42 °C after Red recombination. No DNA (left) or 1,000 bp DNA fragment of catenin gene from H. armigera with 38-bp homologous to ack (right) was electroporated into competent CWE-2 cells. 100 μL of recovered cells were then plated onto agar plates and cultivated at 42 °C. (D) PCR identification of the genomic substitution mentioned in (C). A total of 10 colonies (lane 1 to lane 10) were screened and a 1,116-bp product indicates successful substitution. Lane Con shows a 2,270-bp product in CWE-2. M represents the DNA marker.
Figure 3 Construction and functional testing of thePBAD-E-GmRselection/counter-selection cassette. (A) Schematic of constructing plasmid pKD-EG. DNA fragment in the dotted box indicates the goal region of the constructed PBAD-E-GmRcassette. (B) PCR identification of plasmid pKD-EG. The PCR products were analyzed on a 1% agarose gel. Lane 1 shows a 1,344-bp amplicon in pKD-EG, while no band is observed in the control plasmid pKD46 (Lane Con). M represents the DNA marker. (C) Induction of E in plasmid pKD-EG with arabinose is lethal. GY1 [pKD-EG] was cultured overnight in LB broth without (left) or with the addition of 0.4% arabinose (right). (D) PCR identification of strain GY4. The PCR products were analyzed on a 1% agarose gel. Lane 1 shows a 1,610-bp amplicon in GY4, while no band is observed in the control strain GY1 (Lane Con). M represents the DNA marker. (E) Induction of E in GY4 with arabinose is also lethal. GY14 was cultured overnight in LB broth without (left) or with the addition of 0.4% arabinose (right). (F) Serial spot dilution of GY4 and GY1 [pSim6] were grown in LB agar plates supplemented without (left) or with (right) the addition of 0.4% arabinose.
Figure 4 Genomic s eamless modification in S. marcescens chromosome. (A) Colony formation of GY4 after ssDNA-mediated recombination. 5 μL of recovered cells were cultivated on LB agar plate supplemented with 0.4% arabinose. ssDNA represents GY4 transformed with oligo sspigA-F. Con represents GY4 transformed without DNA. (B) PCR detection of the PBAD-E-GmRdeletion using ssDNA. Lane Con shows a 3,477-bp amplicon in GY4, and a 1,190-bp product indicates successful deletion in Lane 1 to 10. M represents the DNA marker. (C) PCR detection of thePBAD-E-GmR substitution by partial fragment of T7 RNA polymerase gene. Lane Con shows a 1,929-bp amplicon indicates successful substitution in Lane to 10, while no band is observed in the control strain GY5 (Lane Con). M represents the DNA marker.
Figure 5 Improving selection stringency frequency through combining E and kil . Selection stringency frequency ofE , kil and kil-sd-E was analyzed at ack (A),araB (B) and pigA loci (D). Each point represents the mean ± SD of three independent replicates. (C) MG-4A (left) and MG-4B (right) were cultured overnight in LB broth without or with the addition of 0.4% arabinose. (E) Colony formation of GY5 after ssDNA-mediated recombination. 5 μL of recovered cells were cultivated on LB agar plate supplemented with 0.4% arabinose. ssDNA represents GY5 transformed with oligo sspigA-F. Con represents GY5 transformed without DNA. (F) PCR detection of thePBAD-kil-sd-E-GmR deletion using ssDNA. Lane Con shows a 3,713-bp amplicon in GY5, and a 1,190-bp product indicates successful deletion in Lane 1 to 10. M represents the DNA marker. (G) PCR detection of thePBAD-kil-sd-E-GmR substitution by partial fragment of T7 RNA polymerase gene. a 1,929-bp amplicon indicates successful substitution in Lane to 10, while no band is observed in the control strain GY5 (Lane Con). M represents the DNA marker.
Figure 6 Improving selection stringency frequency ofkil-sd-E by introducing plasmid pKDsg-ack. (A) Selection stringency frequency of kil-sd-E at marR-1 , rcsAand hyp-1 loci was analyzed. Each point represents the mean ± SD of three independent replicates. (B) Selection stringency frequency ofkil-sd-E at araB locus in MG1655 [pSim6] and MG1655 [pKDsg-ack] was analyzed. Each point represents the mean ± SD of three independent replicates. (C) Selection stringency frequency ofkil-sd-E at marR-1 , rcsA and hyp-1 loci was analyzed. Each point represents the mean ± SD of three independent replicates. (D) Comparation of selection stringency in S. marcescens carrying plasmid pSim6 or pKDsg-ack. The ratio is determined by dividing the selection stringency of the former by that of the latter.
Figure S1 Schematic of seamless deleting ofPBAD-E-GmR cassette by sspigA-F. PBAD-E-GmR was inserted into the CDS of pigA (between the purple and green boxes). The “//” in sspig-F indicates the junction between the two homologies.
Figure S2 Construction of CWE-3. Schematic of constructing strain CWE-3 is shown in the left. RBS sequence (GAAGGAGATATACC) from pET32a was linked between the CDS of E and kil . DNA fragment in the dotted box indicates the goal region of the constructedkil-sd-E counter-selection cassette. PCR identification of the strain CWE-3 is shown in the right. Lane 1 shows an 851-bp amplicon in CWE-1, while no band is observed in the control strain MG-10 (Lane Con). M represents the DNA marker.
Figure S3 PCR identification of the strain MG-4A and MG-4B. Lane 1 shows an 360-bp (left) or an 600-bp (right) amplicon in MG-4A and MG-4B respectively, while no band is observed in the control strain MG1655 (Lane Con). M represents the DNA marker.
Figure S4 Colony formation of MG655 [pKDsg-ack]. Cells equivalent in quantity to 10−4 μL of Overnight-cultured MG655 [pKDsg-ack] were mixed with 100 μL of fresh LB and then spread onto agar plate without or with the addition of 0.4% arabinose.