FIGURE 4.

Modification of essential genes using a modified two-step strategy. (A) Schematic chart illustrating the modified two-step strategy for editing an essential gene with or without an independent promoter. The whole procedure was similar to the method described above (Figure 1) except that a ribosome binding site sequence (BBa_J61101 in this study) was linked to the ARC. To edit the 5′-end or the internal sequence of an essential gene, the ARC-ribosome binding site should be inserted in front of the CDS to ensure efficient expression of the gene. (B) Modification of the frr gene (b0172), an essential gene in E. coli. The 6 × His-tag-encoding DNA sequence was successfully inserted after the initial codon of frr, which was verified by Sanger DNA sequencing. Sequence alignment was performed using the SnapGene software (GSL Biotech).