Journal: PLOS Genetics

Article Title: High-throughput analyses of a reconstituted diversity-generating retroelement identify intrinsic and extrinsic determinants of diversification

doi: 10.1371/journal.pgen.1012038

Figure Lengend Snippet: (A) Sequences of TR4 and VR4 used for deep sequencing analysis of DGR-mediated adenine mutations. Co-transfer of T from TR4 to replace G in VR4 (colored grey) was used as a signature to distinguish DGR-induced adenine mutations from spontaneous adenine mutations. (B) qPCR analysis of reporter expression levels in E. coli MG1655. Total RNA was extracted from cells harvested at OD 600 ~ 0.6. The VR4 reporter, controlled by a weak (P J23112 ) or strong (P J23118 ) constitutive promoter, was integrated at the 291˚ locus in a derivative of MG1655 strain lacking Δ sbcB Δ mutS . Reporters were inserted in either co-directional or anti-directional orientations with replication, generating the strains HCL124 (P J23112 , co-directional), HCL121 (P J23112 , anti-directional), HCL126 (P J23118 , co-directional) and HCL123 (P J23118 , anti-directional). For each orientation, reporter expression levels were normalized to tufA and then to the expression level of the P J23112 . Error bars denote the standard error of the mean for three biological replicates. (C) Deep-sequencing quantification of DGR-mediated editing rates of the VRs described in A . Cells of the indicated E. coli strains were grown in LB + 0.2% arabinose until OD 600 ~ 2. Genomic DNA was isolated from harvested cells, and the VR regions were PCR amplified for sequencing. The mutagenesis rate of VR4 represents the fraction of sequencing reads with both at least one A-to-N mutation and the designed G-to-T mutation in the TR4 region and then dividing it to the total number of reads in that sample. Error bars denote the standard error of the mean for three biological replicates. Regardless of the transcriptional output, opposing reporters showed significantly different mutational rates: p-values are 0.004 (P J23112 ) and <0.0001 (P J23112 ). (D) Schematic of a plasmid with a p15A origin of replication. The dominant replication fork replicates almost of the entire plasmid (marked by orange arrow). Reporters were inserted in both co-directional and anti-directional orientations relative to the dominant replication fork at the indicated sites, creating pNEB298 (right, anti-directional), pNEB299 (right, co-directional), pNEB300 (left, anti-directional), and pNEB301 (left, co-directional). (E) Repair frequency of reporters described in D . Measurements were performed in the E. coli MG1655 strain using the kanamycin repair assay, with each p15A-based plasmid co-transformed with pDGR2. Bar heights are the mean of n = 6 biological replicates; error bars indicate the standard deviation.

Article Snippet: Total RNA was extracted from log-phase E. coli with Monarch Total RNA Extraction Kit (NEB, T2010).

Techniques: Sequencing, Expressing, Isolation, Amplification, Mutagenesis, Plasmid Preparation, Transformation Assay, Standard Deviation