Fig. 1: A designer neochromosome to harbor essential genes from the left arm of chrXII.
From: Building a eukaryotic chromosome arm by de novo design and synthesis
a, The design of neochromosome. Marker genes, white; TeSS, brown; Insulators, green; URRs, blue. b Arrangements of the ten essential genes (pink) in ptWT10 and ptWT10U. Arrows point to the direction of transcription. The genes with changed transcription direction on ptWT10U are labeled in red. c PFGE and southern blotting analysis of the assembled neochromosomes. M: λ DNA-Mono Cut Mix. Source data are provided as a Source Data file. Images are representative of at least three independent experiments. d Stability test of neochromosomes. BY4742 cells containing ptWT10 or ptWT10U were cultured in SC-His medium for about 125 generations before PCR analysis and whole genome sequencing. e Fitness analysis of strains on different growth conditions. 10-fold gradient dilutions were conducted for cells in this study. WT: BY4741. H2O2: hydrogen peroxide; Noc: nocodazole; MMS: methyl methanesulfonate; Rap: rapamycin; Ben: Benomyl. f–i Full-length transcriptome analysis of BY4742 with ptWT10 or ptWT10U neochromosome. The transcripts on neochromosome (orange) and native transcripts (chrXIIL, blue) were mirrored. On the x-axis, negative number represents distance upstream of ATG and positive values indicate distance downstream of the stop codon. The Y-axis represents the counts of detected transcripts. j, k Transcription analysis of the ten essential genes in ptWT10-10KO or ptWT10U-10KO using DESeq2 to calculate Benjamini-Hochberg adjusted p values and fold change values. WT: BY4742. Differential expression in this study was defined as |log2FC| > 1 and -log10 (Adjusted p-value) >4. FC, fold change. Dashed lines, the threshold defined above. Red, differentially expressed essential genes on neochromosomes. l The copy number of neochromosomes in ptWT10-10KO or ptWT10U-10KO. a,b,c are three independent biological repeats. The mean read-depth of native chromosomes (n = 11,845 1 kb-sized bins) was normalized to 1. For the neochromosomes, n = 36 1 kb-sized bins. The bounds of the box were the upper and lower quartile with the median value in the center. The whiskers indicated 5th and 95th percentile. Source data are provided as a Source Data file. m Normalized expression of genes on ptWT10 (n = 3) and ptWT10U (n = 3). Expression of each gene was normalized to HIS3 on neochromosomes. The data are presented as the mean and SD. A two-tail unpaired Welch’s t-test were employed to calculate the p values, which were adjusted by Benjamini-Hochberg method. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file. The exact p value was listed in Source data.
