Bug mapping and fitness testing of chemically synthesized chromosome X

Abstract

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

Fig. 1. Characterization and fitness testing of synX strain

(A) Design overview of synthetic chromosome X. (B) PCRTag analysis of synX (a pericentromeric region). Analysis of the complete set of PCRTags is shown in fig. S3. (C) Growth fitness of synX strain yYW0115 on various types of media. (D) Competitive growth assay of synX strain yYW0115 and native strain BY4741. Samples were analyzed by flow cytometry to quantify the ratio of RFP-positive to GFP-positive cells. Relative growth rate was calculated based on the ratio of synX to BY4741 at each time point.

Fig. 2. Debugging by pooled PCRTag mapping

(A) Flow diagram of pooled PCRTag mapping (PoPM). Chromosome X strains with patchworks of synthetic and native sequences, generated by incorporation of synthetic DNA minichunks (transformant library) or by backcross with wild-type (WT) cells (spore library), are subjected to phenotype testing under a selective condition (filled circle, high fitness; open circle, low fitness). PCRTagging is carried out on pools of high-fitness colonies as well as pools of low-fitness colonies to enable mapping of the defect to a small segment of synthetic DNA. The purple-shaded region “b” in the PCR indicates the region containing the “bug.” (B) The insertion of a loxPsym site in the 3′ UTR of YJR120W disrupts expression of neighboring gene ATP2, leading to a growth defect on the nonfermentable carbon source glycerol/ethanol (YPGE).

Fig. 3. PCRTag in FIP1 causes a growth defect

(A) The reverse (R) synthetic PCRTag recoded within YJR093C (FIP1) causes a growth defect. (B) FIP1 SYN PCRTag-R introduces a Rap1p recognition site. Percentages indicate similarity of FIP1 SYN PCRTag-R to known Rap1p binding sites. Red letters show differences between synthetic and native PCRTag-R sequences. (C) Growth assay and relative FIP1 RNA expression of codon-by-codon swap strains in FIP1 SYN PCRTag-R. Red letters represent SYN-specific bases; underlined letters represent restored WT codons. RNA level is quantified relative to WT FIP1.

Fig. 4. Large duplications and rearrangements and restoration to desired structure

(A) Karyotypic analysis of synX(A-R), yYW0077 and corrected synX strain, yYW0115 by pulsed-field gel electrophoresis. (B) Two large duplications and rearrangements occurred in the synX(A-R) strain. Sequencing coverage of synX(A-R) strain revealed two large duplications of synthetic fragments. The NotI* site and loxPsym site mediated junction of duplicated segments. (C) Massive duplications and rearrangements occurred during integrative transformation. Intermediate assembly strain semi-synX(A-C) (yYW0007) and synX(A-R) have identical copy numbers in the duplicated region. (D) Meiotic crossover to generate a synX strain lacking amplified segments. (E) Verification of the absence of duplication regions in synX strain yYW0115 by means of junction primers.