De novo design and synthesis of a 30-cistron translation-factor module

Abstract

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

Figure 1.

De novo design of a DNA sequence encoding translation, and method for synthesis of its fragment that encodes translation factors. Synthesized sequences are in red; vector sequences are in green. (A) Modular design of a theoretical DNA sequence encoding all macromolecules necessary and sufficient for protein synthesis (a ‘translatome’ of 121.6 kb) based upon biochemistry central to E. coli. Module sizes are updated from (14) to include recently discovered enzymes (Supplementary Table S1). Dashes represent non-translation sequences of undefined size. The ‘translation factors’ module (red) encodes all aminoacyl-tRNA synthetases and translation factors required for protein synthesis. (B) BioBrick construction of the translation factors module shown in red in (A), but omitting EF-Tu. The final bacterial artificial chromosome (BAC) is shown surrounding the standardized workflow. NdeI sites (N), T7 RNA polymerase promoters (↱), ribosome binding sites (RBS), vesicular stomatitis virus (VSV) and TΦ terminators (⊤⊤) are denoted.

Figure 2.

Design, synthesis and assembly of 30 cistrons into three plasmids for simplified construction of a purified translation system (PURE 3.0). (A) Pre-designed groups of BioBrick genes. Final assembled gene orders are listed from 5′-3′, top-bottom. (B) Pairwise BioBrick assembly into three high copy plasmids. (C) Size verification of plasmids by linearization and pulsed field gel electrophoresis (L, ladder). Further digests are in Supplementary Figure S2. (D) Representative growth curves of duplicate cultures before and after induction at 155 min (small arrows) with 0.5 mM IPTG. Controls (gray) are no plasmid (BLR), pET-EF-G and pET-IF3. (E) Representative patterns of nickel-column-purified proteins from over-expression of each of the three pLD plasmids, as assayed by SDS-PAGE and Coomassie blue staining (L, pre-stained ladder). Plasmid-encoded proteins were assigned (labels on right) based on mass spectrometry of excised bands (Supplementary Table S3). Calculated MWs of encoded proteins in kDa are shown. (F) Approximate relative concentrations of nickel-purified proteins from each plasmid, as estimated from gel densitometries of the gels in (E) with comigrations resolved by mass spectrometry (Supplementary Table S3). Concentrations are from Supplementary Table S5 and are plotted here on a log scale in the order of the genes on the plasmids.

Figure 3.

Assay of purified proteins from pLD plasmids for kinetics of initiation, di- and tripeptide syntheses and for synthesis of full-length proteins (see Materials and Methods). (A) Rate-limiting splitting of vacant 70S ribosomes (initiation) monitored by time course of subsequent non-rate-limiting fMet-Phe dipeptide bond formation using proteins encoded by pLD2 and pLD3. (B) Elongation efficiency measured by templating synthesis of fMet-Phe-Phe tripeptide and monitoring formation of both dipeptide and tripeptide products using proteins encoded by pLD2 and pLD3. The shaded area is a measure of translocation mean time. (C) Comparison of full-length protein yields of DHFR, His₆-IF3 and His₆-AsnRS (predicted 18.0, 21.4 and 53.4 kDa, respectively) templated by uncut plasmids in a commercial PURE system versus our PURE 3.0 containing proteins encoded by pLD1, pLD2 and pLD3. Products were quantitated by incorporation of ³⁵S-Met, separation by SDS-PAGE and phosphoimaging of the gel. A photograph of the pre-stained ladder (L) in lane 5 is superimposed, and the results are reproducible.

Figure 4.

Construction of a 30-cistron translation factor module. (A) Plasmid map of final assembled Translation Factor Module (pTFM1). Linearized low copy number M4F-14-21 (containing cistrons 14–21; red arc) and 22–30 insert (purple arc) were ligated to generate a 14–30 cassette (red-purple arc). This was ligated to M4F-1-13 (black and green arc) to obtain 30 cistrons (117 basic parts) on a single plasmid (pTFM1). BioBrick restriction sites and ligation scars are indicated. Genes are labeled and shown to scale: synthetases are blue; initiation factors are yellow; elongation factors are orange, release factors are pink, and other factors are light grey. (B) Pulsed field gel analysis of linearized intermediate plasmids and final assembled pTFM1. Predicted sizes from L-R are 21.4, 24.8, 39.8, 31.8 and 65.0 kb. The 5 kbp Ladder (right) has increments of 4.9 kb from 9.8 kb. (C) Agarose gel of NdeI+PstI-digested intermediate plasmids and final pTFM1 showing DNA fragments containing each cistron with its 3′ intergenic region (further digests in Supplementary Figure S6). (D) Agarose gel of NdeI+PstI-digested pTFM1 DNA isolated from each of 7 days of culturing in E. coli. (E) In vitro protein expressions from uncut BAC constructs 1–13, 14–21, 22–30 and pTFM1 using a commercial PURE system, ³⁵S-Met and gradient SDS-PAGE. Similar patterns are seen for in vitro synthesis from pTFM1 using a commercial S30 E. coli extract (Supplementary Figure S7).