Terminator Regions, Genetic

Genomic data for the 343 organisms was downloaded on 6 June 2006 from [25 ]. Annotations were taken from the .ptt file accompanying the genomic sequence. Accession and version numbers for the sequences used are available as Additional data file 3. TransTermHP was run using the default search parameters and the -p scoring parameter to produce predictions for the best terminator following each gene in these organisms (available on the web [18 ], --bag option to TransTermHP) as well as the best terminators in each robust tail-to-tail region (Additional data file 2, --t2t-perf option to TransTermHP). The former were used when comparing performance to [3 (link)], while the latter was used to assess the importance of Rho-independent termination within an organism. Experiments were run on a 3.2 GHz Intel Xeon processor running Linux. High-confidence terminators are those with scores ≥ 76.
The C++ source code for TransTermHP is available (Additional data file 1) under an open source license.

In most cases the respective inserts were PCR amplified to create the GreenGate entry modules. The nucleotides 5′-AACA-GGTCTC-A-NNNN (nn)-3′ were added to the forward primer in front of the gene specific sequence. GGTCTC is the BsaI recognition site, AACA was added because the enzyme does not cut if the restriction site is at the extreme ends of PCR products. NNNN represents the module specific overhang and 2 nucleotides (nn) are needed in case of the coding sequence and C-tag modules to bring the modules into frame (NNN represents an in-frame coding triplet in the overhangs). The sequence 5′-AACA-GGTCTC-A-NNNN-3′ was added to the reverse primers, followed by the reverse complement of the sequence of interest. NNNN stands for the reverse complement of the module specific overhang, the coding triplet being underlined.
After amplification, the PCR reactions were separated on agarose gels, the product bands excised, purified with innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany) and digested with BsaI. The respective empty entry modules (∼ 100 ng) were also cut with this enzyme, usually in the same tube (1 h, 37°C). The digestion was purified with the above mentioned kit and ligated with T4 DNA ligase (1 h room temperature, overnight 4°C). After heat-inactivation (10 min, 70°C) the reaction was transformed via heat shock into ccdB sensitive E. coli strains (Mach1™-T1^R, DH5α, XL1-Blue MR). Transformants were checked by colony PCR, plasmid DNA was isolated from positive clones and checked by sequencing and test digestion.
If internal BsaI recognition sites were present in the module sequence, they were removed by nucleotide substitution. For protein-coding sequences, silent mutations were chosen. In promoter and terminator sequences, the nucleotides to be changed were selected at random, but for later constructs we switched to always replace the first guanine by a cytosine. For simplicity, we used scar-free BsaI-cloning to create the substitutions. Primers were designed on both sides of the internal BsaI recognition sites introducing the mismatch and flanked on their 5′-ends by external BsaI recognition sites. The overhangs generated by the external BsaI cut were designed to be part of the gene-specific sequence and being different from the module specific overhangs.
Shorter modules were assembled as oligonucleotide duplices created from overlapping primers with unpaired 5′-overhangs complementary to the module specific overhangs. The oligonucleotides (10 µM or 100 µM) were mixed in equimolar ratios with each other, soused with boiling water, allowed to cool slowly down to room temperature and then ligated into BsaI digested and purified entry vector.

The DNA fragments MMV FLt (-193 to +63 bp), MMV Sgt (-306 to -125 bp), and FMV Sgt (-270 to -63 bp) were chemically synthesized (Gene Universal, Inc., Newark, USA) (Supplemental Table 1). The FM′M promoter was first synthesized as a single DNA fragment. Subsequently, each domain was amplified by PCR using the specific primers MMFg-F, MMSg-F, FsMf-F, FsMf-R, M′FM-R, M′FM-F, and FM′M-R (Supplemental Table 2). To insert the UAS×4 motif and/or the single or double zinc finger binding motifs into the FM′M promoter, the primers FM′M-U-F and FM′M-R or FM′M-US-F and FM′M-US-R or FM′M-UD-F and FM′M-UD-R were used for overlapping PCR (Supplemental Table 2). All promoter fragments were digested with PstI and XbaI, and ligated into pCAMBIA1300, digested with the same restriction endonucleases. The BiP : GFP:HDEL recombinant construct (Islam et al., 2020 (link)) was ligated to each of the hybrid promoters following digestion with the restriction endonucleases XbaI and XhoI. The reporter gene encoding BiP : RBD:SD1:6×His : HDEL (Bangaru et al., 2020 (link)) was digested with the restriction endonucleases XbaI and XhoI, and ligated to the FM′M-UD or CaMV 35S promoters that had previously been digested with the same restriction endonucleases. The recombinant construct, BiP : MP:CBM3:bdSUMO:hIL6:HDEL (Islam et al., 2019 (link)), was ligated to the FM′M-UD or CaMV 35S promoters following digestion with XbaI and XhoI restriction endonucleases. All these constructs contained the RD29B terminator from Arabidopsis thaliana RD29B (D.13044.1) or the recombinant 3PR terminator (Supplemental Table 1). The 3PR terminator sequence was chemically synthesized (Gene Universal, Inc., Newark, United States). These terminators were ligated into the constructs after digestion with restriction endonucleases XhoI and EcoRI. The DNA fragments encoding the transcription factors GAL4:VP16, GAL4:TAC3d2, and ZinC7:TAC3d2 were chemically synthesized (Gene Universal, Inc., Newark, United States), and contained XbaI and XhoI restriction endonuclease sites. The MacT promoter and RD29B terminator were used for the expression of these transcription factors. All the primer sequences used in this study are listed in Supplemental Table 2.

In order to investigate the activity of PrWRI1 visually, the coding sequence of PrWRI1 was cloned into the SacII and BamHI restriction sites of pK34 entry vector, and then the recombinant pK34 vector with double CaMV 35S promoters and a terminator sequence was digested with AscI for entry into plant expression vector, pB110. The vector was then transiently expressed in Nicotiana benthamiana leaves by Agrobacterium-mediated transformation.
The ORF of PrWRI1 were inserted into the vector pCAMBIA1300 under the control of Arabidopsis seed-specific promoter 2S2 by the digestion of KpnI and BamHI. The generated constructs were transformed into Agrobacterium tumefaciens GV3101 using the freeze–thaw method, and then they were used for the transformation of wild-type Arabidopsis by the floral dip method.

Four PhaC amino acid sequences were chosen based on the BLASTP search results. These phaC genes were chemically synthesized with optimized codon usage in E. coli by Eurofins Genomics Co. Ltd. (Tokyo, Japan) for plasmid construction and evaluation. E. coli LSBJ, a fadB fadJ double-deletion strain of E. coli LS5218 [fadR601, atoC (Con)] (Tappel et al., 2012a (link)), was used as the host strain for PHA biosynthesis. This strain is an ideal host for non-native PHA production because of its ability to take up a wide variety of substrates to be incorporated into PHA homo- and copolymers, and bench-level scale-up methodologies available for overall production (Tappel et al., 2012b (link); Levine et al., 2016 (link); Pinto et al., 2016 (link); Fadzil et al., 2018 (link); Furutate et al., 2021 (link); Scheel et al., 2021 (link)). A broad-host-range plasmid pBBR1MCS-2 (Kovach et al., 1995 (link)) harboring the genes encoding the PhaCs to be evaluated, the lac promoter region, the (R)-specific enoyl-CoA hydratase gene from A. caviae (phaJ_Ac), the 3-ketothiolase gene (phaA) from Ralstonia eutropha H16, and the acetoacetyl-CoA reductase gene (phaB) from R. eutropha H16, termed pBBR1-phaCsAB_ReJ_Ac, was used for the expression of PhaCs (Supplementary Figure S1). For phaAB expression, the R. eutropha pha promoter and terminator regions were located upstream and downstream of their genes, respectively. To enhance the supply of 3HHx, 3H4MV, and 3H2MB monomers, the plasmid pTTQ-PCT (Furutate et al., 2017 (link)) containing the propionyl-CoA transferase (PCT) gene from Megasphaera elsdenii (pct) (Taguchi et al., 2008 (link)) was introduced into the E. coli LSBJ strain (Supplementary Figure S1).