Oligonucleotide primers

Coding regions of the mature form of Chia cDNAs were amplified from chicken glandular stomach cDNA [16 (link)] by PCR using KOD Plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) and oligonucleotide primers (Eurofins Genomics, Tokyo, Japan) anchored with the restriction sites for EcoRI and XhoI (Table S1), as described previously [12 (link)]. Amplified cDNA was digested with EcoRI and XhoI and cloned into the same sites of the pEZZ18/Protein A-mouse Chia-V5-His [12 (link)]. The entire nucleotide sequence of the resulting plasmid DNA [pEZZ18/PA-Chia] was confirmed by sequencing (Eurofins Genomics).
To express the CatD or CBD of chicken Chia as a recombinant fusion protein with Protein A and V5-His, these regions were amplified from the chicken full-length Chia-expressing plasmid DNA (pEZZ18/PA-Chia) using oligonucleotide primers (Table S1), as described previously [14 (link)]. Each amplified DNA was then digested with EcoRI and XhoI and subcloned into the pEZZ18 expression vector. The entire nucleotide sequence of the resulting plasmid DNAs [pEZZ18/PA-CatD or pEZZ18/PA-CBD] was confirmed by sequencing (Eurofins Genomics). The recombinant PA-Chia, PA-CatD, and PA-CBD (Figure S1) as well as PA were prepared as described previously [14 (link)].

DNA manipulations were performed following standard protocols⁶⁰ , including transformation of chemically competent E. coli DH5α. Oligonucleotide primers were purchased from Eurofins Genomics (Ebersberg, Germany) while restriction enzymes and T4 DNA ligase for cloning procedures are purchased from NEB (Ipswich, MA, USA) and Thermo Scientific (Waltham, MA, USA). The coding sequence for I. sakaiensis PETase (PETase Uniprot: A0A0K8P6T7; Gene: ISF6_4831; EC:3.1.1.101), together with a Strep-tag at the N terminus, was codon-optimized for the C. reinhardtii chloroplast using the Codon Usage Database (www.kazusa.or.jp/codon) with SapI and SphI sites added for cloning into the pSRSapI transformation vector (Supplementary Fig. S1)^{61 (link)}. The DNA was synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, Iowa-USA), and was cloned into the transformation vector by standard molecular techniques⁶⁰ .

Real time PCR was performed in 20 μl for a set of selected genes using Fast SYBR Green PCR Master Mix (ABI, USA). The list of selected genes and oligonucleotide primers (Eurofins, India) used for each gene are listed in Table S2. oligonucleotide primers for vetiver actin gene (Table S2) were used as the internal control for establishing equal amounts of cDNA in all reactions. The reactions were performed using the following cycle conditions, an initial 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, and the final 5 min extension at 72 °C. After obtaining the ct value for each reaction, the relative expression was calculated by 2^-delta Ct method.