Synthetic dna gene block

Strains used in this study are listed in Table S1. All strains were derived from the S. cerevisiae BY4742 using standard genetic techniques and CRISPR-Cas9 technology (Lee et al., 2015 (link)). C-terminal mAID-3xFlag tags (cdc33-mAID-3xFlag, tif4631-mAID-3xFlag) were generated by amplifying the SG_linker-mAID-3xFlag sequence from pNTI433 with primers that included 40 bp of sequence identity with either side of the insertion, then integrated using CRISPR-Cas9 technology as described by (Brothers and Rine, 2019 (link)). C-terminal mAID-3xV5 tag (tif4632-mAID-3xV5), and P2A-ZEM tags (aro10-P2A-ZEM and pcl5-P2A-ZEM) were constructed in the same manner by amplifying from synthetic DNA gene block (Integrated DNA Technologies) PD552 and pNTI730, respectively. Gene deletions were generated using one-step replacement with marker cassettes, more specifically by PCR amplification of the hygromycin resistance cassette from pNTI730 with flanking sequence homology to the 5’ and 3’ ends of target gene coding sequence (Goldstein and McCusker, 1999 (link); Hentges et al., 2005 (link)). PCL5-ZEM reporters and dCas9-TetR were integrated using NotI-linearized vectors from the EasyClone 2.0 toolkit for yeast genomic integration (Stovicek et al., 2015 (link)). Plasmids and primers used in strain construction are listed in Table S2 and S3, respectively.

The sequence coding for hmGLP1R was ordered as a synthetic DNA geneblock
(Integrated DNA Technologies) bearing HindIII and NotI restriction site for
cloning into a CMV-promoter plasmid (Addgene #60360). Sequences coding for the
hemagglutinin secretion motif and a FLAG Tag were added to the N-terminus of the
GLP1R open reading frame to increase plasma membrane expression and enable
receptor labeling, respectively. Sensor variants were obtained using Gibson
assembly (NEBuilder HiFi DNA Assembly Cloning Kit) (Gibson et al., 2009 (link)). Site-saturated mutagenesis was
performed by PCR using primers bearing randomized codons at specified locations
(NNK). For luminescence-based characterization of G protein and β-arrestin
coupling, the small subunit (i.e. smBit) of NanoLuc (Cannaert et al., 2016 (link)) was PCR-amplified from a
Beta2AR-SmBit donor plasmid and cloned at the C-terminal end of the GLP1R and
GLPLight1 using Gibson assembly. PCRs were performed using a Pfu-Ultra II Fusion
High Fidelity DNA Polymerase (Agilent). All sequences were verified using Sanger
sequencing (Microsynth). For cloning GLPLight1 and GLPLight-ctr into the viral
vector, BamHI and HindIII restriction sites were added flanking the sensor
coding sequence by PCR amplification, followed by restriction cloning into
pAAV-hSynapsin1-WPRE, obtained from the Viral Vector Facility of the University
of Zürich.