Journal: bioRxiv
Article Title: Fine-tuned synthetic transcription factors for production of 3′-phosphoadenosine-5′- phosphosulfate in yeast
doi: 10.64898/2026.02.02.703094
Figure Lengend Snippet: a A schematic demonstrating JUB1-X synTF established in this study. IPTG-inducible synTFs incorporate DBDs originating from the JUB1 TF of the plant, fused to activation domains compatible with yeast . within the DBD of JUB1 to add the so-called X motif to JUB1 DBD. To evaluate the performance of these synTFs in inducing gene expression, chromosomally encoded JUB1-X synTFs were placed under the control of an IPTG-responsive promoter. In the presence of IPTG, IPTG binds to LacI, forming the IPTG-LacI complex that prevents binding to the lacO site in the promoter, thereby activating synTF expression. Next, the nucleus-guided synTF targets its binding site (BS), located in one or more copies upstream of a minimal CYC1 promoter, thereby recruiting RNAP to the promoter. As a result, yeGFP is expressed. Transcriptional output of JUB1-X synTF harboring X motif at b 114 cystein and c 191 cystein. The CDSs of JUB1 harboring the X motif in cysteine sites 114 and 191 were inserted between the N-terminus NLS and GAL4 or EDLL ADs. The generated JUB1-X synTFs were tested for their strength to activate yEGFP reporter gene expression from the CYC1 minimal promoter harboring four (4×) copies of the cognate binding sites. The effector and reporter constructs were chromosomally integrated into the yeast genome. The yEGFP output signal was tested both in the absence and in the presence of the inducer (IPTG). Gray, non-induction medium; green, induction medium. WT, yeast YPH500 wild type control; Pro TDH3 , yeGFP under the control of yeast constitutive promoter TDH3 . Mean fluorescence intensity per cell is given. Data are geometric means ± SD of fluorescence intensity from two cultures, each derived from two independent yeast colonies and measured in three technical replicates. Asterisks indicate statistically significant difference from noninduction medium (Student’s t-test; (∗) p < 0.05; (∗∗) p < 0.01; ns, non-significant). Abbreviations: AD, activation domain; AU, arbitrary units; BS, binding site; IPTG, isopropyl β-D-1-thiogalactopyranoside; JUB1, JUNGBRUNNEN1 transcription factor; lacO , lactose operator; LacI, lactose operon repressor; NLS, nuclear localization signal; Pro, promoter; RNAP, RNA polymerase; JUB1-derived synthetic transcription factor harboring Short tripeptide YVG and GER were placed in the N- and C-terminus of cycteins; yeGFP, yeast-enhanced green fluorescent protein. Full data are shown in Supplementary Data S1 .
Article Snippet: Cells were harvested for RNA extraction (Qiagen RNeasy Kit) and DNase treatment (TURBO DNA-free Kit). cDNA was prepared by reverse transcription on the RNA (NEB LunaScript RT SuperMix Kit), and qPCR analysis was carried out (BIORAD SsoFast EvaGreen supermix with low ROX) using SYBR green with FBA1 as an internal reference gene.
Techniques: Activation Assay, Gene Expression, Control, Binding Assay, Expressing, Generated, Construct, Fluorescence, Derivative Assay
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