Fig. 1
From: Caffeine-inducible gene switches controlling experimental diabetes
Synthetic biology-inspired genetic circuits for caffeine-induced gene expression. a Caffeine-inducible protein dimerization system based on the camelid-derived single-domain antibody aCaffVHH. aCaffVHH homodimerizes in the presence of caffeine and can be used to reconstitute synthetic transcription factors or signaling cascades that fine-tune caffeine-responsive gene expression. b Caffeine-sensing circuit based on the heterodimerization of aCaffVHH-TetR (pDB307) and aCaffVHH-VPminx4 (pDB335), leading to direct transcriptional activation. The caffeine dose-response relationship was quantified with the reporter gene SEAP (PtetO7-SEAP-pASV40, pMF111). c Caffeine-sensing circuit based on the IL13 receptor and the JAK/STAT6 pathway. Caffeine-induced heterodimerization of aCaffVHH-IL13Rα1 (pDB323) and aCaffVHH-IL4Rα (pDB324) leads to phosphorylation of STAT6 (pLS16) by JAK kinases and subsequent transcriptional activation of the STAT6-responsive promoter PSTAT6. The caffeine dose-response relationship was quantified with the reporter gene SEAP (PSTAT6-SEAP-pASV40, pLS12). d Caffeine-sensing circuit based on the MAPK pathway. Caffeine-induced homodimerization of mFGFR1405-822-aCaffVHH (pDB395) led to phosphorylation of MEK1/2 and downstream signaling of the MAPK cascade. Rewiring the signaling cascade through the hybrid transcription factor TetR-Elk1 (MKp37) led to expression of the reporter gene SEAP (PtetO7-SEAP-pASV40, pMF111), enabling quantification of the caffeine dose-response relationship. e Caffeine-sensing circuit based on the Epo receptor and the JAK/STAT3 pathway. Caffeine-induced homodimerization of aCaffVHH-EpoRm-IL-6RBm (pDB306) leads to phosphorylation of STAT3 by JAK kinases and subsequent transcriptional activation of the STAT3-responsive promoter PSTAT3. The caffeine dose-response relationship was quantified with the reporter gene SEAP (PSTAT3-SEAP-pASV40, pLS13). Data in (b–e) are shown as the mean in bar graphs and symbols indicate individual data points. The data displayed represent three independent experiments (n = 3)
