Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii
Fig 5
Effects of intron addition on transgene expression levels of the codon-optimized P. cablin Benth. patchoulol synthase (PcPs) gene.
(A) Vector design of intron-containing construct variants in the modified pOptimized vector (vector A-G, sequence S3 Data). (B) Initial fluorescence screening of 576 isolated transformants. Absolute signals were grouped into three intensity levels: low (850–1500 relative fluorescence units, rfu), medium (1500–3000 rfu) and high (>3000 rfu). Numbers indicate the respective percentage of the initial population. (C) The 20 best transformants from the initial population were isolated and expression was quantified as mean fluorescence per cell, relative mRNA abundance (RTqPCR), protein titer (WB–western blot, α-Strep-tag II with Coomassie Brilliant Blue (CBB) as loading control) and patchoulol production (GC-MS). Cultivations were conducted in microtiter plates and transformants were pooled according to their respective cell densities prior to analysis. Samples of strain UVM4 served as the respective parental control. Error bars represent standard deviations from the mean of triplicate measurements of pooled 20 transformants. PPSAD−promoter and 5’UTR of the C. reinhardtii PSAD gene, mVenus–yellow fluorescent protein variant, StrepII–Strep-tag II epitope.