Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression

Abstract

SUMMARY We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.

Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain

“…The use of a purified, commercially available GFP as reference protein was also our first choice for quantification of the GFP-Fc1 fusion, however this method inflated accumulation measurements almost 9-fold. Heterologous protein expression at these levels should produce a heavy band in the Coomassie gels and a metabolic burden on the transplastomic plants, as our previous experience shows 26 but neither of which occurred here. We found greater accuracy using the previously characterized MRR13 transplastomic tobacco line as a reference for measuring GFP-Fc1 accumulation.…”

“…This is because the N-terminus of IGF-I is important for receptor engagement 23 . To avoid this issue, we look to generate an additional system for oral delivery of therapeutic proteins using the Immunoglobulin G (IgG) fragment crystallizable (Fc) domain for fusion of recombinant protein at the C-terminus, and a chloroplast expression system that consistently delivers high expression levels [24][25][26][27] . We generated chloroplast transformation vectors carrying recombinant proIGF-I and Mstn propeptide fused to the IgG fragment crystallizable (Fc) domain to promote absorption through the epithelial lining of the small intestine 28-30 .…”

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

“…A construct employing the leader sequence of the Bacillus thuringiensis cry9Aa2 gene served as a control; L cry9Aa2 was previously shown to yield only stable dicistronic mRNA( Fig. 1C , pMRR13 ) (Yu et al, 2017; Yu et al, 2020). We previously reported results in tobacco for the monocistronic and dicistronic constructs harboring the maize L atpH GG UTR (pQY3 and pAI5, respectively) (Rojas et al, 2019) but they were analyzed again here as a point of comparison for other constructs.…”

“…3A ). The control, the cry9Aa2 5’ UTR in the aadA - gfp intergenic region, has been shown previously not to yield processed monocistronic RNA in plastids (Yu et al, 2020). The monocistronic gfp mRNA with the wild type tobacco atpH 5’ UTR is ∼4 times more abundant, indicating more efficient protection of the mRNA by the cognate wild type tobacco PPR10 protein ( Di-L atpH Nt , Fig.…”

Genetic Approaches to Enhance Multiple Stress Tolerance in Maize

“…The number of traits and genes that will need to be transformed or crossed into elite maize lines will grow in the future. Besides the current crossbreeding strategy, other advanced techniques for introducing large number of genes are in the pipeline, including the following: polycistronic expression in chloroplast [152], Binary Bacterial Artificial Chromosomes [153] or mini chromosomes [154,155]. However, all of these approaches still await routine use.…”