The Spinach RNA Aptamer as a Characterization Tool for Synthetic Biology

Abstract

8Characterisation of genetic control elements is essential for the predictable engineering 9 of synthetic biology systems. The current standard for in vivo characterisation of control 10 elements is through the use of fluorescent reporter proteins such as green fluorescent 11 protein (GFP). Gene expression, however, involves not only protein production but also 12 the production of messenger RNA (mRNA). Here we present the use of the Spinach 13 aptamer sequence, an RNA mimic of GFP, as a tool to characterise mRNA expression in 14 Escherichia coli. We show how the aptamer can be incorporated into gene expression 15 cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for 16 the first time, simultaneous measurement of mRNA and protein levels from engineered 17 constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding 18 site sequences and promoters and use it to highlight the differences in the temporal 19 behaviour of transcription and translation. 20 21

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

“…In addition to expression in E. coli (46,50,54,63), several RNAs have been expressed as fusion RNAs and imaged in mammalian cells, including 5S, a small ncRNA transcribed by RNA polymerase III that associates with the large ribosomal subunit (46); 7SK, an ncRNA that associates with transcription complexes; and CGG-repeat RNAs, which are linked to fragile X-associated tremor and ataxia syndrome (FXTAS) (57). In each case, fluorescence was not detectable in cells expressing a control RNA, whereas fluorescence was detected in cells expressing the imaging tag (57).…”

Developments in the Tools and Methodologies of Synthetic Biology

“…Recent models of flow cytometers can simultaneously measure cell size, complexity, and up to 17 channels of fluorescence (Basiji et al, 2007; Piyasena and Graves, 2014), each of which could be used to capture data from different reporter outputs. Of the biological reporters available, RNA aptamers are particularly noteworthy, since they have the potential to increase the type and range of biological information that can be measured (Cho et al, 2013; Pothoulakis et al, 2014). For instance, several groups have reported the simultaneous measurement of both transcription (mRNA levels) and translation (protein levels) (Chizzolini et al, 2013; Pothoulakis et al, 2014).…”

“…Of the biological reporters available, RNA aptamers are particularly noteworthy, since they have the potential to increase the type and range of biological information that can be measured (Cho et al, 2013; Pothoulakis et al, 2014). For instance, several groups have reported the simultaneous measurement of both transcription (mRNA levels) and translation (protein levels) (Chizzolini et al, 2013; Pothoulakis et al, 2014). In both cases Spinach, an RNA aptamer that binds a fluorophore (Paige et al, 2011), was incorporated within the 3′ untranslated region (UTR) of a fluorescent reporter protein, either GFP or RFP (Chizzolini et al, 2013; Pothoulakis et al, 2014).…”

“…For instance, several groups have reported the simultaneous measurement of both transcription (mRNA levels) and translation (protein levels) (Chizzolini et al, 2013; Pothoulakis et al, 2014). In both cases Spinach, an RNA aptamer that binds a fluorophore (Paige et al, 2011), was incorporated within the 3′ untranslated region (UTR) of a fluorescent reporter protein, either GFP or RFP (Chizzolini et al, 2013; Pothoulakis et al, 2014). Providing there is no spectral-overlap between fluorophores, this strategy could conceivably be up-scaled to measure entire synthetic pathways, and thus inform operon design strategies (Hiroe et al, 2012; Chizzolini et al, 2013).…”

A Spinach molecular beacon triggered by strand displacement

“…In particular, a RNA aptamer (Spinach) that binds to the fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) has been shown to lead to a large increase in green fluorescence emission. Spinach has been adapted to act as a genetically encoded sensor for RNA transcription in cells (Pothoulakis et al 2013), as an imaging agent, as a reagent for intracellular detection of small molecule analytes such as adenosine 5 ′ -diphosphate (ADP) and S-adenosylmethionine (SAM) (Paige et al 2012), and as a sensor for proteins such as streptavidin, thrombin, and the MS2 coat protein (Song et al 2013).…”