title: Using Spinach aptamer to correlate mRNA and protein levels in Escherichia coli.
abstract: In vivo gene expression measurements have traditionally relied on fluorescent proteins such as green fluorescent protein (GFP) with the help of high-sensitivity equipment such as flow cytometers. However, fluorescent proteins report only on the protein level inside the cell without giving direct information about messenger RNA (mRNA) production. In 2011, an aptamer termed Spinach was presented that acts as an RNA mimic of GFP when produced in Escherichia coli and mammalian cells. It was later shown that coexpression of a red fluorescent protein (mRFP1) and the Spinach aptamer, when included into the same gene expression cassette, could be utilized for parallel in vivo measurements of mRNA and protein production. As accurate characterization of component biological parts is becoming increasingly important for fields such as synthetic biology, Spinach in combination with mRFP1 provide a great tool for the characterization of promoters and ribosome binding sites. In this chapter, we discuss how live-cell imaging and flow cytometry can be used to detect and measure fluorescence produced in E. coli cells by different constructs that contain the Spinach aptamer and the mRFP1 gene.
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London, United Kingdom; Department of Bioengineering, Imperial College London, South Kensington Campus, London, United Kingdom.
authors.forename: Georgios
authors.initials: G
authors.lastname: Pothoulakis
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London, United Kingdom; Department of Bioengineering, Imperial College London, South Kensington Campus, London, United Kingdom. Electronic address: t.ellis@imperial.ac.uk.
authors.forename: Tom
authors.initials: T
authors.lastname: Ellis
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D018724:Spinacia oleracea / Q000235:genetics*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D012313:RNA; D012333:RNA, Messenger; D000097573:Red Fluorescent Protein
keywords: Aptamer; Flow cytometry; Fluorescence; Live-cell imaging; Measurement; Promoter; Ribosome binding site; Spinach RNA