rClone: A Synthetic Biology Tool That Enables the Research of Bacterial Translation

Abstract

Celebrating 20 years of undergraduate research mRNA, called the ribosome binding site (RBS; Figure 1). After the small ribosomal subunit binds to the RBS, the large ribosomal subunit attaches to the small subunit to begin translation of the mRNA into a chain of amino acids. The mRNA bases are read as triplet codons that interact by base pairing with anticodons in transfer RNA (tRNA) molecules, which carry amino acids to the growing protein chain (Malys & McCarthy, 2010). As shown in Figure 1, RNA-RNA base pairing typically involves the Watson-Crick base pairs of G with C, and A with U, but G can also base pair with U. The conventional understanding is that the strength of a given RBS is determined by the strength of its base pairing interactions with the 16S rRNA (Shine & Dalgarno, 1974). In natural bacterial genomes, there is a wide variety of RBS sequences and RBS translational strengths that have resulted from natural selection for global patterns of gene expression. The relationship between RNA base pairing and the strength of an RBS also explains how synthetic RBSs can be produced with widely varying strengths.In addition to intermolecular base pairing, intramolecular base pairing affects the strengths of RBSs. The ability of RNA to engage in intramolecular base pairing is well established (Busan & Weeks, 2013). RBS elements can be disabled by intramolecular RNA folding, as is the case in riboswitches (Breaker, 2012). The RNA in riboswitches adopts an OFF state when the RBS is bound by a complementary anti-RBS sequence within the mRNA. For the ON state, a small molecule ligand binds to the folded RNA and changes the RNA shape so that the RBS is available for interaction with the 16S rRNA.Understanding the function of RBSs informs the discipline of synthetic biology, which uses engineering principles and molecular cloning methods for the construction of parts, devices, and systems, INTRODUCTIONGene expression, the process by which the inherited information of genes is used to direct the function of cells, is regulated in all cells because not all genes are needed all the time or under all circumstances (Hijum, Medema, & Kuipers, 2009). Gene expression begins with transcription, the process by which the DNA base sequence of a gene is converted into RNA sequence information. For genes that encode proteins, the messenger RNA (mRNA) product of transcription is used during translation to encode the sequence of amino acids in a protein. The sequence of bases in mRNA is translated by the ribosome, which is composed of a large (50S) and a small (30S) subunit. Translation is initiated when the 16S ribosomal RNA (rRNA) of the small ribosomal subunit base pairs to a conserved sequence in the Understanding bacterial translation is of interest to many high school and college biology students, but hands-on research of this process has traditionally been inaccessible because of the expertise required for molecular cloning. There is also a need for more and better translational control elements that can be used for genetic ...

A portable epigenetic switch for bistable gene expression in bacteria

“…The antibiotic resistance genes chosen for these experiments were aac3-Ib (henceforth, aac3) and aac(6 ′ )-Ib-cr (henceforth, aac6 ), which encode aminoglycoside acetyl transferases 18 , and bla CTX-M-15 (henceforth, ctxM) , which encodes an extended-spectrum β-lactamase 19 . In these constructs, the native ribosome binding sites were replaced with a stronger RBS, named BI 20 to adjust the sensitivity of the switch to a level that could permit unambiguous detection of the antibiotic resistance phenotype under study, thus facilitating discrimination between OFF and ON cells. Experiments with strains carrying P opvAB :: aac6 :: gfp and P opvAB :: ctxM :: gfp fusions (strains SV9703 and SV9706, respectively) yielded bacterial subpopulations resistant to kanamycin and to cefotaxime, respectively (Fig.…”

“…Modification of the ribosome-binding site of genes under P opvAB control can also contribute to adjust the sensitivity of the switch, facilitating detection of the phenotype under study. For instance, use of the BI ribosome binding site 20 permitted unambiguous detection of aac3 -mediated kanamycin resistance, thereby facilitating discrimination of Km r cells (Fig. 4).…”

rClone Red facilitates bacterial gene expression research by undergraduates in the teaching laboratory

“…In addition to its utility for CUREs, the simple elegance of the rClone Red design also makes it amendable for undergraduates or high schoolers who want to have summer undergraduate research experiences (SUREs) ( 16 , 26 ). There are several interesting areas of investigation that students could explore with rClone Red.…”

“…Because E. coli has more than one 16S rRNA gene, students could design RBS sequences that target one or more of the seven rRNA paralogous operons ( 27 , 28 ). Students could generate libraries of potential RBS sequences by inserting 1-8 N bases during oligonucleotide synthesis and selecting RBS strengths for specific desired applications ( 16 ). Another application of rClone Red is using it for testing the specificity of a riboswitch and its potential for translational actuation ( 29 ).…”

“…In this education research, we describe how rClone Red can be used by students to design and test RBSs. Previously, we described rClone Red in a basic research publication ( 16 ). Independent research students working outside laboratory courses conducted research to examine different RBS designs and ‘anti-RBS’ sequences within the mRNA that reduced reporter protein production.…”