Inducible Genetic Code Expansion in Eukaryotes

Abstract

Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.

Keywords: PylRS; T-REx; Tet-On; amber suppression; unnatural amino acid.

Scheme 1

Overview of T‐REx and Tet‐On systems in combination with GCE technology. Top: Upon addition of tetracycline (tet), the tet‐inducible repressor protein (TetR) unbinds the TetO signals facilitating polymerase (Pol II and Pol III) binding to the promoter. Production of tRNA and translation of the synthetase are inducible in the T‐REx system, illustrated by the flow cytometry schemes. The full‐length reporter can only be expressed in the presence of tet and ncAA (yellow diagonal ellipse), but not upon addition of ncAA alone (red vertical ellipse). The promoter sequence, containing a 2xTetO signal (red box with arrow) leads to expression of the tRNA or synthetase (shown in brown). Bottom: In the Tet‐On system, the reverse Tet‐repressor protein (rtTA) can bind to the TetO signals in response to doxycycline (dox), enhancing the binding of Pol II; this enables inducible translation of the synthetase gene, but not a controllable production of tRNA, as illustrated by the flow cytometry schemes. The yellow ellipse represents full‐length production of the reporter, whereas the red vertical ellipse illustrates the expression of iRFP alone. An 8xTetO‐promoter (blue box with arrow) is positioned in front of the tRNA and synthetase gene (shown in brown).

Figure 1

The inducible T‐REx system. a) Six different Pol III promoter sequences based on the U6 or H1 promoter are shown. TetO signals (violet circles) are added to specific sites in the promoter. U6‐TetO contains two TetO signals, one before and one after the TATA box. 8xTetO‐U6 harbors eight TetO signals in front of the distal and proximal sequence elements (DSE and PSE). The 8xTetO‐U6‐TetO includes the 8xTetO signals before the DSE element and the two TetO signals framing the TATA box. The H1 sequences contain the same pattern as the U6 sequences, but the backbone originating from the H1 promoter. b)–d) HEK Flp‐In T‐REx 293 cells expressing reporter protein iRFP‐GFP^Y39ncAA using the T‐REx system. b) Measuring the geometric mean (GM) of iRFP and GFP by fluorescence flow cytometry (FFC) using the reporter gene (iRFP‐GFP^Y39ncAA), the synthetase under CMV promoter in combination with the different Pol III promoters (U6 and H1) with tet and BocK (dark gray) or only with BocK (light gray). The bar plot visualizes how inducible the different Pol III promoters are. c) Bar plot resulting from FFC measurements of the reporter protein using the inducible synthetase gene with and without an inducible tRNA gene. The error bars in b) and c) indicate the standard error of the mean and are calculated from at least three independent measurements. d) FFC data of reporter gene expressed with inducible synthetase construct (2xTetO‐CMV‐NES‐PylRS^AF) together with noninducible 8xTetO‐U6‐TetO‐tRNA construct. The upper panel shows expression with tet and BocK, the lower just with BocK.

Figure 2

Inducibility of the Tet‐On system in HEK293T cells. a) Bar plot illustrating the ratio between the geometric mean (GM) of GFP and iRFP resulting from FFC data measuring expression of iRFP‐GFP^Y39ncAA with synthetase under CMV promoter in combination with the different Pol III‐driven promoter constructs (U6) with and without dox in presence of BocK. The last two columns show the data for the Tet‐On system in the presence of the 8xTetO‐CMV‐NES‐PylRS^AF construct. None of the three U6 promoters is inducible through the Tet‐On system, whereas synthetase expression can be induced by using the Tet‐On system. The error bars indicate the standard error of the mean and are calculated from at least three independent measurements. b) FFC data showing expression of iRFP‐GFP^Y39ncAA. Left: data from the 8xTetO‐U6 construct with and without dox (top and bottom, respectively) in the presence of BocK. Right: the inducibility of the GCE system when using the 8xTetO‐CMV‐NES‐PylRS^AF construct together with the 8xTetO‐U6 construct.