Glucose monitoring in living cells with single fluorescent protein-based sensors

Glucose is the main source of energy and carbon in organisms and plays a central role in metabolism and cellular homeostasis. However, the sensitive fluctuation of glucose in living cells is difficult to monitor. Thus, we developed a series of ratiometric, highly responsive, single fluorescent protein-based glucose sensors of wide dynamic range by combining a circularly permuted yellow fluorescent protein with a bacterial periplasmic glucose/galactose-binding protein. We used these sensors to monitor glucose transport in living Escherichia coli cells, and found that the cells take up glucose within 10 min to maintain physiological glucose levels, and observed the differences in glucose uptake and glucose metabolism between wild-type and Mlc knockout cells. These sensors can be specific and simple tools for glucose detection in vitro and non-invasive tools for real-time monitoring of glucose metabolism in vivo.


Introduction
Glucose is the main source of energy and carbon in most organisms, from bacteria to humans. Changes in glucose uptake, release, and metabolism are associated with regulation of various physiological and pathological phenomena, such as cell growth, cell proliferation, cell differentiation, cell death, insulin secretion, obesity, and diabetes. 1-3 Therefore, sensitive and selective measurement of glucose has become signicant. Radiolabeling, chromatography, and mass spectrometry have been used effectively to quantify glucose. 4 However, these methods have limited spatiotemporal resolution and are unsuitable for real-time imaging of glucose metabolism in living cells.
Recently, we have reported a series of metabolite sensors, including NADH (Frex), 14 NAD+/NADH ratio (SoNar), 15,16 NADPH (iNap) 17 and histidine (FHisJ) 18 based on circularly permuted yellow uorescent protein (cpYFP). In cpYFP, the original N-and C-termini are fused by a polypeptide linker, and new termini are introduced close to the uorophore, making its uorescence highly sensitive to the protein's conformation. 14-18 Compared with FRET-based sensors, cpYFP-based sensors oen have larger uorescence changes; in specic, Frex, 14 SoNar, 15,16 iNap 17 and FHisJ 18 sensors have more than 500% dynamic range, rendering them capable of tracking subtle metabolic changes.
To maximize the existing technical advantages, we inserted cpYFP into three linker locations of GGBP and developed a series of ratiometric, highly responsive, single uorescent protein-based glucose sensors, denoted as FGBP (uorescent GGBP). Among them, FGBP 1 mM sensor with a binding constant (K d ) of 1.0 mM exhibited sevenfold uorescence ratio changes and t physiological applications.

Plasmid construction and strains
To generate single uorescent protein-based glucose indicators, the MglB DNA sequence coding for the mature GGBP (positions 70-927 relative to ATG) was amplied from E. coli genomic DNA by PCR with the primers P1 and P2 (sequences available in Table S1 †) and cloned into BamHI-HindIII sites in the pRSETb vector (Invitrogen). The GGBP-cpYFP insertion variants were constructed by overlap PCR 19 using wild-type GGBP sequence and cpYFP from Frex. 14 First, the coding sequences of N and C terminal domain of GGBP, and cpYFP were amplied using the primers P1 and PR, PF and P2, and P3 and P4, respectively (sequences available in Table S1 †). Second, the chimeric construct consisting of GGBP and cpYFP was produced using an overlapping PCR with the primers P1 and P2. This product was cloned into the BamHI/HindIII sites of pRSETb (Invitrogen), yielding pRSETb-FGBP (GGBP and cpYFP chimeras) ( Fig. S1A and S1B †). We truncated the N and C terminal amino acid residue of cpYFP to expand the dynamic range of FGBP by PCR (Fig. S1C †). To improve the affinity of the sensors, sited directed mutagenesis of FGBP 27 mM was generated by PCR (Fig. S1D †). The electrophoresis data showed the protein size of different sensors (Fig. S1E †).
Mlc knockout strain is a derivative of E. coli JM109 (DE3). Mlc was deleted according to the method of Datsenko and Wanner 20 using plasmid pKD4 or pKD3, leaving the start codon and seven codons at the 3 0 end of the target gene. The resistance cassettes were eliminated as described previously. 20 To overexpress Mlc, the Mlc gene was amplied from E. coli genomic DNA by PCR and cloned into BamHI-HindIII sites in the pCDFDuet1 vector (Novagen).

Library construction
Preliminary truncations of the N-and C-terminals of cpYFP indicated that deletions beyond ve AAs of the N-and Cterminals (data not shown) caused misfoldings. Therefore, the library was limited to the deletions of four AAs of the N-and Cterminals. Truncation combinations were amplied with Primers STAR HS DNA polymerase (Takara) and fused by T4 DNA ligase (Fermentas). Mutants with different affinities were engineered in FGBP 27 mM , using overlap PCR, 19 and then transferred to the pRSETb vector. The DNA sequence and amino acid sequence of FGBP 1 mM are in the "ESI note †" section.

Protein expression and in vitro characterization
All recombinant proteins with a His 6 -tag were expressed in E. coli JM109 (DE3) by the pRSETb expression plasmid as previously described. 18 Proteins were puried with Ni-NTA His SpinTrap column.
Spectral measurement was performed in 20 mM MOPS buffer (pH 7.4) by using a spectrouorometer (EnSpire). Excitation spectra were recorded between 350 and 510 nm, and emission at 528 nm as previously described. 14,15 For glucose titration, the protein was diluted in 20 mM MOPS buffer (pH 7.4) to a nal concentration of 1 mM. The uorescence value of protein was measured by a lter-based Synergy 2 Multi-Mode microplate reader using 400 BP 10 nm, 485 BP 20 nm excitation, and 528 BP 20 nm emission (BioTek). The ratio (R) was dened as the uorescence intensity at 485 nm divided by the intensity at 400 nm.
The K d of each glucose sensor was determined by tting to a single-site binding isotherm: where S is the fraction of sensor saturation, [L] is the concentration of glucose, R is the uorescence ratio 485/400 of sample, R apo is the ratio in the absence of ligand, and R sat is the ratio at saturation with ligand.

Monitoring glucose transport in E. coli cells
To monitor glucose transport in living cells, E. coli JM109 (DE3) cells carrying pRSETb-FGBP 1 mM were grown in Luria-Bertani medium containing 100 mg ml À1 ampicillin at 37 C until the cultures reached about 0.6-0.8 OD.

Generation of cpYFP-based sensors for glucose
To engineer a cpYFP-based indicator for glucose, bacterial periplasmic GGBP 11-13 was chosen as a suitable glucose-binding detector because of the following ndings: (1) glucose binds to GGBP with high specicity 11,12 and (2) glucose binding to GGBP results in a dramatic conformational change demonstrated by X-ray and NMR analyses, 13 as shown in Fig. 1A. In addition, GGBP was used as a sensor domain of FRET-based glucose indicators as previously reported. 8,9 According to crystallographic structures of GGBP, 13,18 chimeric proteins were generated by inserting cpYFP into the three exible linker regions of GGBP, namely, Gly109-Glu114, Thr253-Asn256, and Val293-Val296 ( Fig. 1A and B). Among them, the chimera with cpYFP inserted between Pro294 and Tyr295 of GGBP showed a 2.1-fold increase in the ratio of uorescence when excited at 485 and 400 nm upon glucose addition (Fig. 1C, Table 1). Fluorescence titration studies showed that the Pro294/Tyr295 chimera named FGBP 3.1 mM had an apparent dissociation constant (K d ) $3.1 mM for glucose at pH 7.4 (Fig. 1D, Table 1). Similar to other cpYFP-based sensors, 17,18 FGBP 3.1 mM has two typical excitation peaks around 420 and 500 nm and one emission peak near 515 nm (Fig. 1E).

Optimization improves the responsiveness and affinity of FGBP sensors
To maximize the response magnitude of sensors, we created a cpYFP-terminal truncation library between GGBP and cpYFP of FGBP 3.1 mM (Fig. 2A) and found that the FGBP 3.1 mM variant N3C4 manifests the most dramatic increase in the presence of glucose, as measured by the ratio of uorescence excited at 485 and 420 nm (Fig. 2B). Fluorescence titration studies showed that the N3C4 variant (named FGBP 27 mM ) had an apparent K d of $27 mM for glucose ( Fig. 2C and Table 1).
Physiological glucose level has been estimated in the range of 0.4-24 mM, such as 2-5 mM in plants, 7 1.5 mM in yeast, 21 3-9 mM in blood, 22 1-10 mM in liver, 23 and 0.4-24 mM in the intestine, 24 far exceeding the dissociation constants of FGBP 27 mM for glucose. To tune the affinity of the FGBP 27 mM sensor, we further created variants of the sensor with single site-directed mutagenesis of three key amino acid residues around the glucose binding pocket (Fig. 3A). [25][26][27] The three mutants, i.e., N256S, L238S, and A213R, had different affinities, with apparent K d values of $380 mM, $1.0 mM, and $3.2 mM, and were denoted as FGBP 380 mM , FGBP 1 mM , and FGBP 3.2 mM , respectively ( Fig. 3B and Table 1). Considering the maximum uorescence ratio change, affinity, and expression level, we chose FGBP 1 mM , which covers the physiological blood glucose range, for further characterization (Table 1). Similar to FGBP 3.1 mM , FGBP 1 mM also has two typical excitation peaks around 420 and 500 nm and one emission peak near 515 nm (Fig. 3C). Upon glucose binding, the uorescence of FGBP 1 mM excited at 485 nm showed a 6.5-fold increase, and the uorescence excited at 400 nm was almost constant (Fig. 3D). Apart from glucose and galactose, none of the other sugars tested induced a signicant change in ratio at 1 and 100 mM concentrations (Fig. 3E), showing the high selectivity of FGBP 1 mM toward glucose. Glucose is expected to be present in signicantly higher concentrations than galactose; thus, FGBP 1 mM is suitable for glucose monitoring in living cells.
Similar to all other genetically encoded sensors based on cpYFP, FGBP 1 mM depended on pH when excited at 485 nm; however, FGBP 1 mM uorescence excited at 400 nm is much more pH resistant (Fig. 4A). At modest pH uctuations, the pH effects of FGBP 1 mM can be corrected by measuring the   uorescence of FGBP 1 mM and cpYFP in parallel, due to their very similar pH responses (Fig. 4B).

Real-time monitoring of intracellular glucose in living cells
The ability of transport glucose across the plasma membrane is a common feature to nearly all cells, from the simple bacterium to the highly compartmented mammalian cells. 28 To test the ability of FGBP 1 mM to report changes in intracellular glucose levels, we expressed the FGBP 1 mM sensor in living E. coli JM109 (DE3) cells. Fluorescence was uniform throughout the cell, suggesting that this sensor was located in the cytoplasm but not cell surface (Fig. 5A). Addition of exogenous glucose into the culture medium induced a rapid, dose-dependent, and saturable increase in the uorescence ratio (Fig. 5B-E), whereas addition of glucose analogs had no effect on uorescence (Fig. 5C). This nding suggested that glucose was readily transported across the cell membrane of these bacteria. Michaelis-Menten tting of FGBP 1 mM 's uorescence ratio versus extracellular glucose concentration produced a K 0.5 of $0.3 mM (Fig. 5E), which is much higher than the K m for ptsG in E. coli, the high-affinity glucose transporter in the plasma membrane. 29 By contrast, only slight changes in the uorescence ratio were observed in E. coli JM109 (DE3) cells expressing cpYFP instead of FGBP 1 mM when glucose was added to the cell culture medium (Fig. 5F and G), excluding the possibilities of uorescence interference of pH variations of the cpYFP domain.
The phosphotransferase system (PTS) is the major sugar transport system in many Gram-positive and Gram-negative bacterial species; however, expression of ptsG is repressed by the Mlc (making large colonies) protein. 30 To investigate the role of Mlc on glucose transport, we constructed Mlc knockout E. coli JM109 (DE3) cells. Compared with wild-type cells, we surprisingly found that glucose-induced the increase of uorescence gradually returned to basal levels as the extracellular glucose was consumed in Mlc knockout cells (Fig. 5D and H), and Mlc   overexpression rendered the similarity in metabolic kinetics of these cells (Fig. 5I). These results imply that Mlc expression level not only regulates glucose uptake but also inuences the rate of glucose metabolism.

Conclusions
In this work, we reported a series of ratiometric, highly specic, highly sensitive, and single uorescent protein-based glucose sensors with different affinities. Among them, FGBP 1 mM can detect glucose in the range of 0.02-40 mM, which covers the physiological glucose concentration in organisms. FGBP 1 mM displays a large dynamic range and is very useful for the realtime tracking of subtle changes in cell metabolism. FGBP 1 mM displays a $700% uorescence change in vitro, almost 10-fold greater than that of previously reported FRET-based glucose sensors, 5-10 rendering it a highly responsive genetic-encoded sensor. Compared with FRET-based glucose sensors, FGBP sensors only have one uorescent protein and are intrinsically ratiometric, allowing the built-in normalization of the uorescence signals irrespective of variations in indicator protein concentrations. Considering the admirable properties of these sensors, we believe that FGBP sensors could be good alternatives to existing methods for intracellular glucose detection.

Conflicts of interest
The authors have declared no conicts of interest.