In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises an amino acid sequence having a 90% sequence identity to EcPhnD. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 95% sequence identity to EcPhnD. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 97% sequence identity to EcPhnD. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 98% sequence identity to EcPhnD. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 99% sequence identity to EcPhnD. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 100% sequence identity to EcPhnD.

In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises an amino acid sequence of at least 290, 295, 300, 305, 310, 315, or 320 amino acids. In some embodiments, the amino acid sequence comprises between 290 and 320 amino acids, between 295 and 315 amino acids, between 300 and 315 amino acids, between 305 and 315 amino acids, or between 310 and 315 amino acids. In some embodiments, the engineered protein biosensor comprises 300, 305, 310, 312, or 315 amino acids. In some embodiments, the amino acid sequence comprises at least 300 amino acids. In some embodiments, the amino acid sequence comprises 312 amino acids.

In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises (i) an amino acid sequence of 312 amino acids having at least a 90% sequence identity to SEQ ID NO: 1, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a sequence identity of between about 85% to about 99%, about 86% to about 99%, about 87% to about 99%, about 88% to about 99%, about 89% to about 99%, about 90% to about 99%, about 91% to about 99%, about 92% to about 99%, about 93% to about 99%, about 94% to about 99%, about 95% to about 99%, about 96% to about 99%, about 97% to about 99%, about 98% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 96%, about 90% to about 95%, about 95% to about 98%, or about 95% to about 97% to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a sequence identity of up to 99%, up to 98%, up to 97%, up to 96%, up to 95%, up to 94%, up to 93%, up to 92%, up to 91%, or up to 90% to SEQ ID NO: 1.

In some embodiments, the engineered protein biosensor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 95% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises an amino acid sequence having a 90% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 95% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 97% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 98% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 99% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered protein biosensor comprises an amino acid sequence having a 100% sequence identity to SEQ ID NO: 1.

In some embodiments, the amino acid sequence comprises 312 amino acids having at least a 90% sequence identity to SEQ ID NO: 1, and wherein the amino acid is asparagine at position 177 of the amino acid sequence. In some embodiments, the amino acid is cysteine at position 126. In some embodiments, the amino acid sequence comprises 312 amino acids having at least a 90% sequence identity to SEQ ID NO: 1, and wherein the amino acid is asparagine at position 177 and the amino acid is cysteine at position 126 of the amino acid sequence.

In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises (i) an amino acid sequence comprising 312 amino acids having at least a 90% sequence identity to SEQ ID NO:1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the amino acid sequence comprising 312 amino acids having at least 92% sequence identity to SEQ ID NO: 1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the amino acid sequence comprising 312 amino acids having at least 95% sequence identity to SEQ ID NO: 1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the amino acid sequence comprising 312 amino acids having at least 97% sequence identity to SEQ ID NO: 1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the amino acid sequence comprising 312 amino acids having at least 98% sequence identity to SEQ ID NO: 1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence. In some embodiments, the amino acid sequence comprising 312 amino acids having at least 99% sequence identity to SEQ ID NO: 1, and wherein the amino acid position 177 is an asparagine and amino acid position 126 is a cysteine, and (ii) a reporter coupled to the amino acid sequence.

In some embodiments, the reporter is coupled to the cysteine at position 126. In some embodiment, the reporter is covalently bound to the cysteine at position 126.

In some embodiments, the engineered protein biosensor having an N-terminal and a C-terminal comprises (i) an amino acid sequence comprising 312 amino acids and having at least a 95% sequence identity to SEQ ID NO: 1; wherein the amino acid at position 177 is an asparagine and the amino acid at position 126 is a cysteine; and (ii) a reporter coupled to the amino acid at position 126 of the amino acid sequence.

In some embodiments, the amino acid sequence further comprises a modification to the C-terminal of the engineered protein. In some embodiments, the C-terminal is truncated to remove the ability of the protein to form a dimer. In some embodiments, the C-terminal is truncated to remove the last nine amino acids of the amino acid sequence. In some embodiments, the C-terminal further comprises a maker. In some embodiments, the marker is a histidine tag.

In some embodiments, the reporter is a fluorescent reporter. In some embodiments, the fluorescent reporter is selected from the group consisting of acrylodan, Coumarin (referred to as (7-Diethylamino-3-(4′-Maleimidylphenyl)-4-Methylcoumarin)), danzyl aziridine, 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole ester (IANBDE), 4-N-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole (IANBDA), Texas Red C2-Maleimide, Lucifer yellow iodoacetamide, Alexafluor 680 maleimide, Kodak X-Sight 670 LSS dye, Texas Red, C5-Bromoacetamide, Alexa Fluor 750 C5-maleimide, and BODIPY 577/618. In some embodiments, the fluorescent reporter is acrylodan or Coumarin.

In some embodiments, the reporter is coupled to the amino acid sequence. In some embodiments, the reporter is covalently bound to the amino acid sequence. In some embodiments, the reporter is covalently bound to the cysteine at position 126 of the amino acid sequence. In some embodiments, the fluorescent reporter is coupled to the amino acid sequence of the engineered protein. In some embodiments, acrylodan is covalently bound to the cysteine at position 126 of the amino acid sequence of the engineered protein biosensor. In some embodiments, Coumarin is covalently bound to the cysteine at position 126 of the amino acid sequence of the engineered protein biosensor.

In some embodiments, an engineered protein biosensor having an N-terminal and a C-terminal, comprises (i) an amino acid sequence having 312 amino acids having at least a 95% sequence identity to SEQ ID NO:1, wherein the amino acid at position 177 is an asparagine and the amino acid at position 126 is a cysteine; and (ii) an acrylodan covalently bound to the cysteine at position 126.

In some embodiments, an engineered protein biosensor having an N-terminal and a C-terminal, comprises (i) an amino acid sequence having 312 amino acids having at least a 95% sequence identity to SEQ ID NO:1, wherein the amino acid at position 177 is an asparagine and the amino acid at position 126 is a cysteine; and (ii) a coumarin covalently bound to the cysteine at position 126.

Method of Detecting Glyphosate with the Biosensor

In some embodiments, a method of detecting glyphosate comprises (i) contacting a sample with an engineered protein biosensor comprising (a) an amino acid sequence and (b) a reporter coupled to the amino acid sequence; and (ii) visualizing the sample for the presence of glyphosate. In some embodiments, a method of detecting glyphosate comprises (i) contacting a sample with an engineered protein biosensor comprising (a) an amino acid sequence and (b) a reporter coupled to the amino acid sequence; (ii) irradiating or exposing the engineered protein biosensor to a light of a wavelength that can excite the reporter; and (iii) detecting the wavelength emitted from the excited reporter. In some embodiments, the detected emitted wavelength will change depending on whether glyphosate is present in the sample. In some embodiments, a shift in the wavelength emitted correlates to the concentration of glyphosate in the sample.

In some embodiments, the method further comprises contacting the engineered protein with an antibody fragment, wherein the antibody fragment is configured to bind to the engineered protein when it is bound to glyphosate. In some embodiments, the method comprises (i) contacting a sample with an engineered protein biosensor comprising (a) an amino acid sequence and (b) a reporter coupled to the amino acid sequence; (ii) providing an antibody fragment configured to bind to the engineered protein biosensor when the engineered protein biosensor is in the closed form configuration; and (iii) detecting the glyphosate bound engineered protein biosensor, wherein the step of detecting occurs through (a) irradiating the sample with light and visualizing the emitted wavelength or (b) an ELISA or universal flow assay. These and other methods that are commonly employed to detect a fluorescent dye, an antibody, or both are to be considered within the scope of the disclosed methods.

In some embodiments, the antibody fragment may be selected from the group consisting of Fab N1 (SEQ ID NO: 6), Fab N2 (SEQ ID NO: 7), Fab N3 (SEQ ID NO: 8), Fab N4 (SEQ ID NO: 9), and Fab N5 (SEQ ID NO: 10). In some embodiments, the antibody fragment is Fab N1. In some embodiments, the antibody fragment is Fab N2. In some embodiments, the antibody fragment is Fab N3. In some embodiments, the antibody fragment is Fab N4. In some embodiments, the antibody fragment is Fab N5. In some embodiments, the antibody fragment is comprises a combination of any one of Fab N1, Fab N2, Fab N3, Fab N4, or Fab N5. In one illustrative embodiment, the antibody fragment is Fab N2.

In some embodiments, the method does not require a consumable reagent or substrate. In some embodiments, the method further comprises the step of dialyzing the engineered protein to remove the ligand making the biosensor reusable. In this embodiment, the ligand is glyphosate. In some embodiments, the engineered protein biosensor is configured to undergo a conformational change when binding glyphosate, that is, from an open to a closed position.

In some embodiments, the sample comprises a solid, liquid, gas, or a combination thereof. In some embodiments, the sample comprises soil or water.

In some embodiments, the method further comprises immobilizing the engineered protein biosensor on a substrate.

SEQUENCES

PhnD 177N

SEQ ID NO: 1

EEQEKALNFG IISTESQQNL KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRFNKVD

IAWYGNLSAM EAVDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAKRKDLTF

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGHETN ALAVANKQVD VATNNT [Image Omitted]

NID

KLKTSAPEKL KELKVIWKSP LIPGDPIVWRKNLSETTKDK IYDFFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA

LSAMSSVSKA VQ

PhnD E177NΔ

SEQ ID NO: 2

EEQEKALNFG IISTESQQNL KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRFNKVD

IAWYGNLSAM EAVDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAKRKDLTF

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGHETN ALAVANKQVD VATNNTNNLD

KLKTSAPEKL KELKVIWKSP LIPGSPIVWR KNLSETTKDK IYDFFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA LSA

PhnD E177N, D205S

SEQ ID NO: 3

EEQEKALNFG IISTESQQNI KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRFNKVD

IAWYGNLSAM EAVDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAKRKDLTF

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGHETN ALAVANKQVD VATNNT [Image Omitted]

NLD

KLKTSAPEKL KELKVIWKSP LIPG [Image Omitted]

PIVWR KNLSETTKDK IYDFFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA

LSAMSSVSKA VQ

PhnD D205N

SEQ ID NO: 4

EEQEKALNFG IISTESQQNL KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRFNKVD

IAWYGNLSAM EAYDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAKRKDLTF

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGEETN ALAVANKQVD VATNNTENLD

KLKTSAPEKL KELKVIWKSP LIPG [Image Omitted]

PIVWR KNLSETTKDK IYDFFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA

LSAMSSVSKA VQ

Wild Type EcPhnD with a 126 C modification

SEQ ID NO: 5

EEQEKALNFG IISTESQQNL KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRFNKVD

IAWYGNLSAM EAVDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAKRKDLTF

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGRETN AIAYANKQVD VATNNT [Image Omitted]

NLD

KLKTSAPEKL KELKVIWKSP LIPGDPIVWR KNLSETTKDK IYDFFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA

LSAMSSVSKA VQ

Fab N1

SEQ ID NO: 6

Light chain:

MKKNIAFLLASMFVFSIATNAYASDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI

YSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGYQSLITFGQGTKVEIKRTVAAPSVFIFPP

SDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

Heavy chain:

FLLASMFVFSIATNAYAEISEVQLVESGGGLVQPGGSLRLSCAASGFNFSYSSIHWVRQAPGKGLEWVASIS

PYYGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYSDSVGYWYSGLDYWGQGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRHHHHHH

Fab N2

SEQ ID NO: 7

Light chain:

MKKNIAFLLASMFVFSIATNAYASDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI

YSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPP

SDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

Heavy chain:

FLLASMFVFSIATNAYAEISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSIHWVRQAPGKGLEWVASISS

SSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQWSSVMEWYIGLDYWGQGTLVTVSSAS

TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS

SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRHHHHHH

Fab N3

SEQ ID NO: 8

Light chain:

MKKNIAFLLASMFVFSIATNAYASDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI

YSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGGWWYPITFGQGTKVEIKRTVAAPSVFIFP

PSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC

Heavy chain:

FLLASMFVFSIATNAYAEISEVQLVESGGGLVQPGGSLRLSCAASGFNVSSYSIHWVRQAPGKGLEWVASIY

SSYGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWSVYYSSYWKAMDYWGQGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRHHHHHH

Fab N4

SEQ ID NO: 9

Light chain:

MKKNIAFLLASMFVFSIATNAYASDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI

YSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYYHMYPITFGQGTKVEIKRTVAAPSVFIFP

PSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC

Heavy chain:

FLLASMFVFSIATNAYAEISEVQLVESGGGLVQPGGSLRLSCAASGFNVSYSSIHWVRQAPGKGLEWVASIY

PSSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQDYFGWYWKTAMDYWGQGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRHHHHHH

Fab N5

SEQ ID NO: 10

Light chain:

MKKNIAFLLASMFVFSIATNAYASDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI

YSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPP

SDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

Heavy chain:

FLLASMFVFSIATNAYAEISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASIS

SSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQYESVWNWYIGLDYWGQGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSRHHHHHH

WT EcPhnD

SEQ ID NO: 11

EEQEKALNEG IISTESQQNL KPQWTPFLQD MEKKLGVKVN AFFAPDYAGI IQGMRENKVD

IAWYGNLSAM EAVDRANGQV FAQTVAADGS PGYWSVLIVN KDSPINNLND LLAEREDLIE

GNGDP [Image Omitted]

STSG FLVPGYYVFA KNNISASDFK RTVNAGHETN ALAVANKQVD VATNNT [Image Omitted]

NLD

KLKTSAPEKL KELKVIWKSP LIPGDPIVWR KNLSETTKDK IYDEFMNYGK TPEEKAVLER

LGWAPFRASS DLQLVPIRQL ALFKEMQGVK SNKGLNEQDK LAKTTEIQAQ LDDLDRLNNA

LSAMSSVSKA VQ

His Tag

SEQ ID NO: 12

GSHHHHHH

The following Examples provide illustrative embodiments of the disclosed engineered proteins, illustrative embodiments of methods to produce these engineered proteins, and illustrative embodiments of methods of detecting glyphosate with the engineered proteins.

Example 1

The crystal structure of PhnD bound to 2-AEP shows that the hydroxyls of residues Tyr47, Try93, Ser127, Thr128 and Ser129 coordinate the phosphonate moiety of 2-AEP, whereas the carboxylates of Glu177 and Asp205 bind to the amino group. Since 2-AEP and glyphosate share the same phosphonate moiety, focus was on replacing Glu177 and/or Asp205 to accommodate the longer glyphosate molecule and its carboxylic acid. Kunkel mutagenesis was used to construct a set of single or double mutants of PhnD. In some embodiments, the mutants also contained an additional cysteine mutation at position 126 for attachment of acrylodan (fluorescent reporter). In some embodiments, the mutants also contained a C-terminal histidine tag (SEQ ID NO: 12) for purification using immobilized metal affinity chromatography.

Example 2

Mutants: PND 177N (SEQ ID NO: 1), 205S (SEQ ID NO: 4), 177N 205S (SEQ ID NO: 3), and PND 177NΔ (SEQ ID NO 2) were constructed, each with a single point mutation in the binding pocket with the goal to increase the affinity for glyphosate. E. coli BL21 DE3 cells were used for transformation with each of the plasmid carrying the specific point mutation. To express each mutant, bacterial cultures were grown on 2×YT media with ampicillin at 37° C. at 300 rpm. IPTG was added at mid-log phase to induce expression of the proteins. Proteins obtained were purified using Immobilized Metal Affinity Chromatography (IMAC) using the AKTA Start Fast Protein Liquid Chromatography (FPLC) system and analyzed on a polyacrylamide gel. Proteins were then buffer exchanged into a low salt working buffer and their concentrations were obtained using UV spectroscopy. Proteins were then dialyzed using a semipermeable membrane to separate out all phosphate present in solution from the protein to limit background noise. PhnD undergoes a conformational change upon binding to its ligand from an open to a closed conformation. This conformational change was used to attach the fluorescent sensor Acrylodan to the protein at a specific site (126C) near the binding pocket.

Example 3

The fluorescent reporter group attached to PhnD changes its emission properties in response to glyphosate binding which can be observed using fluorescent spectroscopy. This allows detection of a ligand (in this case glyphosate), where fluorescence emission can be used to determine the concentration of glyphosate in solution. Two main types of data were collected, Apo vs Saturated spectra showing fluorescence emission with and without glyphosate (not shown), and the other type was titrations done with increasing glyphosate concentrations to determine the K_dvalue for each mutant for glyphosate.

The purified, fluorescently-labeled mutants were tested for their ability to bind to glyphosate. K_dvalues were determined by monitoring the changes in fluorescence as a function of increasing ligand concentrations. Table 1 provides dissociation constants (K_dvalues) of different PhnD variants for glyphosate. All mutants contain a cysteine at position 126 for attachment of acrylodan. Values were obtained by monitoring fluorescence change as a function of ligand concentration using eq. 1.

R
=

R
0

+

R
max

1
+

K
d

[
L
]

eq
.

1

TABLE 1

Dissociation constants of engineered proteins ability to bind glyphosate

Engineered protein
SEQ ID NO
K_dfor glyphosate (μM)

Wild Type (control)
5
650

D205N
4
600

E177N, D205S
3
98

E177N
1
8.0

E177NΔ
2
4.2

The E177N mutant (also referred to as PND177N) showed an 80-fold increase in affinity over the wild-type protein. This was achieved by mutating the glutamic acid residue at position 177 to an asparagine (E177N). The K_dof the mutant for glyphosate is 8.05±0.30 μM (FIG. 1) compared to the wild type PhnD, which has a K_dof ˜650 μM. Based on the structure of PhnD in complex with 2-AEP, changing the glutamic acid residue to an asparagine allows for the larger glyphosate molecule to bind, while replacing a negatively charged side chain with an amide to accommodate the carboxylate of glyphosate.

Additional mutant (PND177NΔ also referred to as E177NΔ) was constructed where the C-terminal 9 amino acids were truncated. These residues have been shown to be involved in dimerization, a property that is unique to PhnD among other PBPs. The truncated mutant improved the affinity for glyphosate with a K_dof 4.2 μM (as shown in Table 1), enhancing the overall binding affinity by over 150-fold.

Example 4

Spectral analysis of the fluorescence change associated with glyphosate binding to the sensor (PND177NΔ) showed a large decrease in fluorescence (FIG. 6). This decrease in the fluorescence is similar to that observed with the wild-type protein, indicating that the mutations did not severely impact the coupling of ligand binding to conformational change. Importantly, the change in fluorescence associated with ligand binding is ratiometric with a larger decrease in emission at lower wavelengths. To determine the K_dvalues, the ratio of fluorescence between two wavelengths (570 nm/450 nm) was monitored as a function of increasing ligand concentrations (FIGS. 2 and 3). The titration data were fit to a single binding isotherm equation (eq. 1), where R is defined as the fluorescence ratio at a given ligand concentration [L], R₀is the fluorescence ratio in the absence of ligand and R_maxis the fluorescence ratio at saturating ligand concentrations.

The equation was also used to determine the affinity of the sensor for commercial ROUNDUP, which contains 18.0% glyphosate isopropylamine as well as “other ingredients.” In contrast with pure glyphosate acid, commercial ROUNDUP contains isopropylamine and other additives that could conceivably interfere with the ability of the sensor to detect the glyphosate content. A titration performed using diluted ROUNDUP solution in working buffer (50 mM MOPS 150 mM NaCl, pH 6.9) revealed a K_dof ˜2 μM (FIG. 3) indicating the ability of the sensor to detect the presence of glyphosate in the commercially available mixture.

Example 5

To examine the specificity of the sensor, we determined the affinity of the sensor for a number of compounds with similar structure to glyphosate. Table 2 shows the specificity of the PhnD-E177NΔ sensor for glyphosate, ROUNDUP and related molecules. The sensor exhibited high affinity for ROUNDUP, while the affinity for 2-AEP was greatly reduced compared to the wild-type protein by more than 3 orders of magnitude. The sensor also showed moderate affinities for phosphate and arsenate.

TABLE 2

Comparison of binding affinity between WT (SEQ ID NO: 5) and

PND177Δ (SEQ ID NO: 2)

Ligand
WT K_d
PND177NΔ K_d

Glyphosate
650 μM
4.29 μM

RoundUp
ND
1.95 μM

2-Aminoethyl Phosphate (2-AEP)
0.002 μM
5.61 μM

Phosphate
260 μM
26.1 μM

Arsenate
230 μM
10.7 μM

Example 6

To test the ability of the sensor to detect the presence of glyphosate in samples contaminated with ROUNDUP, test solutions with different glyphosate concentrations were prepared by diluting ROUNDUP in working buffer. First, the initial fluorescence ratio of the sensor was recorded and used as R₀(equation 1). Then, a sample of the test solution was added to the sensor and the fluorescence ratio was recorded. Saturating amounts of glyphosate were then added to the sensor following each measurement to establish the Rmax. The glyphosate concentration in each test sample was calculated using a rearrangement of equation 1. The results show a linear correlation between the theoretical concentrations of glyphosate and those determined using the sensor. Further, the sensor can detect concentrations as low as 500 nM, well below the permitted limit for drinking water in the United States.

Example 7

Antibody fragments that bind specifically to the closed (glyphosate-bound) form of EcPhnD were engineered. A phage display selection was performed by using a Fab library to select for specific antibody fragments (Fabs) that can bind to the closed form of PhnD. A fab is an antibody fragment protein that binds to a specific spot on a target protein. Fab binding can be very specific and target one conformational state of a target protein. Conformation-specific Fabs can, therefore, distinguish between the open and closed form of PhnD. According to Le Chatelier's principle, Fabs that bind specifically to the closed form of PhnD would drive the equilibrium from the open form to the closed form. By stabilizing the bound form of the protein, these Fabs would further enhance the affinity of PhnD for glyphosate. Using the phage display selection technique, we were able to generate a set of 5 Fabs: Fab N1 (SEQ ID NO: 6), Fab N2 (SEQ ID NO:7), Fab N3 (SEQ ID NO: 8), Fab N4 (SEQ ID NO: 9), and Fab N5 (SEQ ID NO: 10) that recognized closed form of PhnD. Using fluorescence spectroscopy, data showing Apo and saturated form with fab were obtained as well as titration of PhnD in the presence of Fabs to determine the Kd of the proteins for glyphosate.