Figure 2. Liquid culture of the triple knockout Escherichia coli strain left: overexpressing Aes and right: without overexpressing Aes; after addition of magenta-butyrate.

Figure 3.1. Colonies of Escherichia coli overexpressing the Aes. Colonies produce magenta color after addition of magenta butyrate.

Figure 3.2. Colonies of Escherichia coli overexpressing the hydrolase Aes. Upon addition of magenta caprylate, a magenta color is produced after approximately 30 minutes incubation at 37°C.

Chromogenic assay
To assess color development after reaction of the enzyme with the chromogenic substrate, a liquid culture of our triple knockout Escherichia coli strain overexpressing Aes was grown until an OD₆₀₀ of 0.4 - 0.6 was reached before addition of 5-Bromo-6-Chloro-3-indolyl butyrate (magenta butyrate, dissolved in acetone) to a final concentration of 100 µM (Figure 2). To study the color development in the actual Colisweeper game setup, colonies were plated by pipetting 1.5 µl of the triple knockout Escherichia coli liquid culture (OD₆₀₀ of 0.4 - 0.6) on an M9 agar plate and incubated overnight. Addition of 1.5 µl of 20 mM magenta butyrate onto each colony results in color generation visible after two to three minutes at room temperature (Figure 3.1).
Unfortunately, substrate tests in our triple knockout strain without Aes overexpression have shown that when using the butyrate as a substrate, there is background activity observable, meaning that enzymes other than the Aes can catalyze hydrolysis of the butyrate substrate. Therefore, 5-Bromo-6-Chloro-3-indolyl caprylate (magenta caprylate) was chosen as an alternative substrate, which has shown to be more specifically cleaved by the Aes in our knockout strain. However, when using magenta caprylate as the substrate, color on colonies developed only after at least half an hour of incubation at 37°C, or overnight at room temperature (Figure 3.2).
Because the player of Colisweeper is not expected to incubate plates after each move, we made an assay comparing time differences of the color development in colonies overexpressing and not overexpressing Aes, using different concentrations of the substrates as well. Results have shown that with the butyrate substrate (20 mM in DMSO) and at room temperature (RT), colonies overexpressing Aes already developed color within the first few minutes, whereas colonies not overexpressing Aes did not show color until six hours after addition of the substrate. Even at twelve hours after adding the substrate, only a faint color was observed in the colonies not overexpressing Aes. Therefore, we continued using magenta butyrate for further tests and finally for the game, due to speed and intensity of color development compared to magenta caprylate.

Figure 4. Aes catalyzed hydrolysis reaction of magenta butyrate or magenta caprylate.

Enzyme kinetics

Figure 5. Cell lysate of Escherichia coli overexpressing Aes after reaction with 4-MU-butyrate.

For the kinetics assay, the Escherichia coli lysate overexpressing Aes was tested with the substrate 4-MU-butyrate which after cleavage gives a fluorescent product. The picture on the right (Figure 5) was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 6. Enzymatic reaction of Aes with 4-MU-butyrate.

To characterize the enzyme, we conducted Michaelis Menten kinetics to obtain the K_m value. Escherichia coli cells were harvested and lysed, and the cell free extract (CFX) was then collected for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. The plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm and determined a K_m value of 31.5 ± 12.5 μM. The obtained data was then evaluated and fitted to Michaelis-Menten-Kinetics with SigmaPlot™:

Figure 7. Michaelis-Menten-Kinetics of cell lysate from Escherichia coli overexpressing Aes with the chromogenic substrate: plots velocity versus substrate concentration (10 μL, 30 μL, 100 μL, 200 μL, 400 μL) in 20 mM Tris buffer of pH 8. A kinetic value for K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m = 31.5 ± 12.5 μM. All assays were carried out in triplicates, results are presented as means.

Figure 8. Liquid culture from Escherichia coli overexpressing PhoA or PhoA-His respectively after reacting with pNPP. The negative control tube contains liquid culture without pNPP.

Chromogenic assay
To test the functionality of this PhoA, a liquid culture of Escherichia coli overexpressing this hydrolase was grown until an OD₆₀₀ of 0.4 - 0.6 was reached before addition of p-nitrophenyl phosphate (pNPP, dissolved in DEA) to a final concentration of 5 mM. This substrate gives rise to yellow color after hydrolysis by PhoA (Figure 8). In the Colisweeper game, substrates are pipetted onto colonies, Figure 10 shows color development on colonies five minutes after addition of 1.5 µl of a 0.5 M pNPP solution.

Figure 9. Reaction of PhoA catalyzed hydrolysis of pNPP (1) and BCIP (2).

Figure 10.1. Colonies of Escherichia coli overexpressing the Citrobacter PhoA. Colonies produce yellow color within two minutes at RT after addition of pNPP.

Figure 10.2. Colonies of Escherichia coli overexpressing the Citrobacter PhoA. Colonies produce blue color after addition of BCIP.

Characterization of PhoA-HIS tagged protein

Figure 11. PhoA-HIS-tag characterization by western blotting. The red stripe indicates the anti-anti-HIS antibody carrying the red fluorescent dye. The white circles indicate the PhoA-HIS protein in the SDS-PAGE gel as well as on the western blot membrane. The molecular weight of this protein is 53kDa.

We introduced a HIS tag to the PhoA enzyme. The HIS tag enables to purify the enzyme and therby e.g. making a full Michalis Menten studies which can give exact values for the model. Adding of a HIS tag can lead to an unfunctional enzyme, so we first tested the functionality of both PhoA and PhoA HIS tag (see Figure 8) with our coloremetric substrat. In a second step we confirmed the presents of the HIS tag by Western Blot: The PhoA and PhoA with HIS-tag were overexpressed in the Escherichia coli BL21 DE3 strain, induced by 5 μM IPTG (iso-propy-β-D-1-thiogalactopyranoside) and finally harvested in order to obtain the cell lysate by lysozyme lysis. This cell lysate of both cultures were analyzed next to each other in two SDS-PAGE gels, one for comassie staining (blue gel in the picture) and one for western blotting (black picture) with Anti-6X His tag® antibody from mouse and a second reporter goat anti mouse antibody with a IRDye 680RD. In the picture we can distinguish the PhoA His (53 kDa) on the western blot as well as on the SDS-PAGE gel (see white circles).

Enzyme kinetics

Figure 12. Cell lysate from Escherichia coli overexpressing PhoA-HIS (left) and PhoA (right) after reaction with 4-MU-phosphate.

For the kinetics assay, we tested both the Citrobacter phoA and phoA-HIS encoded proteins with the fluorescent substrate 4-MU-phosphate. The image in Figure 12 was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 13. Enzymatic reaction of PhoA with 4-MU-phosphate.

To characterize the enzymes, we conducted fluorometric assays to obtain K_m values. Escherichia coli were harvested and lysed, and the cell free extract (CFX) was then collected for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. The plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm, determining a K_m values of 105.9 ± 5.3 μM for PhoA and 72.0 ± 7.3 μM for Pho-HIS.
The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:

Figure 14. Michaelis-Menten-Kinetics of cell lysate from Escherichia coli overexpressing the Citrobacter PhoA or PhoA-HIS: plots velocity versus substrate concentration (2.5 μL, 5 μL, 10 μL, 30 μL, 60 μL, 120 μL, 240 μL) in 20 mM Tris buffer of pH 8. A kinetic value for K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m (PhoA) = 105.9 ± 5.3 μM and K_m (PhoA-HIS) = 72.0 ± 7.3 μM. All assays were carried out in triplicates, results are presented as means.

Figure 15. Liquid cultures of the triple knockout Escherichia coli strain; the natively expressed LacZ reacts with the added substrates Green-Gal (left) and X-Gal (middle).

Chromogenic assay
The β-Galactosidase is natively expressed in the triple knockout Escherichia coli strain used in Colisweeper. Its commonly used chromogenic substrate is 5-Bromo-4-Chloro-3-indolyl-β-D-Galactopyranoside (X-Gal), which yields a blue-colored precipitate (Figure 15, middle). In Colisweeper, this protein catalyzes hydrolysis of the flagging substrate, which is N-Methyl-3-indolyl-β-D-Galactopyranoside (Green-β-D-Gal) and produces a green color upon cleavage of the chromophore (Figure 15, left tube). For the color assay in liquid culuture, we used Escherichia coli cultures grown to an OD₆₀₀ of 0.4 - 0.6, then added the substrates to a final concentration of 50 mM.

Figure 17. Colonies of our triple knockout strain Escherichia coli in the hexagonal grid setup. Colonies natively produce LacZ, and addition of X-Gal gives rise to green color.

Figure 16. Hydrolysis of Green-β-D-Gal (1) or X-Gal (2) under catalysis of LacZ

Figure 18. Liquid culture from Escherichia coli overexpressing GusA; after reaction with Salmon-Gluc.

Chromogenic assay
To characterize the reaction of GusA with its substrate, a liquid culture of Escherichia coli overexpressing GusA was grown until an OD₆₀₀ of 0.4 - 0.6 was reached before addition of 6-Chloro-3-indolyl-β-glucuronide (Salmon-Gluc), a substrate for GusA which produces a rose/salmon color after cleavage (Figure 18). The final concentration of the substrate in liquid culture was 1 mM and the conversion of this substrate was done by incubation at 37°C.

Figure 19. Enzymatic reaction of GusA with the chromogenic substrate Salmon-Gluc.

Enzyme kinetics

Figure 20. Cell lysate from Escherichia coli overexpressing GusA, after reaction with 4-MU-β-D-Glucuronide.

For the kinetics assay, we tested both gusA and gusA-HIS encoded proteins with the fluorescent substrate 4-MU-β-D-Glucuronide. The picture on the right (Figure 20) was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 21. Enzymatic reaction of GusA with the fluorogenic substrate 4-MU-β-D-Glucuronide.

For the enzyme kinetics of GusA, we conducted fluorometric assays to obtain the K_m value. Escherichia coli overexpressing GusA were harvested and lysed, and the cell free extract (CFX) was then collected for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm, and determined a K_m value of 141.1 ± 5.3 μM for GusA, and 201.9 ± 51.1 μM for GusA-HIS. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:

Figure 22. Michaelis-Menten-Kinetics of Escherichia coli overexpressing GusA and GusA-HIS: plots velocity versus substrate concentration (8 μL, 16 μL, 32 μL, 65 μL, 130 μL, 260 μL, 520 μL)) in 20 mM Tris buffer of pH 8. A kinetic value for K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m (GusA) = 141.1 ± 5.3 μM and K_m (GusA-HIS) = 201.9 ± 51.1 μM. All assays were carried out in triplicates, results are presented as means.

Figure 23. Liquid culture of the triple knockout Escherichia coli strain overexpressing NagZ; after reaction with pNP-GluNAc (yellow), Magenta-GluNAc (magenta) and X-GluNAc (blue).

Chromogenic assay
NagZ was proposed to be constitutively expressed in the mine colonies, processing its blue color-yielding substrate 5-Bromo-4-Chloro-3-indolyl-β-N-acetylglucosaminide (X-GluNAc). However, we could not initially manage to get color from the reaction with this enzyme-substrate pair. Using p-nitrophenyl-β-N-acetylglucosaminide (pNP-GluNAc) at an end concentration of 0.01 mM in an Escherichia coli liquid culture with an OD₆₀₀ of 0.4 - 0.6, development of yellow color could be observed (Figure 23), confirming that NagZ is expressed in our mine cells.
According to Orenga et al. (1), the substrate incorporating the indoxyl chromophore is less preferentially hydrolyzed by bacterial NagZ in comparison to yeast NagZ, and more preferentially hydrolyzes the alizarine-based substrate they applied. To increase hydrolysis of the indoxyl-based substrate, we added the substrate to a liquid culture supplemented with BSA for stabilization of the enzyme (2), which enhanced hydrolysis by NagZ dramatically. The end concentration in liquid culture of both X-GluNAc and 5-Bromo-6-Chloro-3-indolyl-β-N-acetylglucosaminide (Magenta-GluNAc), a magenta color-yielding substrate for NagZ, were at 1 mM (Figure 23).

Figure 24. Colonies of the triple knockout Escherichia coli strain overexpressing NagZ; after addition of the NagZ substrates pNP-GluNAc (yellow colonies), Magenta-GluNAc (pink colonies) and X-GluNAc (blue colonies).

Figure 25. β-N-Acetylglucosaminidase catalyzed hydrolysis of the chromogenic substrates X-β-N-acetylglucosaminide (1), Magenta-GluNAc (2) and pNP-GluNAc (3).