Enzyme-substrate reactions

Figure 1: Colorimetric output. This image shows liquid cultures of our mutant Escherichia coli strain expressing the enzymes PhoA, NagZ, LacZ, GusA or Aes to convert specific chromogenic substrates into the color outputs needed for our project. There is a possibility to create more colors, since our hydrolases have a large spectrum of substrates to convert to give different colorimetric outputs. As an example of multi-substrate conversion, there is the LacZ which can convert X-Gal into a blue output and Green-β-D-Gal into a green output.

To generate visible output by adding substrates to colonies in Colisweeper, we made use of orthogonal enzyme-substrate reactions. A set of chromogenic substrates was chosen to produce different colors depending on the abundant hydrolases and thereby to uncover the identity of each colony to the player.
Chromogenic substrates incorporate a chromophore whose absorbance properties change after the enzyme reaction, and the color signal produced then is directly related to the enzyme-catalyzed reaction.
Indican is a chromogenic glycoside hydrolase substrate which belongs to a family of natural glycosides found in plants. Cleavage of the glycosidic bond forms an unstable hydroxyindole intermediate, which dimerizes by oxidation to form indigo as a blue precipitate:

Numerous enzyme substrates have been designed following this natural product example, giving rise to many colored phenols that are used to detect enzyme activities.

As shown in Figure 1, some of the hydrolases used in the Colisweeper reporter system can catalyze hydrolysis of various substrates, with different chromophores that give rise to a wide range of colors. This variety of substrates and color outputs enables change of positions and function of these enzymes. Other possible substrates that can be used for the enzymes of the Colisweeper reporter system can be found in the Hydrolases section. In Colisweeper, the set of enzyme-substrate pairs are chosen as illustrated in the following table:

Acetyl esterase (Aes)

Figure 1. Liquid culture from Escherichia coli overexpressing acetyl esterase after addtion of magenta-butyrate.

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 magenta butyrate to a final concentration of 250 µM. To study the color development in the actual Colisweeper game setup, colonies were plated by pipetting 1.5 µl of triple knockout Escherichia coli liquid culture (OD₆₀₀ of 0.4 - 0.6) on an M9 agar plate. Addition of 1.5 µl of 20 mM magenta butyrate onto each colony results in color generation visible after a few minutes at room temperature.

Figure 2. Acetyl esterase catalyzed hydrolysis reaction of magenta butyrate.

Figure 3. Cell lysate from E.Coli overexpressing Aes after reacting with 4-MU-butyrate.

Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-butyrate. The picture was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

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

To characterize the enzyme they conducted fluorometric assays to obtain K_m values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://https://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used 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. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

Figure 5. Michaelis-Menten-Kinetics of Aes cell lysate from E.Coli overexpressing Aes: 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.

Alkaline phosphatase(PhoA)

To test the functionality of this alkaline phosphatase, 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) to a final concentration of 5 µM. This substrate gives rise to yellow color after hydrolysis by the alkaline phosphatase. In the Colisweeper game, substrates are pipetted onto colonies. Figure Bla shows color development on colonies five minutes after addition of 1.5 µl of a 0.5 M pNPP solution.

Figure 1. Liquid culture from Escherichia coli overexpressing PhoA or PhoA-His respectively after reacting with pNPP. The negative control tube contains liquid culture without pNPP.

Figure 2. Colonies of Escherichia coli overexpressing the Citrobacter alkaline phosphatase. Colonies produce yellow color after addition of pNPP.

Figure 3. Reaction of alkaline phosphatase catalyzed hydrolysis of pNPP.

Purification of PhoA-HIS tagged protein

Figure 1. 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.

Enzyme kinetics

Figure 3. 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 picture on the right was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

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

To characterize the enzymes, we conducted fluorometric assays to obtain K_m values. Bacterial 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. A plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:

Figure 5. Michaelis-Menten-Kinetics of alkaline phosphatase cell lysate from Escherichia coli overexpressing the Citrobacter alkaline phosphatase: 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 = 105.9 ± 5.3 μM. All assays were carried out in triplicates, results are presented as means.

Beta-galactosidase(LacZ)

Chemical conversion

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Beta-glucuronidase(GusA)

Figure 1. Liquid culture from Escherichia coli overexpressing β-glucuronidase after reacting with Salmon-β-glucuronide.

To test the functionality of β-glucuronidase, a liquid culture of Escherichia coli overexpressing β-glucuronidase was grown until an OD₆₀₀ of 0.4 - 0.6 was reached before addition of Salmon-β-glucuronide, a substrate for β-glucuronidase which produces a rose color after cleavage. The final concentration of the substrate in liquid culture was 1 mM and the full conversion of this substrate was reached by overnight incubation at 37°C.

Figure 2. Enzymatic reaction of β-glucuronidase with the chromogenic substrate Salmon-β-glucuronide.

Enzyme kinetics

Figure 3. Cell lysate from Escherichia coli overexpressing β-glucuronidase after reacting 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 was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

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

To characterize the enzymes, we conducted fluorometric assays to obtain K_m values. Bacterial 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. A plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:

Figure 5. 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 = 141.1 ± 5.3 μM. All assays were carried out in triplicates, results are presented as means.

β-N-Acetylglucosaminidase(NagZ)

Chemical conversion

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

We thank our sponsors: