Team:ETH Zurich/Experiments 3
From 2013.igem.org
Line 17: | Line 17: | ||
<h1>Acetyl esterase (Aes)</h1> | <h1>Acetyl esterase (Aes)</h1> | ||
- | [[File:Aes_colored.png|frame|right|200px| <b>Figure | + | [[File:Aes_colored.png|frame|right|200px| <b>Figure 2.</b> Liquid culture from <i>Escherichia coli</i> 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 <i>Escherichia coli</i> strain overexpressing Aes was grown until an OD<sub>600</sub> 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 <i>Escherichia coli</i> liquid culture (OD<sub>600</sub> 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. | To assess color development after reaction of the enzyme with the chromogenic substrate, a liquid culture of our triple knockout <i>Escherichia coli</i> strain overexpressing Aes was grown until an OD<sub>600</sub> 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 <i>Escherichia coli</i> liquid culture (OD<sub>600</sub> 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. | ||
- | [[File:Aes and Magenta Butyrate.jpeg|thumb|right|200px| <b>Figure | + | [[File:Aes and Magenta Butyrate.jpeg|thumb|right|200px| <b>Figure 3.</b> Colonies of ''Escherichia coli'' overexpressing the acetyl esterase. Colonies produce magenta color after addition of magenta butyrate.]]<br> |
- | [[File:AesReaction.png|thumb|center|800px| <b>Figure | + | [[File:AesReaction.png|thumb|center|800px| <b>Figure 4.</b> Acetyl esterase catalyzed hydrolysis reaction of magenta butyrate.]]<br> |
<br><b>Enzyme kinetics</b> | <br><b>Enzyme kinetics</b> | ||
- | [[File:Aes_fluorescent.png|thumb|right|200px|<b>Figure | + | [[File:Aes_fluorescent.png|thumb|right|200px|<b>Figure 5.</b> Cell lysate of <i>Escherichia coli</i> overexpressing acetyl esterase after reaction with 4-MU-butyrate.]] |
For the kinetics assay, the ''aes'' encoded protein was tested with the fluorescent substrate 4-MU-butyrate. The picture on the right was taken with a common single lens reflex camera mounted on a dark hood at λ<sub>Ex</sub> 365 nm. | For the kinetics assay, the ''aes'' encoded protein was tested with the fluorescent substrate 4-MU-butyrate. The picture on the right was taken with a common single lens reflex camera mounted on a dark hood at λ<sub>Ex</sub> 365 nm. | ||
- | [[File:AES_reaction.png|thumb|center|400px|<b>Figure | + | [[File:AES_reaction.png|thumb|center|400px|<b>Figure 6. Enzymatic reaction of Aes with 4-MU-butyrate.</b>]]<br> |
To characterize the enzymes, we conducted fluorometric assays to obtain K<sub>m</sub> 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<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | To characterize the enzymes, we conducted fluorometric assays to obtain K<sub>m</sub> 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<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | ||
The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. | The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. | ||
- | [[File:Hydrolase AES.png|thumb|center|400px|<b>Figure | + | [[File:Hydrolase AES.png|thumb|center|400px|<b>Figure 7.</b> Michaelis-Menten-Kinetics of cell lysate from <i>Escherichia coli</i> overexpressing acetyl esterase: 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<sub>m</sub> obtained by fitting the raw data to standard the Michaelis Menten equation; K<sub>m</sub> = 31.5 ± 12.5 μM. All assays were carried out in triplicates, results are presented as means.]] |
<br clear="all"/> | <br clear="all"/> | ||
- | <h1>Alkaline phosphatase(PhoA)</h1> | + | <h1>Alkaline phosphatase (PhoA)</h1> |
+ | To test the functionality of this alkaline phosphatase, liquid culture of <i>Escherichia coli</i> overexpressing this hydrolase was grown until an OD<sub>600</sub> 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 10 shows color development on colonies five minutes after addition of 1.5 µl of a 0.5 M pNPP solution.<br clear="all"/> | ||
+ | [[File:PhoA_PhoA_his_compared_colored.png|thumb|left|200px|<b>Figure 8.</b> Liquid culture from <i>Escherichia coli</i> overexpressing PhoA or PhoA-His respectively after reacting with pNPP. The negative control tube contains liquid culture without pNPP.]] | ||
- | + | [[File:PhoA and pNPP.jpeg|thumb|right|200px| <b>Figure 10.</b> Colonies of ''Escherichia coli'' overexpressing the ''Citrobacter'' alkaline phosphatase. Colonies produce yellow color after addition of pNPP.]] | |
- | [[File: | + | |
- | + | [[File:PhoAReaction2.png|thumb|600px|center|<b>Figure 9.</b> Reaction of alkaline phosphatase catalyzed hydrolysis of pNPP.]] | |
- | + | ||
- | [[File:PhoAReaction2.png|thumb|600px|center|<b>Figure | + | |
<br clear="all"/> | <br clear="all"/> | ||
<b>Characterization of PhoA-HIS tagged protein</b> | <b>Characterization of PhoA-HIS tagged protein</b> | ||
- | [[File:Phoahis.png|left|400px|thumb|<b>Figure | + | [[File:Phoahis.png|left|400px|thumb|<b>Figure 11. PhoA-HIS-tag characterization by western blotting.</b> 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.]] |
<br clear="all"/> | <br clear="all"/> | ||
<br><b>Enzyme kinetics</b> | <br><b>Enzyme kinetics</b> | ||
- | [[File:PhoA_fluorescent.png|thumb|right|200px|<b>Figure | + | [[File:PhoA_fluorescent.png|thumb|right|200px|<b>Figure 12.</b> Cell lysate from <i>Escherichia coli</i> 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<sub>Ex</sub> 365 nm. | 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<sub>Ex</sub> 365 nm. | ||
- | [[File:phoa_fluorescent_reaction.png|frame|center|<b>Figure | + | [[File:phoa_fluorescent_reaction.png|frame|center|<b>Figure 13. Enzymatic reaction of PhoA with 4-MU-phosphate.</b>]] |
To characterize the enzymes, we conducted fluorometric assays to obtain K<sub>m</sub> 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<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | To characterize the enzymes, we conducted fluorometric assays to obtain K<sub>m</sub> 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<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | ||
The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™: | The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™: | ||
- | [[File:PhoA and phoAHIS kinetics.png|frame|center|500px|<b>Figure | + | [[File:PhoA and phoAHIS kinetics.png|frame|center|500px|<b>Figure 14.</b> Michaelis-Menten-Kinetics of alkaline phosphatase cell lysate from <i>Escherichia coli</i> 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<sub>m</sub> obtained by fitting the raw data to standard the Michaelis Menten equation; K<sub>m</sub> = 105.9 ± 5.3 μM. All assays were carried out in triplicates, results are presented as means.]] |
<br clear="all"/><br> | <br clear="all"/><br> | ||
Line 60: | Line 59: | ||
<h1>β-Galactosidase (LacZ)</h1> | <h1>β-Galactosidase (LacZ)</h1> | ||
- | + | The β-Galactosidase is natively expressed in the triple knockout Escherichia coli strain used in Colisweeper. Therefore, this protein catalyzes hydrolysis of the flagging substrate, which is Green-β-D-Galactoside and produces a green color upon cleavage of the chromophore. | |
- | [[File:LacZ and GreenGal.jpeg| | + | [[File:LacZ and GreenGal.jpeg|thumb|right|200px| <b>Figure 3.</b> Colonies of our triple knockout strain ''Escherichia coli''. Colonies natively produce LacZ, and addition of Green-β-D-Galactoside gives rise to green color.]] |
[[File:LacZReaction.png|center]] | [[File:LacZReaction.png|center]] | ||
<br clear="all"/> | <br clear="all"/> | ||
Line 81: | Line 80: | ||
<h1>β-N-Acetylglucosaminidase (NagZ)</h1> | <h1>β-N-Acetylglucosaminidase (NagZ)</h1> | ||
- | + | In Colisweeper, the β-N-acetylglucosaminidase is constitutively expressed in the mine cells. Addition of X-β-N-acetylglucosaminide would result in a blue color precipitate, as shown in Figure Bla. However, addition of the X-β-N-acetylglucosaminide substrate that we have did not result in any color production. Control assays with p-nitrophenyl-β-N-acetylglucosaminide on the other hand gave rise to yellow color, which confirms that NagZ is expressed in our mine cells. | |
- | [[File:NagZReaction.png|center]] | + | [[File:NagZReaction.png|frame|center|<b>Figure 2.</b> β-N-Acetylglucosaminidase catalyzed hydrolysis of the chromogenic substrate X-β-N-acetylglucosaminide.]] |
<br clear="all"/> | <br clear="all"/> | ||
Revision as of 02:55, 5 October 2013
Contents |
Enzyme-substrate reactions
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)
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.
Enzyme kinetics
For the kinetics assay, the aes encoded protein was tested with the fluorescent substrate 4-MU-butyrate. The picture on the right was taken with a common single lens reflex camera mounted on a dark hood at λEx 365 nm.
To characterize the enzymes, we conducted fluorometric assays to obtain Km 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 λEx 365 nm and λEm 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™.
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 10 shows color development on colonies five minutes after addition of 1.5 µl of a 0.5 M pNPP solution.
Characterization of PhoA-HIS tagged protein
Enzyme kinetics
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 λEx 365 nm.
To characterize the enzymes, we conducted fluorometric assays to obtain Km 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 λEx 365 nm and λEm 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:
β-Galactosidase (LacZ)
The β-Galactosidase is natively expressed in the triple knockout Escherichia coli strain used in Colisweeper. Therefore, this protein catalyzes hydrolysis of the flagging substrate, which is Green-β-D-Galactoside and produces a green color upon cleavage of the chromophore.
β-Glucuronidase (GusA)
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.
Enzyme kinetics
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 λEx 365 nm.
To characterize the enzymes, we conducted fluorometric assays to obtain Km 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 λEx 365 nm and λEm 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™:
β-N-Acetylglucosaminidase (NagZ)
In Colisweeper, the β-N-acetylglucosaminidase is constitutively expressed in the mine cells. Addition of X-β-N-acetylglucosaminide would result in a blue color precipitate, as shown in Figure Bla. However, addition of the X-β-N-acetylglucosaminide substrate that we have did not result in any color production. Control assays with p-nitrophenyl-β-N-acetylglucosaminide on the other hand gave rise to yellow color, which confirms that NagZ is expressed in our mine cells.