Team:ETH Zurich/Data Page

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<h1>Gene circuit</h1>
<h1>Gene circuit</h1>
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<html>
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<p><b>Feel free to click on parts to go to the according registry entry</b></p>
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<p><b>Feel free to click on parts that links to the registry entries</b></p>
<h1>Our favorite new parts</h1>
<h1>Our favorite new parts</h1>
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1. [http://parts.igem.org/Part:BBa_K1216002 Main Page] - '''Acetyl esterase (Aes) BBa_K1216002: '''is a hydrolase originated from ''Escherichia Coli'' which can be used as a reporter enzyme in synthetic biology. Different butyrate-substrate can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on your requirements. We characterized the enzyme by using the blue 5-Bromo-6-Chloro-3-indoxyl butyrate and the fluorescent 4-Methylumbelliferone. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing Aes <br>(K<sub>m</sub>=31.47 &plusmn; 12.51 &mu;M). <br><br>
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Our candidate for best natural biobrick: <br>
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1. [http://parts.igem.org/Part:BBa_K1216002 Main Page] - '''Acetyl esterase (Aes), BBa_K1216002''': is a hydrolase that originated from ''Escherichia Coli'' which can be used as a reporter enzyme in biology. Different butyrate or caprylate substrates can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on the requirements. We characterized the enzyme by using the blue 5-Bromo-6-Chloro-3-indoxyl butyrate and the fluorescent 4-Methylumbelliferyl-butyrate. Therein we did a Michaelis Menten kinetic of the cell lysate of ''E.Coli'' overexpressing Aes <br>(K<sub>m</sub>=31.47 &plusmn; 12.51 &mu;M). <br><br>
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2. [http://parts.igem.org/Part:BBa_K1216005 MainPage] - '''Alkaline phosphatase with His tag and TEV cleavage site (PhoA), BBa_K1216002''': is an improved version of the [http://parts.igem.org/Part:BBa_J61032 BBa_J61032] PhoA. This hydrolase is originated from ''Citrobacter'' which can be used as reporter enzyme in synthetic biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reaction to various alcohols. We prooved that the His-tag doesn't affect the protein function. Therefore we tested the enzymatic reaction with 4-Nitrophenylphosphate and the fluorescent 4-MU-phosphate which were both converted to the respective colorimetric output. To characterize the PhoA with His tag we did a Michaelis-Menten Kinetic of the cell lysate overexpressing PhoA-His (K<sub>m</sub>= 72.0  &plusmn; 7.3 &mu;M). In order to physically proove the presence of a His-tag we did an SDS-PAGE gel and a western blot using an anti-His antibody from mouse and an anti mouse antibody carrying a red fluorescent dye to detect the His-Tag.<br><br>
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2. [http://parts.igem.org/Part:BBa_K1216005 Main Page] - '''Alkaline phosphatase (PhoA) with His tag and TEV restriction site, BBa_K1216002''': is an improved version of the [http://parts.igem.org/Part:BBa_J61032 BBa_J61032] PhoA. This hydrolase is originated from ''Citrobacter'' which can be used as reporter enzyme in biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reactions to various alcohols. We proved that the His-tag doesn't affect the protein function. To this end we tested the enzymatic reaction with yellow 4-Nitrophenylphosphate, the blue 5-Bromo-4-Chloro-3-indolyl phosphate and the fluorescent 4-MU-phosphate which were both converted to the respective colorimetric or fluorescent output. To characterize the PhoA with His tag we did a Michaelis Menten kinetic of the cell lysate of ''E.Coli'' overexpressing PhoA-His (K<sub>m</sub>= 72.0  &plusmn; 7.3 &mu;M). In order to physically prove the presence of a His-tag we did an SDS-PAGE gel and a western blot using an anti-His antibody from mouse and an anti mouse antibody carrying a red fluorescent dye to detect the His-Tag.<br><br>
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3. [http://parts.igem.org/Part:BBa_K1216007 MainPage] - '''pluxR mutated promoter, BBa_K1216007''': is a mutated version of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] pluxR wild type promoter. The engineered device is used as a high pass filter in our system. We characterize the promoter by establishing a OHHL dose response curve in liquid culture as well as in agar plates. We could fit an EC<sub>50</sub> of 6.462 nM in liquid culture and 12'555 nM in agar plates. For comparison the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] plux wild type has an EC<sub>50</sub>=0.02 nM in liquid culture and 4.45 nM in agar plates (EC<sub>50</sub> is the half maximal effective concentration). This correspond to a 300'000 fold shift of sensitivity in liquid culture and 2800 fold shift of sensitivity in agar plates. We did the characterization on microtiter plates and in single cell analysis. We also modelled the expression of different reporters under the control of the wild type and the pluxR variant (G1).</p>
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3. [http://parts.igem.org/Part:BBa_K1216007 Main Page] - '''P<sub>LuxR</sub> mutated promoter, BBa_K1216007''': is a mutated version of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] P<sub>LuxR</sub> wild type promoter. The engineered part is used as a high pass filter in our system. We characterized the promoter by establishing an AHL dose response curve in liquid culture as well as in agar plates. We could fit an EC<sub>50</sub> of 6.462 nM in liquid culture and 12'555 nM in agar plates. For comparison the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] P<sub>LuxR</sub> wild type has an EC<sub>50</sub> of 0.02 nM in liquid culture and 4.45 nM in agar plates (EC<sub>50</sub> is the half maximal effective concentration). This correspond to a 300'000 fold shift of sensitivity in liquid culture and 2800 fold shift of sensitivity in agar plates. We did the characterization on microtiter plates and in single cell analysis. We also modelled the expression of different reporters under the control of the wild type and the P<sub>LuxR</sub>  variant (G1).</p>
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<h1>Characterized pre-existing parts :</h1>
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<h1>Used and characterized pre-existing parts:</h1>
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1. [http://parts.igem.org/Part:BBa_R0062:Experience Experience] - '''pluxR wild type, BBa_R0062''': Antiquity (2003-01-31) : the promoter has an EC<sub>50</sub> of 0.02 nM in liquid culture and 4.45 nM on agar plates (EC<sub>50</sub> is the half maximal effective concentration). This is a 220 fold shift in sensitivity between liquid culture and agar plates. We did the characterization on microtiter plates and in single cell analysis (FACS).<br><br>
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1. [http://parts.igem.org/Part:BBa_R0062:Experience Experience] - '''P<sub>LuxR</sub> wild type, BBa_R0062''': Antiquity (2003-01-31): the promoter has an EC<sub>50</sub> of 0.02 nM in liquid culture and 4.45 nM on agar plates (EC<sub>50</sub> is the half maximal effective concentration). This is a 220 fold shift in sensitivity between liquid culture and agar plates. We did the characterization on microtiter plates and in single cell analysis (FACS).<br><br>
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2. [http://parts.igem.org/Part:BBa_J61032:Experience Experience] - '''Alkaline phosphatase, BBa_J61032''': Arkin Lab(2006-11-10) : is a hydrolase originated from ''Citrobacter'' which can be used as a reporter in synthetic biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reaction to various alcohols. We characterized the enzyme by using the yellow 4-Nitrophenylphosphate and the fluorescent 4-MU-phosphate. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing PhoA (K<sub>m</sub>=105.89 &mu;M &plusmn; 5.46 &mu;M). </p>
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2. [http://parts.igem.org/Part:BBa_J61032:Experience Experience] - '''Alkaline phosphatase (PhoA), BBa_J61032''': Arkin Lab (2006-11-10): is a hydrolase originated from ''Citrobacter'' which can be used as a reporter in biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reactions to various alcohols. We characterized the enzyme by using the yellow 4-Nitrophenylphosphate, the blue 5-Bromo-4-Chloro-3-indolyl phosphate and the fluorescent 4-MU-phosphate. Therefore we did a Michaelis Menten kinetic of the cell lysate of ''E.Coli'' overexpressing PhoA (K<sub>m</sub>=105.89 &plusmn; 5.46 &mu;M).
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<br><br>
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3. [http://parts.igem.org/Part:BBa_K330002:Experience Experience] - '''β-Glucuronidase (GusA), BBa_K330002''': iGEM10_HKUST  (2010-10-10): is a hydrolase originated from ''Bacillus subtilis'' which can be used as a reporter enzyme in biology. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten kinetic of the cell lysate ''E.Coli'' overexpressing GusA (K<sub>m</sub>=141.11 &plusmn; 5.27 &mu;M).<br><br></p>
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4. [http://parts.igem.org/Part:BBa_R0063:Experience Experience] - '''P<sub>LuxL</sub>, BBa_R0063''': Antiquity (2003-01-31): the P<sub>LuxL</sub> promoter is repressed by the LuxR/AHL complex at high AHL concentrations. We use this promoter for the optimization of our circuit. We use it to drive expression of LacI which represses the expression of LuxR in the uninduced state. At high AHL concentration LacI expression is reduced and LuxR is expressed constitutively from P<sub>Lac</sub>.
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<br><br>
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5. [http://parts.igem.org/Part:BBa_J09855:Experience Experience] - '''P<sub>Lac</sub> LuxR P<sub>LuxR</sub>, BBa_J09855''': iGEM2005  (2006-10-26): we used this construct in various experiments. For example for all the promoter library we mutated the BBa_R0062 promoter in the BBa_J09855 construct. We used this construct in our receiver cells, beeing very modulable for reporters, we could clon GFP, RFP and the hydrolases in front of this construct to express the reporter by AHL dependent activation.
<h1>Characterized new parts</h1>
<h1>Characterized new parts</h1>
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1. [http://parts.igem.org/Part:BBa_K1216000 MainPage] - '''β-Glucuronidase (gusA), BBa_K1216000''': is a hydrolase originated from ''Bacillus subtilis'' which can be used as a reporter enzyme in synthetic biology. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA(K<sub>m</sub>=141.11 &mu;M &plusmn; 5.27 &mu;M).<br><br>
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1.- '''P<sub>LuxR</sub> promoter library, [http://parts.igem.org/Part:BBa_K1216007 BBa_K1216007], [http://parts.igem.org/Part:BBa_K1216008 BBa_K1216008], [http://parts.igem.org/Part:BBa_K1216009 BBa_K1216009], [http://parts.igem.org/Part:BBa_K1216010 BBa_K1216010], [http://parts.igem.org/Part:BBa_K1216011 BBa_K1216011], [http://parts.igem.org/Part:BBa_K1216012 BBa_K12160012], [http://parts.igem.org/Part:BBa_K1216013 BBa_K1216013], [http://parts.igem.org/Part:BBa_K1216014 BBa_K1216014]''': is a library of mutated versions of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] P<sub>LuxR</sub> wild type promoter. BBa_K1216008 is used as a high pass filter in our system. We characterized the promoters by establishing an AHL dose response curve on agar plates. EC<sub>50</sub>: BBa_K1216007: 12'555 nM, BBa_K1216008: 81.4 nM, BBa_K1216009: 92.8 nM, BBa_K1216010: 53.8 nM, BBa_K1216011: 62.7 nM, BBa_K1216012: 645 nM, BBa_K1216013: 346 nM, BBa_K1216014: 81.4 nM. For comparison the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] P<sub>LuxR</sub> wild type has an EC<sub>50</sub> of 4.45 nM, on agar plates (EC<sub>50</sub> is the half maximal effective concentration). We also modelled the expression of different reporters under the control of the wild type and the promoter library.</p>
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2. [http://parts.igem.org/Part:BBa_K1216004 MainPage] -''' β-Glucuronidase (gusA) with HIS-Tag and TEV cleavage site, BBa_K1216004''': is a hydrolase originated from ''Bacillus subtilis'' which can be used as a reporter enzyme in synthetic biology. Different D-glucuronic acids substrate can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on your requirements. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA-His (K<sub>m</sub>=201.94 &mu;M &plusmn; 51.14 &mu;M).<br><br>
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2. [http://parts.igem.org/Part:BBa_K1216004 Main Page] -''' β-Glucuronidase (GusA) with HIS-Tag and TEV restriction site, BBa_K1216004''': is a hydrolase originated from ''Bacillus subtilis'' which can be used as a reporter enzyme in synthetic biology. Different D-glucuronic acids substrate can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on your requirements. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA-His (K<sub>m</sub>=201.94 &plusmn; 51.14 &mu;M).<br><br>
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3. [http://parts.igem.org/Part:BBa_K1216003 MainPage] -''' β-N-Acetylglucosaminidase (nagZ), BBa_K1216003''': is a hydrolase originated from ''Escherichia coli'' which can be used as a reporter enzyme in synthetic biology. We characterized the enzyme by using the yellow p-nitrophenyl-β-N-acetylglucosaminide.<br></p>
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3. [http://parts.igem.org/Part:BBa_K1216003 Main Page] -''' β-N-Acetylglucosaminidase (NagZ), BBa_K1216003''': is a hydrolase originated from ''Escherichia coli'' which can be used as a reporter enzyme in synthetic biology. We characterized the enzyme by using the yellow p-nitrophenyl-β-N-acetylglucosaminide, the magenta 5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide and the blue 5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide.<br>
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Latest revision as of 23:27, 28 October 2013

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Contents

Gene circuit

Feel free to click on parts that links to the registry entries

Our favorite new parts

Our candidate for best natural biobrick:
1. [http://parts.igem.org/Part:BBa_K1216002 Main Page] - Acetyl esterase (Aes), BBa_K1216002: is a hydrolase that originated from Escherichia Coli which can be used as a reporter enzyme in biology. Different butyrate or caprylate substrates can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on the requirements. We characterized the enzyme by using the blue 5-Bromo-6-Chloro-3-indoxyl butyrate and the fluorescent 4-Methylumbelliferyl-butyrate. Therein we did a Michaelis Menten kinetic of the cell lysate of E.Coli overexpressing Aes
(Km=31.47 ± 12.51 μM).

2. [http://parts.igem.org/Part:BBa_K1216005 Main Page] - Alkaline phosphatase (PhoA) with His tag and TEV restriction site, BBa_K1216002: is an improved version of the [http://parts.igem.org/Part:BBa_J61032 BBa_J61032] PhoA. This hydrolase is originated from Citrobacter which can be used as reporter enzyme in biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reactions to various alcohols. We proved that the His-tag doesn't affect the protein function. To this end we tested the enzymatic reaction with yellow 4-Nitrophenylphosphate, the blue 5-Bromo-4-Chloro-3-indolyl phosphate and the fluorescent 4-MU-phosphate which were both converted to the respective colorimetric or fluorescent output. To characterize the PhoA with His tag we did a Michaelis Menten kinetic of the cell lysate of E.Coli overexpressing PhoA-His (Km= 72.0 ± 7.3 μM). In order to physically prove the presence of a His-tag we did an SDS-PAGE gel and a western blot using an anti-His antibody from mouse and an anti mouse antibody carrying a red fluorescent dye to detect the His-Tag.

3. [http://parts.igem.org/Part:BBa_K1216007 Main Page] - PLuxR mutated promoter, BBa_K1216007: is a mutated version of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] PLuxR wild type promoter. The engineered part is used as a high pass filter in our system. We characterized the promoter by establishing an AHL dose response curve in liquid culture as well as in agar plates. We could fit an EC50 of 6.462 nM in liquid culture and 12'555 nM in agar plates. For comparison the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] PLuxR wild type has an EC50 of 0.02 nM in liquid culture and 4.45 nM in agar plates (EC50 is the half maximal effective concentration). This correspond to a 300'000 fold shift of sensitivity in liquid culture and 2800 fold shift of sensitivity in agar plates. We did the characterization on microtiter plates and in single cell analysis. We also modelled the expression of different reporters under the control of the wild type and the PLuxR variant (G1).

Used and characterized pre-existing parts:

1. [http://parts.igem.org/Part:BBa_R0062:Experience Experience] - PLuxR wild type, BBa_R0062: Antiquity (2003-01-31): the promoter has an EC50 of 0.02 nM in liquid culture and 4.45 nM on agar plates (EC50 is the half maximal effective concentration). This is a 220 fold shift in sensitivity between liquid culture and agar plates. We did the characterization on microtiter plates and in single cell analysis (FACS).

2. [http://parts.igem.org/Part:BBa_J61032:Experience Experience] - Alkaline phosphatase (PhoA), BBa_J61032: Arkin Lab (2006-11-10): is a hydrolase originated from Citrobacter which can be used as a reporter in biology. It catalyzes production of free inorganic phosphate and phosphoryl transfer reactions to various alcohols. We characterized the enzyme by using the yellow 4-Nitrophenylphosphate, the blue 5-Bromo-4-Chloro-3-indolyl phosphate and the fluorescent 4-MU-phosphate. Therefore we did a Michaelis Menten kinetic of the cell lysate of E.Coli overexpressing PhoA (Km=105.89 ± 5.46 μM).

3. [http://parts.igem.org/Part:BBa_K330002:Experience Experience] - β-Glucuronidase (GusA), BBa_K330002: iGEM10_HKUST (2010-10-10): is a hydrolase originated from Bacillus subtilis which can be used as a reporter enzyme in biology. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten kinetic of the cell lysate E.Coli overexpressing GusA (Km=141.11 ± 5.27 μM).

4. [http://parts.igem.org/Part:BBa_R0063:Experience Experience] - PLuxL, BBa_R0063: Antiquity (2003-01-31): the PLuxL promoter is repressed by the LuxR/AHL complex at high AHL concentrations. We use this promoter for the optimization of our circuit. We use it to drive expression of LacI which represses the expression of LuxR in the uninduced state. At high AHL concentration LacI expression is reduced and LuxR is expressed constitutively from PLac.

5. [http://parts.igem.org/Part:BBa_J09855:Experience Experience] - PLac LuxR PLuxR, BBa_J09855: iGEM2005 (2006-10-26): we used this construct in various experiments. For example for all the promoter library we mutated the BBa_R0062 promoter in the BBa_J09855 construct. We used this construct in our receiver cells, beeing very modulable for reporters, we could clon GFP, RFP and the hydrolases in front of this construct to express the reporter by AHL dependent activation.

Characterized new parts

1.- PLuxR promoter library, [http://parts.igem.org/Part:BBa_K1216007 BBa_K1216007], [http://parts.igem.org/Part:BBa_K1216008 BBa_K1216008], [http://parts.igem.org/Part:BBa_K1216009 BBa_K1216009], [http://parts.igem.org/Part:BBa_K1216010 BBa_K1216010], [http://parts.igem.org/Part:BBa_K1216011 BBa_K1216011], [http://parts.igem.org/Part:BBa_K1216012 BBa_K12160012], [http://parts.igem.org/Part:BBa_K1216013 BBa_K1216013], [http://parts.igem.org/Part:BBa_K1216014 BBa_K1216014]: is a library of mutated versions of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] PLuxR wild type promoter. BBa_K1216008 is used as a high pass filter in our system. We characterized the promoters by establishing an AHL dose response curve on agar plates. EC50: BBa_K1216007: 12'555 nM, BBa_K1216008: 81.4 nM, BBa_K1216009: 92.8 nM, BBa_K1216010: 53.8 nM, BBa_K1216011: 62.7 nM, BBa_K1216012: 645 nM, BBa_K1216013: 346 nM, BBa_K1216014: 81.4 nM. For comparison the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] PLuxR wild type has an EC50 of 4.45 nM, on agar plates (EC50 is the half maximal effective concentration). We also modelled the expression of different reporters under the control of the wild type and the promoter library.

2. [http://parts.igem.org/Part:BBa_K1216004 Main Page] - β-Glucuronidase (GusA) with HIS-Tag and TEV restriction site, BBa_K1216004: is a hydrolase originated from Bacillus subtilis which can be used as a reporter enzyme in synthetic biology. Different D-glucuronic acids substrate can be used to detect either a fluorescent or colorimetric signal after cleavage, depending on your requirements. We characterized the enzyme by using the salmon 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt and the fluorescent 4-MU-β-D-Glucuronide. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA-His (Km=201.94 ± 51.14 μM).

3. [http://parts.igem.org/Part:BBa_K1216003 Main Page] - β-N-Acetylglucosaminidase (NagZ), BBa_K1216003: is a hydrolase originated from Escherichia coli which can be used as a reporter enzyme in synthetic biology. We characterized the enzyme by using the yellow p-nitrophenyl-β-N-acetylglucosaminide, the magenta 5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide and the blue 5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide.