Gene circuit

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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 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
(K_m=31.47 ± 12.51 μM).

2. [http://parts.igem.org/Part:BBa_K1216005 MainPage] - Alkaline phosphatase (PhoA) with His tag and TEV cleavage 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 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_m= 72.0 ± 7.3 μM). 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.

Our candidate for best engineered biobrick:
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 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] plux wild type has an EC₅₀=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).

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 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_m=105.89 ± 5.46 μM).

Characterized new parts

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_m=141.11 ± 5.27 μM).

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_m=201.94 ± 51.14 μM).

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.

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