Gene circuit

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Our favorite new parts

1. 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 (Km=31.47±12.51 uM).

2. MainPage - Alkaline phosphatase with His tag and TEV cleavage site (PhoA), BBa_K1216002: is an improved version of the BBa_J61032 PhoA. This hydrolase is originated from _____ which can be used as reporter enzyme in synthetic biology. We prooved that the His-tag doesn#t affect the protein function. Therefore we tested the enzymatic reaction with 4-Nitrophenylphosphate and the fluorescent _______ 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 (Km=____±____uM). 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.

3. MainPage - pluxR mutated promoter, BBa_K1216007: is a mutated version of the 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 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 4000 fold shift of sensitivity in agar plates. The data were obtained by single cell analysis using a FACS device. We also modelled the expression of GusA and Aes under the control of the wild type and the pluxR variant (G1).

Characterized pre-existing parts :

1. 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. The data were obtained by single cell analysis using a FACS device.

2. 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. We characterized the enzyme by using the yellow 4-Nitrophenylphosphate and the fluorescent ______. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing PhoA (Km=____±_____ uM).

Characterized new parts

1. Main Page - Acetyl esterase (AES)with His-Tag and TEV cleavage site, BBa_K1216006 : '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-His (Km=____±___uM).

2. MainPage - β-Glucuronidase (gusA), BBa_K1216000 :is a hydrolase originated from ______ which can be used as a reporter enzyme in synthetic biology. . We characterized the enzyme by using the yellow _______ butyrate and the fluorescent _______. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA(Km=____±___uM).

3. MainPage - β-Glucuronidase (gusA)with HIS-Tag and TEV cleavage site, BBa_K1216004 :is a hydrolase originated from ______ 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 salmon ______ and the fluorescent_______. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing GusA-His (Km=____±___uM).

4. MainPage - β-N-Acetylglucosaminidase (nagZ), BBa_K1216003 : is a hydrolase originated from ______ which can be used as a reporter enzyme in synthetic biology. We characterized the enzyme by using the purple ___________ and the fluorescent _______. Therefore we did a Michaelis Menten Kinetic of the cell lysate overexpressing NagZ (Km=____±___uM).

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