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 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
(K_m=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 (K_m= 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] - P_LuxR mutated promoter, BBa_K1216007: is a mutated version of the [http://parts.igem.org/Part:BBa_R0062 BBa_R0062] P_LuxR wild type promoter. The engineered part is used as a high pass filter in our system. We characterized the promoter by establishing an 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] P_LuxR 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 P_LuxR variant (G1).

Used and characterized pre-existing parts:

1. [http://parts.igem.org/Part:BBa_R0062:Experience Experience] - P_LuxR 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 (K_m=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 (K_m=141.11 ± 5.27 μM).

4. [http://parts.igem.org/Part:BBa_R0063:Experience Experience] - P_LuxL, BBa_R0063: Antiquity (2003-01-31): the P_LuxL 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_Lac.

5. [http://parts.igem.org/Part:BBa_J09855:Experience Experience] - P_Lac LuxR P_LuxR, 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.- P_LuxR 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_LuxR 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] P_LuxR 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 (K_m=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.

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