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Team:ETH Zurich/Data Page
From 2013.igem.org
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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 (Km=31.47±12.51 uM). <br><br> | 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 (Km=31.47±12.51 uM). <br><br> | ||
| - | 2. [http://parts.igem.org/Part:BBa_K1216005 MainPage] - '''Alkaline phosphatase with His tag and TEV cleavage site( | + | 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 ''_____'' 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 blott using an anti-His antibody from mouse and an anti mouse antibody carrying a red fluorescent dye to detect the His-Tag. We also sequenced the gene. <br><br> |
3. [http://parts.igem.org/Part:BBa_K1216007 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 EC<sub>50</sub> of 6.462 nM in liquid culture and 12'555 nM in agar plates. For comparison the 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 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).</p> | 3. [http://parts.igem.org/Part:BBa_K1216007 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 EC<sub>50</sub> of 6.462 nM in liquid culture and 12'555 nM in agar plates. For comparison the 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 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).</p> | ||
Revision as of 20:51, 4 October 2013
Contents |
Gene circuit
Feel free to click on parts to go to the according registry entry
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 blott using an anti-His antibody from mouse and an anti mouse antibody carrying a red fluorescent dye to detect the His-Tag. We also sequenced the gene.
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) : phoA gene originated from Citrobacter. We did Michealis-Menten kinetics of the phoA enzyme and also showed the conversion of PNP (para-nitrophenol phosphate) in a yellow analog precipitate
Characterized new parts
1. Main Page - Acetyl esterase (AES)with His-Tag and TEV cleavage site, BBa_K1216006 : We did Michealis-Menten kinetics with cell free extract and showed that the hydrolase can convert into an indigo analog precipitate
2. MainPage - β-Glucuronidase (gusA), BBa_K1216000 :We did Michealis-Menten kinetics with cell free extract and showed that the hydrolase can convert into an red analog precipitate.
3. MainPage - β-Glucuronidase (gusA)with HIS-Tag and TEV cleavage site, BBa_K1216004 :We did Michealis-Menten kinetics with cell free extract and showed that the hydrolase can convert into an red analog precipitate.
4. MainPage - β-N-Acetylglucosaminidase (nagZ), BBa_K1216003 : We did Michealis-Menten kinetics with cell free extract and showed that the hydrolase can convert into a blue analog precipitate.
