Difference between revisions of "Part:BBa K1216002"
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− | The | + | The acetyl esterase, apart from its hydrolase activity also binds competitively to malT, a transcription activator for the maltose operon, thus inhibiting regulation of genes required for maltose catabolism[[Part:BBa_K1216002#References|<sup>[2]</sup>]] [[Part:BBa_K1216002#References|<sup>[3]</sup>]]. |
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==Characterization== | ==Characterization== | ||
− | <b>The final construct was sequenced.</b> | + | <b>The final construct was sequenced.</b><br> |
− | ===Colorimetric response=== | + | |
− | [[File:Aes MagentaButyrate.JPG|thumb|right|200px|<b>Figure | + | Jump to: <br> |
+ | *[[Part:BBa_K1216002#Colorimetric and fluorometric response|Colorimetric and fluorometric response]] | ||
+ | *[[Part:BBa_K1216002#Kinetics|Kinetics]] | ||
+ | *[[Part:BBa_K1216002#Crosstalk|Crosstalk]] | ||
+ | <br> | ||
+ | |||
+ | ===Colorimetric and fluorometric response=== | ||
+ | [[File:Aes MagentaButyrate.JPG|thumb|right|200px|<b>Figure 2. Liquid culture of the triple knockout ''Escherichia coli'' strain overexpressing Aes; after reaction with magenta butyrate.</b> The negative control (right) is a liquid culture without the substrate added.]] | ||
+ | [[File:MagentaCaprylate.png|thumb|right|200px|<b>Figure 3. Colonies of the Δ''aes''Δ''gusA''Δ''nagZ'' ''Escherichia coli'' strain overexpressing Aes on M9 agar.</b> Magenta caprylate was added to colonies (purple).]] | ||
+ | |||
[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used Aes in their project as reporter enzyme. | [http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used Aes in their project as reporter enzyme. | ||
− | To test the functionality of the enzyme, liquid culture of | + | To test the functionality of the enzyme, liquid culture of the Δ''aes''Δ''gusA''Δ''nagZ'' ''Escherichia coli'' strain overexpressing Aes was incubated with its chromogenic substrate magenta butyrate (Figure 2). Another suitable substrate for detection of this enzyme is magenta caprylate (Figure 3).<br> |
− | + | [[File:AesSubstrates.png|thumb|center|396px|<b>Figure 1. Enzymatic reaction of Aes with magenta butyrate or magenta caprylate.</b>]] | |
− | [[File: | + | Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-butyrate. The picture in Figure 5 was taken with a single lens reflex camera mounted on a dark hood at λ<sub>Ex</sub> 365 nm. |
− | Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-butyrate. The picture was taken with a | + | |
− | + | ||
[[File:AES_reaction.png|thumb|center|396px|<b>Figure 4. Enzymatic reaction of Aes with 4-MU-butyrate.</b>]] | [[File:AES_reaction.png|thumb|center|396px|<b>Figure 4. Enzymatic reaction of Aes with 4-MU-butyrate.</b>]] | ||
+ | [[File:Aes_fluorescent.png|thumb|center|206px|<b>Figure 5. Cell lysate of the <i>E. coli</i> triple knockout strain overexpressing Aes after reacting with 4-MU-butyrate.</b>]] | ||
+ | |||
+ | <html><table border="1" bordercolor="silver" style="float:left;margin-top:10px;font-size:12px;font-family:verdana;border-collapse:collapse"> | ||
+ | <tr> | ||
+ | <th width="95px" >Hydrolase</th> | ||
+ | <th width="220px" >Substrate</th> | ||
+ | <th width="120px" >Absorption λ<sub>max</sub> or Excitation/Emission</th> | ||
+ | <th width="145px" >Stock solution</th> | ||
+ | <th width="145px" >Liquid culture: end concentration</th> | ||
+ | <th width="145px" >Colonies: 1.5 μl of substrate solution</th> | ||
+ | <th width="145px" >Response time</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td rowspan="3" align="center">Aes</td> | ||
+ | <td bgcolor="#F5CCE0">5-Bromo-6-Chloro-3-indoxyl butyrate (Magenta butyrate) | ||
+ | </td> | ||
+ | <td>Magenta,<br>565 nm</td> | ||
+ | <td>0.5 M in Acetone</td> | ||
+ | <td>0.1 mM</td> | ||
+ | <td>20 mM</td> | ||
+ | <td><b>~ 2 minutes</b></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td bgcolor="#F5CCE0">5-Bromo-6-Chloro-3-indoxyl caprylate (Magenta caprylate) | ||
+ | </td> | ||
+ | <td>Magenta,<br>565 nm</td> | ||
+ | <td>0.2 M in Acetone</td> | ||
+ | <td>1 mM</td> | ||
+ | <td>0.2 M</td> | ||
+ | <td><b>overnight</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> 4-MU-butyrate</td> | ||
+ | <td>Blue (fluorescent),<br>372 nm (λ<sub>Ex</sub>), <br>445 nm (λ<sub>Em</sub>)</td> | ||
+ | <td>50 mM in DMSO</td> | ||
+ | <td>150 μM</td> | ||
+ | <td>-</td> | ||
+ | <td><b>~ 5 minutes</b></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
+ | <br clear="all"><br> | ||
+ | <hr /> | ||
+ | |||
+ | ===Kinetics=== | ||
To characterize the enzyme they conducted fluorometric assays to obtain K<sub>m</sub> values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λ<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | To characterize the enzyme they conducted fluorometric assays to obtain K<sub>m</sub> values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λ<sub>Ex</sub> 365 nm and λ<sub>Em</sub> 445 nm. | ||
The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. | The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. | ||
See the resulting graph below. | See the resulting graph below. | ||
− | [[File:Hydrolase AES.png|thumb|center|650px|<b>Figure | + | [[File:Hydrolase AES.png|thumb|center|650px|<b>Figure 6. Michaelis-Menten-Kinetics of Aes cell lysate from <i>E.Coli</i> overexpressing Aes:</b> plots velocity versus substrate concentration (10 μL, 30 μL, 100 μL, 200 μL, 400 μL) in 20 mM Tris buffer of pH 8. A kinetic value for K<sub>m</sub> obtained by fitting the raw data to standard the Michaelis Menten equation; K<sub>m</sub> = 31.5 ± 12.5 μM. All assays were carried out in triplicates, results are presented as means.]] |
The experimental procedure was as following: | The experimental procedure was as following: | ||
#Prepare buffers | #Prepare buffers | ||
Line 73: | Line 126: | ||
#*Detection of fluorescence in suitable plate reader (λ<sub>Ex</sub> 365 nm, λ<sub>Em</sub> 445 nm) | #*Detection of fluorescence in suitable plate reader (λ<sub>Ex</sub> 365 nm, λ<sub>Em</sub> 445 nm) | ||
− | + | <br> | |
− | + | ||
− | + | ||
− | + | ||
<hr /> | <hr /> | ||
+ | |||
===Crosstalk=== | ===Crosstalk=== | ||
+ | <p align="justify">To ensure specificity of the enzyme-substrate pairs used in [http://2013.igem.org/Team:ETH_Zurich Colisweeper] (ETH Zurich 2013), a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.</p> | ||
− | [[File: | + | [[File:Crosstalk.png|thumb|right|450px| <b>Figure 7.</b> Liquid cultures of the Δ''aes''Δ''gusA''Δ''nagZ'' <i>Escherichia coli</i> strain overexpressing Aes, GusA, NagZ or none in a 96-well plate, with substrates indicated on the left added horizontally.]] |
− | + | ||
− | + | ||
+ | <p align="justify">This crosstalk test was done in a 96-well plate, each well containing 200 μl from liquid cultures of our Δ''aes''Δ''gusA''Δ''nagZ'' <i>Escherichia coli</i> strain overexpressing either Aes, GusA, NagZ or none, each distributed among the column-wells of the plate. Horizontally, the chromogenic substrates were pipetted to the liquid cultures in the same order as their corresponding hydrolase. If specificity of the chosen enzyme-substrates pairs were given, we would expect an output as shown in the figure below (Figure 6). As Figure 7 shows, the overexpressed hydrolases cleave only the substrates they were expected to. | ||
+ | </p> | ||
+ | [[File:CrosstalkExpected.png|frame|center| <b>Figure 6. Expected outcome.</b> Added substrates should be specifically cleaved by their hydrolases.]] | ||
+ | <br clear="all"/> | ||
+ | <hr /> | ||
===References=== | ===References=== |
Latest revision as of 16:34, 30 October 2013
Acetyl esterase (aes) from Escherichia Coli
Acetyl esterase - This is a cytosolic hydrolase that can catalyze hydrolysis of esters of p-nitrophenyl derivatives
A form of this protein with added TEV and poly-HIS tags can be found here.
Usage and Biology
The hydrolase capacity of acetyl esterase makes it suitable for reporter application with substrates like acetylated xylan, ethyl acetate, cephalosporin C and derivatives[1] . Further it can be used for desacteylation of β-Lactam antibiotics[1] .
The acetyl esterase, apart from its hydrolase activity also binds competitively to malT, a transcription activator for the maltose operon, thus inhibiting regulation of genes required for maltose catabolism[2] [3].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 27
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 951
Characterization
The final construct was sequenced.
Jump to:
Colorimetric and fluorometric response
[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used Aes in their project as reporter enzyme.
To test the functionality of the enzyme, liquid culture of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes was incubated with its chromogenic substrate magenta butyrate (Figure 2). Another suitable substrate for detection of this enzyme is magenta caprylate (Figure 3).
Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-butyrate. The picture in Figure 5 was taken with a single lens reflex camera mounted on a dark hood at λEx 365 nm.
Hydrolase | Substrate | Absorption λmax or Excitation/Emission | Stock solution | Liquid culture: end concentration | Colonies: 1.5 μl of substrate solution | Response time |
---|---|---|---|---|---|---|
Aes | 5-Bromo-6-Chloro-3-indoxyl butyrate (Magenta butyrate) | Magenta, 565 nm |
0.5 M in Acetone | 0.1 mM | 20 mM | ~ 2 minutes |
5-Bromo-6-Chloro-3-indoxyl caprylate (Magenta caprylate) | Magenta, 565 nm |
0.2 M in Acetone | 1 mM | 0.2 M | overnight | |
4-MU-butyrate | Blue (fluorescent), 372 nm (λEx), 445 nm (λEm) |
50 mM in DMSO | 150 μM | - | ~ 5 minutes |
Kinetics
To characterize the enzyme they conducted fluorometric assays to obtain Km values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λEx 365 nm and λEm 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.
The experimental procedure was as following:
- Prepare buffers
- Lysis buffer: 10 mg/ml Lysozyme, 20 mM Tris buffer, pH 8
- Reaction buffer: 20 mM Tris buffer, pH 8
- NOTE: For other enzymes than the ones we tested (Aes,GusA,NagZ,PhoA) you might need different buffers
- Cell culture
- Inoculate bacteria in 20 mL of LB with antibiotics
- Let grow at 37°C shaking(200 rpm) to an OD600 of 0.6
- Induce enzyme expression (100nM AHL in our case)
- Let grow at 37°C shaking(200 rpm) for 4-5h
- Cell lysis
- Transfer to 50 mL Falcon™ tube
- Spin down at 4°C for 5 min with 4 rcf
- Resuspend in lysis buffer, 1 μL/mg pellet
- Transfer to eppendorf tubes
- Incubate at room temperature for 10 min at 220 rpm
- Spin down at 4°C for 10 min with max. speed
- Transfer the supernatant to new tubes, discard pellets
- Cell free extract can be stored at -20°C or continue processing
- Dilution
- The following values were provided by Johannes Haerle
- Aes: Dilute CFX 1:100 in reaction buffer
- GusA: Dilute CFX 1:100 in reaction buffer
- NagZ: Use pure
- PhoA: Dilute CFX 1:10 in reaction buffer
- The following values were provided by Johannes Haerle
- Hydrolysis reaction
- Perform this measurement in a 96 well plate or similar
- Perform 3 replicates for each substrate concentration
- Present 41.6 μL reaction buffer in each well
- Add 8 μL diluted CFX (the further dilution ocurring here is intended)
- Add 30.4 μL of corresponding substrate
- Detection of fluorescence in suitable plate reader (λEx 365 nm, λEm 445 nm)
Crosstalk
To ensure specificity of the enzyme-substrate pairs used in [http://2013.igem.org/Team:ETH_Zurich Colisweeper] (ETH Zurich 2013), a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.
This crosstalk test was done in a 96-well plate, each well containing 200 μl from liquid cultures of our ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing either Aes, GusA, NagZ or none, each distributed among the column-wells of the plate. Horizontally, the chromogenic substrates were pipetted to the liquid cultures in the same order as their corresponding hydrolase. If specificity of the chosen enzyme-substrates pairs were given, we would expect an output as shown in the figure below (Figure 6). As Figure 7 shows, the overexpressed hydrolases cleave only the substrates they were expected to.
References
- [http://www.cpcbiotech.it/EN/c/d/enzyme-portfolio/enzymes/acetyl-esterase-lyophilized CPC Biotech]
- [http://www.ecocyc.org/new-image?type=GENE&object=EG11101 Ecocyc]
- [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=MONOMER0-158&orgids=ECOLI Ecocyc]