Generator

Part:BBa_K1216002

Designed by: Parvathi Chandran   Group: iGEM13_ETH_Zurich   (2013-08-29)
Revision as of 16:08, 30 October 2013 by Angela a (Talk | contribs) (→‎Usage and Biology)

Acetyl esterase (aes) from Escherichia Coli

Acetyl esterase - This is a cytosolic hydrolase that can catalyze hydrolysis of esters of p-nitrophenyl derivatives

3D representation of the acetyl esterase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=4KRX RCSB]

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 27
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 951


Characterization

The final construct was sequenced.

Colorimetric and fluorometric response

Figure 2. Liquid culture of the triple knockout Escherichia coli strain overexpressing Aes; after reaction with magenta butyrate. The negative control (right) is a liquid culture without the substrate added.
Figure 3. Colonies of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes on M9 agar. 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. 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).

Figure 1. Enzymatic reaction of Aes with magenta butyrate or magenta caprylate.

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.

Figure 4. Enzymatic reaction of Aes with 4-MU-butyrate.
Figure 5. Cell lysate of the E. coli triple knockout strain overexpressing Aes after reacting with 4-MU-butyrate.

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.

Figure 6. Michaelis-Menten-Kinetics of Aes cell lysate from E.Coli overexpressing Aes: 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 Km obtained by fitting the raw data to standard the Michaelis Menten equation; Km = 31.5 ± 12.5 μM. All assays were carried out in triplicates, results are presented as means.

The experimental procedure was as following:

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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.

Figure 7. Liquid cultures of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes, GusA, NagZ or none in a 96-well plate, with substrates indicated on the left added horizontally.

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.

Figure 6. Expected outcome. Added substrates should be specifically cleaved by their hydrolases.


References

  1. [http://www.cpcbiotech.it/EN/c/d/enzyme-portfolio/enzymes/acetyl-esterase-lyophilized CPC Biotech]
  2. [http://www.ecocyc.org/new-image?type=GENE&object=EG11101 Ecocyc]
  3. [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=MONOMER0-158&orgids=ECOLI Ecocyc]
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