Generator

Part:BBa_K1216002

Designed by: Parvathi Chandran   Group: iGEM13_ETH_Zurich   (2013-08-29)
Revision as of 01:38, 28 October 2013 by Angela a (Talk | contribs) (→‎Characterization)

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 acetyle 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.

Figure 1. Liquid culture from E.Coli overexpressing Aes after reacting with Magenta butyrate (left). The negative control shows an Escherichia coli liquid culture without substrate added.

[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 E.Coli overexpressing Aes was incubated with Magenta butyrate.

Figure 2. Enzymatic reaction of Aes with Magenta butyrate.
Figure 3. Cell lysate from E.Coli overexpressing Aes after reacting with 4-MU-butyrate.

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 common single lens reflex camera mounted on a dark hood at λEx 365 nm.

Figure 4. Enzymatic reaction of Aes with 4-MU-butyrate.

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 5. 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)

To check the functionality of our reporter construct, we tested the functionality of the enzyme by using the following enzymatic reaction resulting in a violet color.

This reaction was tested in liquid culture as well as on colonies.


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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