Difference between revisions of "Part:BBa K1216004"

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Cell lysate for the assay described below was tested for active enzyme with the fluorescent substrate 4-MU-β-D-Glucuronide (Figure 1). The picture in Figure 2 was taken with a common single lens reflex camera mounted on a dark hood at &lambda;<sub>Ex</sub> 365 nm.
 
Cell lysate for the assay described below was tested for active enzyme with the fluorescent substrate 4-MU-β-D-Glucuronide (Figure 1). The picture in Figure 2 was taken with a common single lens reflex camera mounted on a dark hood at &lambda;<sub>Ex</sub> 365 nm.
 
[[File:gusa_fluorescent_reaction.png|frame|center|<b>Figure 1.  Enzymatic reaction of GusA with 4-MU-β-D-Glucuronide.</b>]]
 
[[File:gusa_fluorescent_reaction.png|frame|center|<b>Figure 1.  Enzymatic reaction of GusA with 4-MU-β-D-Glucuronide.</b>]]
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<th width="95px" >Hydrolase</th>
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<th width="220px" >Substrate</th>
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<th width="120px" >Excitation/Emission</th>
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<th width="145px" >Stock solution</th>
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<th width="145px" >Apllication: end concentration</th>
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<th width="145px" >Response time</th>
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<td rowspan="2" align="center">GusA
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<td> 4-MU-β-D-Glucuronide</td>
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<td>Blue (fluorescent),<br>372 nm (&lambda;<sub>Ex</sub>), <br>445 nm (&lambda;<sub>Em</sub>)</td>
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<td>50 mM in DMSO</td>
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<td>100 &mu;M</td>
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<td><b>~ 5 minutes</b></td>
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Revision as of 16:17, 30 October 2013

β-Glucuronidase (gusA) from Bacillis Subtilis with TEV and poly-HIS tags

gusA (also called uidA[1]) encodes β-Glucuronidase, an intracellular enzyme that catalyzes the hydrolysis of β-D-glucuronides.
3D representation of the β-Glucuronidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=3K46 RCSB]
The poly-HIS tag can be used for protein purification (IMAC)[2]. The TEV tag can then be used to have the TEV protease specifically cleave off the poly-HIS tag from the purified protein [3].


A form of this protein without TEV and poly-HIS tags can be found here.

Usage and Biology

β-Glucuronidase is used as a fusion protein marker in higher plants, due to them lacking intrinsic β-Glucuronidase activity[4]. Generally it can be used as reporter enzyme with detection by biochemical activity assays, immunological assays or by histochemical staining of tissue sections or cells[5].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 538
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The final construct was sequenced.

Fluorometric response

Figure 2. Cell lysate from the triple knockout E. coli strain overexpressing GusA-His after reacting with 4-MU-β-D-Glucuronide.

Cell lysate for the assay described below was tested for active enzyme with the fluorescent substrate 4-MU-β-D-Glucuronide (Figure 1). The picture in Figure 2 was taken with a common single lens reflex camera mounted on a dark hood at λEx 365 nm.

Figure 1. Enzymatic reaction of GusA with 4-MU-β-D-Glucuronide.

Hydrolase Substrate Excitation/Emission Stock solution Apllication: end concentration Response time
GusA
4-MU-β-D-Glucuronide Blue (fluorescent),
372 nm (λEx),
445 nm (λEm)		 50 mM in DMSO 100 μM ~ 5 minutes


Kinetics

To prove that the enzyme retained functionality with an additional His-tag 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 3. Michaelis-Menten-Kinetics of GusA-His cell lysate from E.Coli overexpressing GusA-His: plots velocity versus substrate concentration (8 μL, 16 μL, 32 μL, 65 μL, 130 μL, 260 μL, 520 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 = 201.9 ± 51.1 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)

References

  1. [http://ecoliwiki.net/colipedia/index.php/uidA:Gene ecoliwiki]
  2. Loghran ST, "Purification of poly-histidine-tagged proteins.",Methods Mol Biol. 2011;681:311-35. doi: 10.1007/978-1-60761-913-0_17.[http://www.ncbi.nlm.nih.gov/pubmed/20978973]
  3. [http://homepage.univie.ac.at/nikos.pinotsis/tev_protease.html University of Vienna TEV Protease info]
  4. Jefferson A R, "GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.", EMBO J. 1987 December 20; 6(13): 3901–3907 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC553867/]
  5. [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/gusabul.Par.0001.File.tmp/gusabul.pdf Sigma Aldrich]