Difference between revisions of "Part:BBa K1216000"

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==Characterization==
 
==Characterization==
−
[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used GusA in their project as reporter enzyme. To make predictions about their system, the model obviously needed parameters, so they decided to conduct fluorometric assays in order 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 their system]). After another 4-5h of growth, cells were harvested and lysed, the cell free extract 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. 365nm and ΛEm. 445nm.  
+
[[File:GusA_colored.png|frame|right|<b>Figure 1.  Cell lysate from <i>E.Coli</i> overexpressing GusA after reacting with Salmon-Gluc.</b>]]
 +
[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used GusA in their project as reporter enzyme.
 +
To test the functionality of the enzyme, cell lysate of <i>E.Coli</i> overexpressing GusA was incubated with Salmon-Gluc.
 +
[[File:gusa_color_reaction.png|thumb|center|396px|<b>Figure 2.  Enzymatic reaction of GusA with Salmon-Gluc.</b>]]
 +
[[File:GusA_fluorescent.png|thumb|right|206px|<b>Figure 3.  Cell lysate from <i>E.Coli</i> overexpressing GusA after reacting with 4-MU-β-D-Glucuronide.</b>]]
 +
Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-β-D-Glucuronide. The picture was taken with a common single lens reflex camera mounted on a dark hood at &lambda;Ex 365 nm.
 +
[[File:gusa_fluorescent_reaction.png|frame|center|<b>Figure 4.  Enzymatic reaction of GusA with 4-MU-β-D-Glucuronide.</b>]]
 +
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&trade;.
 
The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot&trade;.
 
See the resulting graph below.
 
See the resulting graph below.
−
[[File:gusa_menten.png|center|551x376px|Michaelis-Menten-Kinetics of GusA with 4-MU-β-D-glucuronide.]]
+
[[File:gusa_menten.png|frame|center|500px|<b>Figure 5.  Michaelis-Menten-Kinetics of GusA cell lysate from <i>E.Coli</i> overexpressing GusA:</b> plots velocity versus substrate concentration (2.5 &mu;L, 5 &mu;L, 10 &mu;L, 30 &mu;L, 60 &mu;L, 120 &mu;L, 240 &mu;L) 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 = 141.1 ± 5.3 &mu;M. All assays were carried out in triplicates, results are presented as means.]]
 +
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 OD<sub>600</sub> of 0.6
 +
#*Induce enzyme expression (100nM AHL in our case)
 +
#*Let grow at 37°C shaking(200 rpm) for 4-5h<br>
 +
#Cell lysis
 +
#*Transfer to 50 mL Falcon&trade; tube
 +
#*Spin down at 4°C for 5 min with 4 rcf
 +
#*Resuspend in lysis buffer, 1 &mu;L/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
 +
#Hydrolysis reaction
 +
#*Perform this measurement in a 96 well plate or similar
 +
#*Perform 3 replicates for each substrate concentration
 +
#*Present 41.6 &mu;L reaction buffer in each well
 +
#*Add 8 &mu;L diluted CFX (the further dilution ocurring here is intended)
 +
#*Add 30.4 &mu;L of corresponding substrate
 +
#*Detection of fluorescence in suitable plate reader (&lambda;Ex 365 nm, &Lambda;Em 445 nm)
 +
 
  
 
===References===
 
===References===

Revision as of 19:38, 4 October 2013

β-Glucuronidase (gusA) from Bacillis Subtilis

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]


A form of this protein with added 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[2]. 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[3].


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

Figure 1. Cell lysate from E.Coli overexpressing GusA after reacting with Salmon-Gluc.

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used GusA in their project as reporter enzyme. To test the functionality of the enzyme, cell lysate of E.Coli overexpressing GusA was incubated with Salmon-Gluc.

Figure 2. Enzymatic reaction of GusA with Salmon-Gluc.
Figure 3. Cell lysate from E.Coli overexpressing GusA after reacting with 4-MU-β-D-Glucuronide.

Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-β-D-Glucuronide. 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 GusA with 4-MU-β-D-Glucuronide.

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 GusA cell lysate from E.Coli overexpressing GusA: plots velocity versus substrate concentration (2.5 μL, 5 μL, 10 μL, 30 μL, 60 μL, 120 μL, 240 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 = 141.1 ± 5.3 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. 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/]
  3. [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/gusabul.Par.0001.File.tmp/gusabul.pdf Sigma Aldrich]