Difference between revisions of "Part:BBa K1216004"

Latest revision as of 16:36, 30 October 2013

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

3D representation of the β-Glucuronidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=3K46 RCSB]

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

Characterization

The final construct was sequenced.

Jump to:

• Fluorometric response
• Kinetics

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 λ_Ex 365 nm.

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

Kinetics

To prove that the enzyme retained functionality with an additional His-tag they conducted fluorometric assays to obtain K_m 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_Ex 365 nm and λ_Em 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 K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m = 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 (λ_Ex 365 nm, λ_Em 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]