Difference between revisions of "Part:BBa K330002:Experience"

Latest revision as of 17:13, 30 October 2013

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Applications of BBa_K330002

Promoter measurement

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used GusA in their project as a reporter enzyme. See here for their experience.

Characterization of BBa_K330002

Outlines:

1. Introduction

2. Qualitative characterization with GUS substrate X-Gluc

3. Quantitative characterization with GUS substrate 4-NPG

4. Reference

1. Introduction

PBI121(Kmr) containing gusA reporter gene which encoded GUS was used as the GUS producer. The promoter driving gusA was CaMV 35S, a constitutive promoter in E.coli. BioBrick BBa_I746101 which contained agrC gene but no gusA reporter gene was used as the control. E.coli DH10B competent cell was used as the bacteria host for the characterization. E.coli has gusA gene as well in its genome, though, the expression of this gene is much less than that in PBI121.

2. Qualitative characterization with GUS substrate X-Gluc

X-Gluc, 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, is a commonly used GUS substrate which turns blue when induced by GUS.

Experiment I: Assay by applying X-Gluc to lysed cell.

Due to the defective X-Gluc permeability, E.coli K12 derivates (including DH10B and DH5α) need to be lysed or perforated on cell membrane. In this assay, lysozyme and sonication were used to lyse E.coli.

Procedure:

a. Transformed cells were grown 6-16h at 37°C in LB broth with kanamycin added.

b. Lysozyme was added to get a final concentration of 1mg/ml. The cell mixture was incubated at 37℃ for 1h.

c. The mixture was lysed by sonication.

d. X-Gluc solved in DMSO was added to reach the final concentration of 0.5mg/ml. The cell mixture was incubated at 37℃ for 15min.

Results:

The PBI121 group turned blue, while the agrC group did not.

Experiment II: Assay by applying X-Gluc to permeability increased cells

Acetone-Toluene was used to increase the permeability of E.coli instead of cell lysis.

Procedure:

a. Transformed cells were grown 6-16h at 37°C in LB broth with kanamycin added.

b. Cells were harvested and collected by centrifugation and then resuspended in 400ul of 50mM NaPi buffer (pH 7.0).

c. The remaining cell suspensions were used in GUS assay. To each cell suspension, 25 ul of acetone–toluene (9:1 v/v) was added, and the mixture was incubated at 37°C for 40 min for cell permeabilization.

d. The mixture were added with X-Gluc to 0.5mg/ml at final concentration

Involved reagents:

NaPi (50mM, PH=7.0)

Acetone–toluene (9:1 vol/vol)

GUS buffer (50mM NaPi, pH7.0, 10mM β-mercaptoethanol, 1mM EDTA, 0.1% Triton X-100)

Results:

Both the PBI121 group and the agrC group turned blue. Yet the PBI121 group had darker blue, which suggested there were more GUS in the PBI121 group.

3. Quantitative characterization with GUS substrate 4-NPG

4-NPG, 4-Nitrophenyl β-D-glucuronide, is commonly used for spectrophotometric GUS assay.

Procedure:

a. Transformed cells were grown 6-16h at 37°C in LB broth with kanamycin added.

b. Cells were harvested. OD600 was measured.

c. Collect cells by centrifugation and then resuspended in 400ul of 50mM NaPi buffer (pH 7.0).

d. The remaining cell suspensions were used in GUS assay. To each cell suspension, 25 ul of acetone–toluene (9:1 v/v) was added, and the mixture was incubated at 37°C for 40 min for cell permeabilization.

e. Volumes of 50ul of each cell mixture were immediately applied to the GUS assay by adding 200ul of GUS buffer (50mM NaPi, pH7.0, 10mM b-mercaptoethanol, 1mM EDTA, 0.1% Triton X-100) and 20 ul of GUS substrate (make final concentration 0.5mg/ml) ( para-nitrophenyl-b-D-glucuronic acid in 50mM NaPi buffer; Sigma).

f. Mixtures were incubated at 37°C for X (X=10, 20, 30)min, and each reaction was stopped by the addition of 200 ul of 200mM Na2CO3.

g. Volumes of 200ul of each mixture were then monitored spectrophotometrically at OD405, and obtained values of GUS were used to determine specific GUS activity per optical cell density (OD405/OD600).

Involved reagents:

NaPi (50mM, PH=7.0)

Acetone–toluene (9:1 vol/vol)

GUS buffer (50mM NaPi, pH7.0, 10mM β-mercaptoethanol, 1mM EDTA, 0.1% Triton X-100)

GUS substrate ( para-nitrophenyl-β-D-glucuronic acid in 50mM NaPi buffer)

Na2CO3 (200mM)

Results:

GUS activity=OD405/OD600

4. Reference

Johnsborg, O., Diep, D. B. & Nes, N. F. (2003). Structural analysis of the peptide pheromone receptor plnB, a histidine protein kinase from Lactobacillus plantarum. Journal of Bacteriology, 185 (23), 6913–6920.

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ETH Zurich 2013 Experience

Colorimetric and fluorometric response

Figure 2. Liquid culture of the triple knockout Escherichia coli strain overexpressing GusA; after reaction with Salmon-Gluc. The negative control (right) is a liquid culture without the substrate added.

[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, liquid cultures of our ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing GusA was incubated with Salmon-Gluc (Figure 2).

Figure 1. Enzymatic reaction of GusA with Salmon-Gluc.

Figure 4. Cell lysate from the ΔaesΔgusAΔnagZ Escherichia coli strain 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 in Figure 4 was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 3. Enzymatic reaction of GusA with 4-MU-β-D-Glucuronide.

Kinetics
To characterize the enzyme 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 5. Michaelis-Menten-Kinetics of GusA cell lysate from E.Coli overexpressing GusA: 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 = 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 (λ_Ex 365 nm, λ_Em 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.

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