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Applications of BBa_K330002

Promoter measurement

Characterization of BBa_K330002

[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. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

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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