Part:BBa_K330002:Experience

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Applications of BBa_K330002

Promoter measurement

Characterization of BBa_K330002

Outlines:

1. Introduction

2. Qualitative characterization with GUS substrate X-Gluc

3. Quantitative characterization with GUS substrate 4-NPG

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 reduced 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.

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