Difference between revisions of "Part:BBa K1509001"

Revision as of 11:50, 17 October 2014

A bi-directional promoter affected by SmtB protein

smt O-P is a promoter which has two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between gene smtA and gene smtB. The full promoter/operator DNA binds two SmtB dimmers forming a S1/S2-SmtB:SmtB-S3/S4 bridge complex. After binding with smtB, smt O-P represses the expression of downstream genes.

Usage and Biology

In our project, we wanted to use pigments as reporters in our designed genetic construction which can be recognizable by the naked eyes. According to the previous work, we have chosen an identified pigment in the Registry: the biobrick of amilCP (BBa_K592009) was used as reporter gene in our metal detection device. After exchanging the biobrick part with SmtA in smtB-OP-smtA device, the pigment gene was under control of metal-induced promoter (smtB-OP).

Result

fig.1. Growth of cells containing smtB-OP-smtA element in LB medium supplemented with 2μM Cd2+. Cells were inoculated at a density of 1x106 cells ml-1, and growth was monitored by measuring the OD540 value. Data points represent the mean values from three separate cultures with SD.

fig.2. A. Metal-induced expression of the pigments (RFP), Rosetta-plysS (1x106 cells ml-1) carrying the smtB-OP-RFP element were grown with Cd2+ and Zn2+ (1-50μM) supplement for 2h immediately before assay and expression was monitored by measuring the OD450 value; B. Metal-induced expression of the pigments (RFP). Rosetta-plysS carrying the smtB-OP-RFP element were grown with Cd2+ and Zn2+ (2μM) supplement for 1-12h immediately before measurement; C. Cadmium-induced expression of the pigment (RFP) at different concentrations. Rosetta-plysS (1x107 cells ml-1) carrying the smtB-OP-RFP element were grown with Cd2+ (1-20μM) supplement for 2h. The data points shown in A and B represent the means of three separate assays with SD.

fig.3. A. Cadmium-induced expression of the pigment (amilCP) at constant concentration. Rosetta-plysS (1x106 cells ml-1) carrying the smtB-OP-amilCP element were grown with Cd2+ (1μM) supplement for 1-2h immediately before assay and the expression was monitored by measuring the OD600 value; B. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS carrying the smtB-OP-amilCP device were grown with Cd2+ (1-100μM) supplement for 1h immediately before assay；C. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS (1x107 cells ml-1) carrying the smtB-OP-amilCP element were grown with Cd2+ (10, 20, 50, 100, 200 and 500 μM) supplement for 2h. The data points shown in A and B represent the means of three separate values with SD.

Sequence and Features