Difference between revisions of "Part:BBa K2705008"

Revision as of 05:39, 17 October 2018

P_liaG&43

P_liaG&43 is a constitutive promoter which is an artificial combination of the σA recognition site of P_liaG with the σB recognition site of P₄₃.

Sequence and Features

The original part P_liaG(BBa_K823000)

P_liaG(BBa_K823000) is a weak, constitutive promoter from B. subtilis. It’s responsible for the transcription of the last four genes of the liaIHGFSR locus and therefore for the production of the components of the LiaRS system, which is important for the detection of cell wall antibiotics (Jordan et al., 2006). P_liaG was evaluated with the lux operon as well as the lacZ as reporter. For more details, visit the Data page of the LMU-Munich Team 2012 or get an overview of the whole project Beadzillus.(http://2012.igem.org/Team:LMU-Munich)

Design

We intended to improve the part P_liaG (BBa_K823000). Inspired by the relatively strong constitutive promoter P₄₃(BBa_K143013), we chose to mimic its structure, which can be recognized by both sigma factor 55 (the major sigma factor A) and sigma factor 37 (the lag phase sigma factor B). As the promoter P_liaG is a constitutive Bacillus subtilis σA promoter, we replaced some part of the promoter P_liaG sequence with the σB recognition site at the same relative position which the σB recognition site to the σA recognition site of the promoter P₄₃ to create the new promoter P_liaG&43.

Luminescence measurements

The constitutive promoters P_liaG&43 (BBa_K2705008) was evaluated in the reporter vector pHT01-P_x-GFP. The promoter activity results in gene expression and production of the green fluorescence protein(GFP). The fluorescence intensity(FI) was measured by the Multimode Plate Reader Enspire(PerkinElmer). All clones showed a normal growth behavior. As the σB recognition site of P₄₃ we added is for the lag phase, we tested the FI at the very beginning (the lag phase, Fig.1) and at the very last(the stationary phase , Fig.1b) of its life. P_liaG&43 outperformed the original promoter P_liaG at the lag phase, whose activity is about 1.5 times as high as that of P_liaG.

Fig. 1 Luminescence measurement of the constitutive Bacillus promoters P_liaG&43-2 and P_liaG in the reporter vector pHT01-P_x-GFP. The strains were cultured in LB medium with 5 µg/mL chloramphenicol in dark for 2 hours. Fluorescence intensity of GFP and the OD₆₀₀ was measured. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates. **Very significantly different (P ‹ 0.01) by Student's t-test. .