Difference between revisions of "Part:BBa K2705011"

Revision as of 15:28, 17 October 2018

P_liaG&43-3

P_liaG&43-3 is a promoter which is an artificial combination of P_liaG and P₄₃ with partly modification.

Sequence and Features

The original part P_liaG(BBa_K823000)

P_liaG(BBa_K2705000) 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 improved the part P_liaG(BBa_K2705000) which is a weak, constitutive promoter of Bacillus subtilis. 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 σ^A promoter, we replace some part of the promoter P_liaG sequence with the σ^B recognition site at the same relative position which the σ^B recognition site to the σ^A recognition site of the promoter P₄₃ to create the new promoter P_liaG&43. On the basis of this, we made 2 adjustments to create another new promoter P _liaG&43-3. The first one is to adjust the sequence of the -35 region of P_liaG (from CGTATC to TTGACA). The other one is adjustments to the spacing between -35 region and -10 region from 19 bp to 17 bp.

Luminescence measurements

The constitutive promoters P_liaG&43-3 (BBa_K2705011) was evaluated in the reporter vector pHT01-P_x-GFP. The promoter activity results in gene expression and production of the protein 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 σ^B recognition site of P₄₃ we added is for the lag phase, we tested the FI at the very beginning (the lag phase, Fig.1) of its life. P_liaG&43-3outperformed the original promoter P_liaG at the lag phase, whose activity is about 1.34 times as high as that of P_liaG.

Fig. 1 Luminescence measurement of the constitutive Bacillus promoters P_liaG&43-3 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 was measured, which was normalized against OD₆₀₀. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates. **Very significantly different (P ‹ 0.01) by Student's t-test.