The 2018 NKU_CHINA team improved 2 existing parts, the promoter P_liaG (BBa_K823000) of Bacillus subtilis (B. subtilis) and the tetracycline resistance protein TetA(C) (BBa_J31006). Then we created TetA (optimized for LL3) (BBa_K2705007), P_liaG&43 (BBa_K2705008), P_liaG&43-1 (BBa_K2705009), P_liaG&43-2 (BBa_K2705010), P_liaG&43-3 (BBa_K2705011).

1. P_liaG Improvements

Usage and Biology

P_liaG (BBa_K823000) is a weak, constitutive promoter from B. subtilis. It is responsible for the transcription of the last four genes of the liaIHGFSR cluster 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.

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 σ^A promoter, we replace part of the promoter P_liaG sequence with the σ^B recognition site at the same relative position as 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 some adjustments to the spacing between -35 region and -10 region, the sequence of -35 region and created P_liaG&43-1(BBa_K2705009), P_liaG&43-2(BBa_K2705010), P_liaG&43-3 (BBa_K2705011).

Luminescence Measurements

The constitutive promoters P_liaG&43 (BBa_K2705008), P_liaG&43-1 (BBa_K2705009), P_liaG&43-2 (BBa_K2705010), P_liaG&43-3 (BBa_K2705011) were 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 σ^B recognition site of P₄₃ we added is a lag phase sigma factor binding site, we tested the FI at the very beginning (the lag phase, Fig.1a) of its life. All the modified promoters except for the promoter P_liaG&43-2 (BBa_K2705010) outperformed the original promoter P_liaG at the lag phase. The activity of the best promoter, P_liaG&43 is about 1.5 times as high as the activity of P_liaG. The promoter P_liaG&43-2 (BBa_K2705010) still has a slightly higher activity than the promoter P_liaG (Fig.1)

Fig. 1 Luminescence measurement of the constitutive _Bacillus sp. promoters P_liaG&43, P_liaG&43-1, P_liaG&43-2, 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.

Result

As these promoters are all constitutive promoters, the activity enhancement at the lag phase can surely enhance the promoters' effect, which has achieved the goal of promoter enhancement as well as the part P_liaG (BBa_K823000)'s improvement.

2. TetA Improvements

Usage and Biology

The tetracyclines (Tcs) is a group of antibiotics that have been widely used since the 1940s against both Gram-negative and Gram-positive bacteria. Resistance to Tcs has many mechanisms including the active efflux system of tetracycline, target gene mutations, enzymatic degradation of antibiotics, decreased drug permeability and so on. The tetracycline efflux pumps are encoded by several genes, including tetA, tetC, tetE, tetG, and tetH, which have been reported a major mechanism of Tcs resistance.

Design

Here we improved tetA (BBa_J31006) by codon optimizing for Bacillus amyloliquefaciens, which could result in Tcs resistance by encoding membrane-associated proteins that pump Tcs out of the cell, reducing intracellular drug concentrations and protecting intracellular ribosomes.

Here we entrust Genescript to help us do the optimization. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in Bacillus amyloliquefaciens FZB42 by upgrading the CAI from 0.69 to 0.92. (Fig. 2) GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. (Fig. 3) The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, our optimization process has screened and successfully modified several negative cis-acting sites.