Designing a dual selection system
+We constructed a dual selection system based on plasmid + gYB2a-pobRWT-mCherry-codA-cmr. This plasmid mainly contains three parts: +
+1. The pobR coding sequence (CDS) which can express PobR protein;
+2. The cytosine deaminase(codA) gene which can express CDase protein;
+3. An engineered operon consisting of the red fluorescent protein(mCherry) + and the chloramphenicol(Cm) resistant gene (cmr).
+How this plasmid acts as a dual selection system is as follows:
+1. Negative selection: Been activated by PobRWT, PpobA + activates the expression of CDase which is available for conversion of + exogenously added 5-FC to 5-FU, causing cell death;
+2. Positive selection: Without no additional 5-FC, adding chloramphenicol and an + aromatic compound to cultivate E. coli cells will get a library of PobR + mutants. Strains with PobRmut that recognize the aromatic compound + are expected to grow in the LB agar medium.
+To further understand each part, please click + here to view our design + part.
+To constructed gYB2a-pobRWT-mCherry-codA-cmr, we first tried to + generate plasmid gYb2a-PobR-PpobA*2-mcherry-SacB-Cmr by Goldengate + assembly. However, when applying the plasmid + gYb2a-PobR-PpobA*2-mcherry-SacB-Cmr to achieve functions as a dual + screen system, the results are not ideal.
+We obtain the two target fragments, gYb2a-PobR-PpobA*2-mcherry-Cmr and + CD by the method of PCR. Then the two fragments were ligated by using Goldengate + assembly and transformed into E. coli BWΔcodA competent cells and + the plasmid gYB2a-pobRWT-mCherry-codA-cmr was constructed.
+The result of DNA sequencing showed that our + gYB2a-pobRWT-mCherry-codA-cmr plasmid was constructed.
+Directed evolution of PobR
+Aiming to get strains that respond to different aromatic compounds by the dual + selection system, a large PobR mutant library which is able to include as many + mutants situations as possible is crucial. Therefore, we used error-prone PCR to + construct the PobR mutant library.
+The generated library with highly random PobR mutants was transformed into + E.coli BWΔcodA to obtain transformants containing mutant plasmids. + The PobR mutant library was transformed into BWΔcodA competent cells and + transferred to M9 medium for culturing in shaking flasks. Ten clones were + randomly picked to sequence their PobR CDS regions for the quality control, + which revealed diverse mutants with an average mutants rate of about 0.36%. +
Fluorescence assay and screening of the PobR mutant library
+Another part which is essential to get the new ligands specificities and + detection range of PobRmut is the screening part.
+In the negative selection, the obtained strains were cultivated in liquid culture + supplemented by 4HB and 5-FC. In the initial negative selection, we used a + constant 4HB concentration of a relatively high level, 0.5 g/L, and then tested + different concentrations of 5-FC to inhibit both the pseudo-positive and + 4HB-responsive strains. In this selection step, we first used 50 mg/L 5-FC, and + observed insufficient inhibition of the bacterial growth. Thus, we increased the + 5-FC concentration to 200 mg/L, improving the selection effectiveness.
+In the positive selection, we added seven aromatic compounds to LB agar medium + and cultivated E. coli cells harboring a library of PobR mutants in this + medium for strain selection. As for the aromatic compounds, we chose + phenylethanol (2-PE), mandelate (MA), 4-hydroxymandelate (HMA), phenylpyruvate + (PPA), 4-hydroxyphenylpyruvate (HPP), phenylacetaldehyde (PAld) and p-Coumaric + acid.
+After culturing for 30 hours, a few pink colonies were picked and cultured in a + 96 deep-well plate. Since the expression of the reporter gene mCherry was + positively correlated with responsiveness, all that required is to transfer the + pink colonies to LB media containing only Amp after activation and used 0.5 g/L + of the test ligands for initial characterization. At the same time, negative + control without the addition of test ligands was used to avoid a few biosensor + variants with a strong fluorescence response in the absence of any ligand. After + performing fluorescence measurement，we got some strains that are highly + responsive to aromatic compounds.
+Besides, we used the dilution coating method to estimate the number of mutants + which are capable of aromatic compounds per screen. The selection capacity for + each compound was more than 900，000 clones (with at least four plates, the + original density of the two-round selected bacteria was 450,000 CFU/mL.)
+Please click here to + view our notebook.
+The specificity and detection range of PobR mutants responsive to new ligands +
+We obtained several responsive strains, of which the fluorescence intensity was + 1.5-fold higher than that of the negative control. To further evaluate the PobR + mutants obtained above, the second round of characterization experiments were + carried out to individually examine their responsiveness to each candidate + ligand. For each ligand, we selected a mutant strain with the highest responsive + profiles and plotted the curve for their ligand response. In total, 9 potential + biosensors were isolated after the second round of characterization, and all + these variants were sequenced to determine the mutations in their primary + sequences. Amino acids at positions 163, 177 and 234 are located near the ligand + binding pocket of PobR, and amino acid at position 40 is located in the DNA + binding domain.
+Modeling and docking
+To better understand and analyze the effects of amino acid substitutions on the + response of the PobR protein, we tried to use software to simulate the structure + of the protein and build a molecular docking between the PobR protein and its + inducer.
+We first use Homologous Model website SWISS-MODEL to construct the PobR mutant + model using the PobR wild type as a template.
+Secondly, we obtained the structure files of the ligands 4HB, 2-PE, MA, HMA, + PAld, HPP and PPA from the organic small molecule database Pubchem. For more + details you can click here.
+Based on the structures of PobR monomer and ligands, the docking engine Autodock + is used to simulate molecular docking. Autodock search space coordinates were + set + as center_x = -4.672, center_y = 3.331, center_z = -2.213. Dimensions of the + search space were set as size_x = 40, size_y = 40, size_z = 40, and + exhaustiveness was set at 15. The 15 conformational conditions in a score based + on the lowest binding energy were listed as the docking results. To examine the + accuracy of our docking, we used Ligplus to check the interaction between the + small molecule and predicted protein receptors. Finally, the three-dimensional + schematics of the protein and its ligand were portrayed using PyMol Version + 2.2.0.
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