Our project aims to develop a dual selection system consisting of negative and + positive selections to identify PobR mutants responsive to different aromatic + compounds.
+Biosensor
++ An allosteric TF (aTF), PobR +
+An aTF-based biosensor can respond to a specific compound, or a series of + compounds with similar structures, and subsequently activate reporter gene + expression [1-6]. The TF PobR belongs to + the iclR superfamily, and is a positive transcriptional regulator of the + pobA + gene involved in 4-hydroxybenzoic acid (4HB) metabolism in Acinetobacter [7]. +
+Last year, our team developed a PobR-based biosensor responsive to a 4HB analog, + hydroxymandelic acid (HMA) [8]. These results strongly + indicated that PobR had the potential to be modified to become biosensors for a + variety of aromatic compounds.
+Directed evolution
++ Construction of the PobR mutant library +
+In order to construct a PobR library with random mutagenesis, we employed the + approach of error-prone PCR amplification. The generated PobR mutants were + subcloned into the vector containing PpobA, mCherry, + codA and cmr. +
+
+ + The dual selection system +
+After random mutagenesis to generate a PobR library, we wanted to eliminate the + PobR mutants that were either still responsive to 4HB or constitutively active + in driving the PpobA independent of any ligand. For this purpose, we + designed a dual selection system.
+We first constructed a negative selection system consisting of the promoter + PpobA and the CD enzyme coding sequence. The CD enzyme could convert + exogenously added 5-FC to 5-FU that killed bacteria, and this mechanism allowed + us to eliminate both PobR mutants still responsive to 4HB and pseudo-positive + mutants.
+Secondly, we constructed a positive selection system through inserting the + chloramphenicol resistance gene downstream of the PpobA promoter to + help us select the PobR mutants with desired ligand specificity.
+We also added a reporter gene, mCherry, encoding a red fluorescence, in + the selection system. The fluorescence intensity was proportional to the + ligand + binding affinity of PobR mutants that subsequently activated the + PPobA promoter.
+
+ In the negative selection, the obtained bacteria were cultivated a liquid medium + supplemented by 4HB and 5-FC.
+In the positive selection, bacteria were cultured in a selective liquid medium + supplemented with different aromatic compounds and chloramphenicol.
+Next, we further characterized the responsiveness of selected PobR mutants to any + aromatic compound through evaluating the induction of downstream genes and + obtained mutants that were responsive to different aromatic compounds.
+
+ Test
++ Identification of amino acid mutations in selected PobR mutants +
+DNA sequencing of the selected PobR mutants was carried out to determine their + mutation sites and classify the mutated amino acids.
++ Modeling and docking +
+To envision the effects of amino acid substitutions on the response of PobR + proteins and their interactions with small molecule ligands, we used SWISS-MODEL + [9] to + simulate the protein structures and Autodock [10] to mimic the docking of + different aromatic compounds to the proteins. +
+Using bioinformatic methods to process molecular docking, we evaluated how an + amino acid mutation could affect ligand association with the binding pocket of + the PobR mutant and predicted the alteration of binding affinity.
+
+ References
+-
+
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