In this part of our project, we aimed to engineer new colors for the chromoprotein AmilCP by mutating position N157, a key residue influencing the chromophore and its color. Additionally, we worked to enhance the maturation time of the green variant, which is slower compared to its blue counterpart. By introducing mutations modeled after similar proteins, we sought to accelerate its maturation. To validate these changes, we developed a novel measurement method.
We investigated environmental strains to discover new chromophores or identify known ones in previously uncharacterized strains. Through a series of experiments,including cell lysis, size-exclusion chromatography (SEC), and semi-native SDS-PAGE,we aimed to isolate and concentrate potential chromophores. Complementing our lab work, our bioinformatics team developed a program called ChromoSearch to support and confirm our experimental findings.
Our pipeline offers a streamlined approach for identifying bacterial chromoproteins by analyzing genomic data. It starts with sequenced genomic data in .fasta format and uses Prodigal to identify potential protein-coding sequences, which are translated into putative proteins. These are then compared against databases of known chromoproteins or pigment-producing enzymes using pBLAST, followed by a more detailed Smith-Waterman alignment for high-scoring matches. The molecular weight, length, and a normalized score are calculated for each protein to assess their plausibility, and a Gumbel distribution is used to model rare hits. The pipeline automates these steps, providing an efficient way to identify potential chromoproteins for further validation.
To evaluate the maturation kinetics of the mutated chromoproteins, we developed a novel assay that monitors color development over time under controlled conditions. This new maturation assay offers significant advantages over previously employed methods, as its automated design enables near-continuous monitoring of intensity changes, unlike the discrete measurements often employed in the lab. This streamlined approach also requires less manual effort from lab staff, making the determination of maturation time for chromoproteins both more accurate and less arduous.