Difference between revisions of "Part:BBa K4140010"

Latest revision as of 17:27, 10 October 2022

Permease

Part Description

The bacterium Escherichia coli naturally transports phenylalanine across membranes via PheP (Phenylalanine-specific permease). PheP is a single, integral membrane transporter that uses the proton motive force to antiport L-phenylalanine and L-tyrosine. This transporter's activity under natural expression is known to range between 9 and 17,5.

Usage

PheP, an intrinsic membrane transporter that employs the proton motive force to antiport L-phenylalanine and L-tyrosine, naturally transports phenylalanine across membranes via phenylalanine-specific permease. We employ the component to increase the intracellular concentration of phenylalanine, which improves the permeability of cells to phenylalanine and triggers our circuit to express PAH in the therapeutic circuit and beta-galactosidase in the diagnostic circuit as shown in figure 1.

Figure(1) Shows an SBOL demonstrating the usage of permease in our whole cel-based biosensor

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the premease protein. The (H37K) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (A153L) had the least evolutionary fitness for premease protein. As displayed in Figure(2)

Figure 2. (shows the mutational landscape of the Permease protein.)

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Literature Characterization

The mutant PheP after changing each proline residue to alanine using synthetic oligonucleotides was sequenced by the team in study. After that the replicative form of M13mp18 DNA containing the mutations was isolated, and the EcoRI-SalI fragment containing pheP was cloned into the corresponding sites on pLG339. The uptake of phenylalanine ( 10 M) was measured in transformants of E. coli JP6488 (aroP pheP) with plasmid pLG339 carrying the mutated pheP gene as shown i figure 3.

Fig. 3 shows the measurements of the uptake of phenylalanine (10 M) in transformants of E. coli JP6488 (aroP pheP) with plasmid pLG339 carrying the mutated pheP gene.

The results of these tests are shown in the figure above. Only P54, P442, and P341 had significant effect on the transport activity. P54 and P341 are two highly conserved proteins in the family, and only P341 seems to play a critical role in functioning PheP. The alanine-substituted mutants of other highly conserved residues, such as P238, P269, P279, and P329, showed little or no loss of activity, and in the case of P279, they showed increased activity.

Experimental Characterization

This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 7. The run part (ordered from IDT) included Lac promoter - Permease.

References

1. Pi, J., Dogovski, C., & Pittard, A. J. (1998). Functional consequences of changing proline residues in the phenylalanine-specific permease of Escherichia coli. Journal of bacteriology, 180(21), 5515-5519.‏

Sequence and Features