Difference between revisions of "Part:BBa K1983014"

Latest revision as of 12:21, 22 October 2016

PheP under constitutive promoter, high strength RBS and terminator

Overview

PheP (Phenylalanine-specific permease) is a natural phenylalanine membrane transporter of the bacterium Escherichia coli. PheP is a single integral membrane transporter which selectively transports L-phenylalanine and L-tyrosine as an antiporter using proton motive force. The activity of this transporter under natural expression is known to be 9 and 17,5 nmol/mgDW(cells) for L-phenylalanine and L-tyrosine respectively [1]. This biobrick part of PheP is labeled with N-6XHis-Tag and put under a constitutive promoter (BBa_K823005), high strength RBS 9BBa_J61132) and terminator (BBa_B0017).

Experiments and Results

Cloning

The received sequences were amplified using Uni-FW/RV primers and digested with EcoRI and SpeI. The fragments containing mutant genes were cloned into pSB1C3 vector containing BBa_B0017 digested with EcoRI and XbaI. Transformant colonies were PCR-screened using VF2/VR primers and positive clone plasmids were sequenced prior to further usage.

Characterization in vivo

Vilnius-Lithuania iGEM team has proven this parts effectiveness as a transporter by using an enzyme that breaks down L-phenylalanine to form ammonia and trans-cinnamic acid (tCA) inside the cell. As this enzyme, phenylalanine ammonia lyase (PAL), is expressed inside the cell, and the L-phenylalanine is in the outer medium, additional expression of PheP would facilitate the flux through the membrane and drive the enzymatic reaction, thus raising the levels of formed tCA.

As can be seen in the figure 1, PheP provides facilitated transport for L-phenylalanine through the membrane. This result is achieved using a composite biobrick part BBa_K1983014 - PheP under constitutive promoter, high strength RBS and terminator.

Figure 1. Activity of PAL and PAL with PheP in vivo Activity of PAL and PAL with PheP (BBa_K1983014) in E. coli cell over a period of 20 minutes in 7.4 pH. The activity is evaluated by production of tCA. The initial amount of L-phenylalanine in reaction mixture was 1.1 g. Every system which was tested during this experiment was transformed into E. coli TOP10. Control - E. coli TOP10 strain without PAL and pheP biobricks. The total mass of recombinant cells in the reaction mixture was 5 grams.

References

1. Cosgriff, A. J., G. Brasier, et al. (2000). "A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport." J Bacteriol 182(8): 2207-2217.

Sequence and Features