Difference between revisions of "Part:BBa K1983014"
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<h2>Overview</h2> | <h2>Overview</h2> | ||
− | 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. | + | 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 ([https://parts.igem.org/Part:BBa_K823005 BBa_K823005]), high strength RBS 9[https://parts.igem.org/Part:BBa_J61132 BBa_J61132]) and terminator ([https://parts.igem.org/Part:BBa_B0017 BBa_B0017]). |
<h2>Experiments and Results</h2> | <h2>Experiments and Results</h2> |
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]