Composite

Part:BBa_K4140025

Designed by: Ahmed Gamal Mohamed Mattar   Group: iGEM22_AFCM-Egypt   (2022-09-28)
Revision as of 04:45, 12 October 2022 by Mohamed-Ab0elghare (Talk | contribs) (→‎Part Description)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


PAH releasing device

Part Description

It's a Coding sequence for an enzyme that causes phenylalanine to be hydroxylated in order to produce tyrosine. PAH is a hydroxylase that needs biopterin. It's linked to KP-SP ISA secreting peptide signal at the N-terminus of the protein to transmit it from intra-cellular to extracellular

Usage

We are developing a tunable therapeutic approach to give the body the ability to produce phenylalanine hydroxylase (PAH) by implementing our designed therapeutic circuit in a cell-based system that produces PAH according to phenylalanine and tyrosine levels as shown in figure 1. In the presence of phenylalanine (Phe), the TyrR will activate tyrP, accordingly tyrP will enhance the production of PAH gene. While the inhibitory effect of the ParoF will not be initiated, thus L7Ae will be expressed, preventing the expression of the dcas12g.

Right

Figure 1. (shows the E.coli-based system in our therapeutic platform.)

Characterization by mathematical modeling

We are using mathematical modeling to detect the increased level of phenylalanine (phe) in phenylketonuria patients in our therapeutic platform. It depends on E.coli-based system through a cascade of reactions to finally end by formation of PAH that is deficient in PKU patients as shown in graph (1).


Capture6.png


Graph(1) illustrates a direct relation between phenylalanine and PAH ,so as the biomarker increases, the released amount of the enzyme increases till it reaches constant value after about 30 time units. Therefore, the maximum amount of the biomarker releases the maximum amount of PAH.

Experimental Characterization

Tube121.png








This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 10 (the last one). The run part (ordered from IDT) included KP-SP - PAH.













Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 383
    Illegal BamHI site found at 910
    Illegal XhoI site found at 620
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None