Kian+C.

[|SensoLyte® pNPP Protein Phosphatase Assay Kit *Colorimetric*] -Note: activity assay kit to use later

=__Week 7 & 8__= __ Mutant DNA Analysis __

=__Week 5 & 6__=

__DH5α Transformation with Mutant EhPTP pNIC-Bsa4 __
Figures 9-14. Transformation with 100 μl of product compared to a 1:10 dilution of the product in SOC growth media following site-directed mutagenesis with 25 ng of wild-type template DNA.

Yes, sample A and B of the wildtype EhPTP sequence (in pNIC28-Bsa4) from the start codon (green) to the pLIC-rev site (pink). I used DNA from sample A for mutagenesis on because it had an AvgQ score of 41, greater than that of sample B (34). Does the score difference necessarily matter? This afternoon, I plated transformed DH5-alpha with the control plasmid, EhMut125 (final and 1:10), EhMut126 (final and 1:10), and EhMut125-126 (final and 1:10) - each hopefully containing a Y to F codon substitution(s). Currently, the plates are incubating so tomorrow morning we'll be able to see if the mutants cultured! - Kian 07/05/17 = = =__Week 3 & 4__= __DNA Analysis__
 * Nice work Kian. I assume the below sequences are just the unmodified plasmid? - DR. B 07052017**

__ Midiprep __

=__Week 1 & 2__=

__PyMOL Mutagenesis__
//PDB ID// 3JS5 Figures 5 & 6. Mutagenesis of tyrosine residues 125 & 126 to non-phosphorylatable phenylalanine in PyMOL. //Note:// Through Y to F substitution, the change of polarity due to the removal of the hydroxyl group (as seen above) should mimic the properties of Tyr residues 125 & 126 after undergoing dephosphorylation and their role in EhPTP activity, enabling a functional model of EhPTP pre- and post-phosphorylation of the two residues - individually and together - when compared to the activity of the wildtype strain in subsequent phosphatase assays.

__ Primer Design __ Caplan_EhMut125-126, forward and reverse primers Required primers were designed using NEBaseChanger to substitute TACTAC (1054-1059), encoding Tyr 125 & 126, with TTCTTC to encode Phe 125 & 126.
 * **Name_For/Rev** || **Oligo** || **Len** || **% GC** || **Tm** || **Ta*** ||
 * Caplan_EhMut125-126_For || TCCGGACCCGttcttcGGTGGTGAAA || 26 || 58 || 69 || 66 ||
 * Caplan_EhMut125-126_Rev || ACTTCGGTGGTTTTGATGGTG || 21 || 48 || 65 || 66 ||

Caplan_EhMut125, forward and reverse primers Required primers were designed using NEBaseChanger to substitute TAC (1054-1056), encoding Tyr 125, with TTC to encode Phe 125.
 * **Name_For/Rev** || **Oligo** || **Len** || **% GC** || **Tm** || **Ta*** ||
 * Caplan_EhMut125_For || TCCGGACCCGttcTACGGTGGTG || 23 || 65 || 72 || 68 ||
 * Caplan_EhMut125_Rev || ACTTCGGTGGTTTTGATGGTGG || 22 || 50 || 67 || 68 ||

Caplan_EhMut126, forward and reverse primers Required primers were designed using NEBaseChanger to substitute TAC (1057-1059), encoding Tyr 126, with TTC to encode Phe 126.
 * **Name_For/Rev** || **Oligo** || **Len** || **% GC** || **Tm** || **Ta*** ||
 * Caplan_EhMut126_For || GGACCCGTACttcGGTGGTGAAA || 23 || 57 || 68 || 65 ||
 * Caplan_EhMut126_Rev || GGAACTTCGGTGGTTTTGATG || 21 || 48 || 63 || 65 ||

__PyMOL__
//PDB ID// 3JS5
 * Residues of interest:** Tyrosine 125 & 126, as discussed in the conclusion section of Linford and colleagues (2014).
 * Reference: A.S. Linford et al. / Molecular & Biochemical Parasitology 193 (2014) 33–44
 * "The E. histolytica LMW-PTP protein sequence has the conserved DPYY loop containing the general acid/base asparagine and the two tyrosines that can be phosphorylated to regulate phosphatase activity or allow binding of adapter proteins"
 * "To address if phosphorylation of the DPYY loop tyrosines of the LMW- PTP affects its activity, for example, mutant forms of the LMW-PTP with substituted non-phosphorylatable residues at those positions could be expressed in vivo or in vitro and then assayed for phosphorylation and phosphatase activity; in vitro testing of these mutants alongside the recombinant wild-type LMW-PTP for phosphatase activity on pNPP after phosphorylation with Src kinase could be performed."
 * Amino acid substitution:**
 * Tyr to Phe 125 & 126
 * [[image:vdsstream/Tyr_phe.gif]]
 * Phenylalanine is a known non-phosphorylatable, phosphotyrosine mimic

__DH5α Transformation with EhPTP pNIC-Bsa4__


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=Past EhPTP Work (Spring 2017)=

__DNA Analysis__


3JS5:A | PDBID | CHAIN | PROTEIN SEQUENCE MAHHHHHHMGTLEAQTQGPGSMKLLFVCLGNICRSPAAEAVMKKVIQNHHLTEKYICDSAGTCSYHEGQQADSRMRKVGKSRGYQVDSISRPVVSSDFKNFDYIFAMDNDNYYELLDRCPEQYKQKIFKMVDFCTTIKTTEVPDPYYGGEKGFHRVIDILEDACENLIIKLEEGKLIN