phospholipase+C+(clostridium+perfrigens)

 ***Protein ID (NP or XP #) or Wolbachia#:** **2WY6 **  ***Organism (including strain):** **clostridium perfrigens **  **Etiologic Risk Group (see link below):**  ***/** **Disease Information**  **(sort of like the Intro to your Mini**  **Research Write**  **up):** Clostridium perfringens alpha toxin is a [|toxin]  produced by the <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt; text-decoration: none;">[|bacterium]  <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt;"> [|Clostridium perfringens] (C. perfringens) and is responsible for <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt; text-decoration: none;">[|gas gangrene]  <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt;"> and <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt; text-decoration: none;">[|myonecrosis]  <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt;"> in <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt; text-decoration: none;">[|infected] <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt; text-decoration: none;">[|tissues]  <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10.5pt;">. <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Link to TDR Targets page (if present):** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">None ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Essentiality of this protein:** <span style="font-family: Arial,sans-serif; font-size: 10pt;">Is it a monomer or multimer as biological unit**? (make prediction at** <span style="font-family: Arial,sans-serif; font-size: 10pt;">@http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html <span style="font-family: Arial,sans-serif; font-size: 10pt;">): <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Complex of proteins?:** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">monomer ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***EC#:** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">3.1.4.3 ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Link to BRENDA EC# page:** <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;"> http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.4.3&Suchword=&organism%5B%5D=Clostridium+perfringens <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **--** Show screenshot of BRENDA enzyme mechanism schematic <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Enzyme Assay** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**information (spectrophotometric, coupled assay ?, reagents):** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">colorimetric ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **-- link to Sigma page for assay** <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/phospholipaseccper.pdf <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **--- List cost and quantity of substrate reagents, supplier, and catalog #** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Structure (PDB or Homology model)** <span style="font-family: Arial,sans-serif; font-size: 10pt;">-- PDB #: <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">2WY6 <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Current Inhibitors:** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">21 ** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Purification Method** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**:** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Image of protein (PyMol with features delineated and shown separately):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***Amino Acid Sequence** <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">WDGKIDGTGTHAMIVTQGVSILENDLSKNEPESVRKNLEILKENMHELQLGSTYPDYDKNAYDLYQDHFWDPDIDNNFSK <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">DNSWYLAYSIPDTGESQIRKFSALARYEWQRGNYKQATFYLGEAMHYFGDIDTPYHPANVTAVDSAGHVKFETFAEERKE <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">QYKINTAGCKTNEAFYTDILKNKDFNAWSKEYARGFAKTGKSIYYSHASMSHSWDDWDYAAKVTLANSQKGTAGYIYRFL <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">HDVSEGNDPSVGKNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMTGSKDTYTFKLKDENLKIDD <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">IQNMWIRKRKYTAFPDAYKPENIKIIANGKVVVDKDINEWISGNSTYNIK
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">*Target (protein/gene name): ****<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">Phospholipase C (alpha toxin) **
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">*NCBI Gene # or RefSeq#: **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 9pt;">4201274
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">Phospholipase C from clostridium perfringens **
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">P7633-25UN; CAS: 0009001869; $75.10 (Sigma) **
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">Alkaline Phosphatase **
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">10713023001; CAS:0000000000; $91.99 (Sigma) **
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Expression Information (has it been expressed in bacterial cells): **
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">Alpha toxin and N-terminal domain expressed in Escherichia coli **
 * <span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">Affinity chromatography, gel filtration, 2500fold purification from culture filtrate **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***length of your protein in Amino Acids:** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">370 ** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Molecular Weight** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of your protein in kiloDaltons using the** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> [|**Expasy ProtParam**] **<span style="font-family: Arial,sans-serif; font-size: 10pt;">website ****<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">42546.0 ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Molar** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Extinction coefficient** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of your protein at 280 nm wavelength:** **<span style="color: #548235; font-family: Arial,sans-serif; font-size: 10pt;">93740 M-1 cm-1 at 280nm ** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **TMpred graph Image** ( <span style="font-family: Arial,sans-serif; font-size: 10pt;">@http://www.ch.embnet.org/software/TMPRED_form.html <span style="font-family: Arial,sans-serif; font-size: 10pt;">).

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***CDS** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Gene Sequence** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**(paste as text only):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene:** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***CDS Gene Sequence (codon optimized) - copy from** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**output** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Primer Design** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">**Protocol (paste as text only):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene (codon optimized):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;">Do Not Need this info for <span class="r7mb967" style="font-family: Arial,sans-serif; font-size: 10pt;">Spring <span style="font-family: Arial,sans-serif; font-size: 10pt;">(but still copy these lines to your Target page for now) <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **(** **<span style="color: #008000; font-family: Arial,sans-serif; font-size: 10pt;">link to DNA Works output ** **<span style="color: #008000; font-family: Arial,sans-serif; font-size: 10pt;">text file ** **<span style="color: #008000; font-family: Arial,sans-serif; font-size: 10pt;">- ** <span style="font-family: Arial,sans-serif; font-size: 10pt;">that should be saved in your <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">Google Docs <span style="font-family: Arial,sans-serif; font-size: 10pt;">folder after you did the primer design protocol) <span style="font-family: Arial,sans-serif; font-size: 10pt;">-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">you are not <span style="font-family: Arial,sans-serif; font-size: 10pt;">sure how to.

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Primer design results for 'tail' primers (this is just 2 sequences):**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">**