Target-+phosphoserine+phosphatase+(Vibrio+cholerae)


 * Targe**t: //IEC224// Phosphoserine phosphatase
 * NCBI Gene #**: 11915261
 * Protein ID#**: YP_005334106.1 (Used Dax's Oligo Design to order primers. Couldn't find Will's DNA Works Output on GDocs. Julia's first primer is small..? - Dr. B 090313)


 * organism**: //Vibrio cholerae//
 * etiologic risk group**: Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia


 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**
 * Vibrio cholerae is a bacteria that can cause a disease known as cholera depending on the strain encountered. The disease was first discovered in 1817 when it was spread by using trading routes. Several pandemics have occured through the mass spreading of the bacteria. In addition to several antibacterial treatments for extreme cases of the disease, vaccines are available to prevent transmission. However, the main treatment used is a process called oral rehydration therapy.**


 * Cholera is spread when a host consumes food or drink that has been contaminated with the waste of an infected person. Some of the main symptoms include diarrhea and vomiting. Symptoms typically occur between one and five days from becoming infected. Water is usually the cause of cholera in developing countries and although rare, the cause of cholera in developed regions of the world is seafood particularly plankton and shellfish. The nickname for this disease is "blue death" due to it causing blue rashes on the body of an infected person. Currently, there are around 3-5 million people experiencing cholera worldwide.**

.
 * To become infected, the average ingestion of the bacteria must be at around 100 million. Of course this number is lowered in people with weak immune systems and certain diseases. Some of the most infected parts of the world include India, Haiti, certain parts of Africa, and a few others.**


 * Essentiality of this protein: essential**
 * Complex of proteins?: works alone with current information available**
 * Druggable Target: yes**


 * *EC#: 3.1.3.3 **
 * Link to BRENDA EC# page:** http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.3


 * --** Show screenshot of BRENDA enzyme mechanism schematic

http://www.sigmaaldrich.com/catalog/search?interface=All&term=phosphoserine&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286 []
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link (or citation) to paper that contains assay information**

http://www.rcsb.org/pdb/explore/explore.do?structureId=3N28
 * -- List cost and quantity of substrate reagents and supplier**
 * phosphoserine 63.50**
 * Structure Available (PDB or Homology model)**

[] MSLDALTTLPIKKHTALLNRFPETRFVTQLAKKRASWIVFGHYLTPAQFEDMDFFTNRFNAILDMWKVGRYEVALMDGEL TSEHETILKALELDYARIQDVPDLTKPGLIVLDMDSTAIQIECIDEIAKLAGVGEEVAEVTERAMQGELDFEQSLRLRVS KLKDAPEQILSQVRETLPLMPELPELVATLHAFGWKVAIASGGFTYFSDYLKEQLSLDYAQSNTLEIVSGKLTGQVLGEV VSAQTKADILLTLAQQYDVEIHNTVAVGDGANDLVMMAAAGLGVAYHAKPKVEAKAQTAVRFAGLGGVVCILSAALVAQQ KLSWKSKEGHHHHHH
 * Current Inhibitors: none found**
 * Expression Information (has it been expressed in bacterial cells): under Brenda, this enzyme has been expressed in //porphyromonas gingivalis//**
 * Purification Method:**
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

__ Julia C. __ Dr. B said that we should use the NCBI sequence instead of the PDB one. Here's the 'correct' filtered through aa sequence: mdalttlpikkhtallnrfpetrfvtqlakkraswivfghyltpaqfedmdfftnrfnai ldmwkvgryevalmdgeltsehetilkaleldyariqdvpdltkpglivldmdstaiqie cideiaklagvgeevaevteramqgeldfeqslrlrvsklkdapeqilsqvretlplmpe lpelvatlhafgwkvaiasggftyfsdylkeqlsldyaqsntleivsgkltgqvlgevvs aqtkadilltlaqqydveihntvavgdgandlvmmaaaglgvayhakpkveakaqtavrf aglggvvcilsaalvaqqklswkskp

atgagcctggatgcgctgaccaccctgccgattaaaaaacataccgcgctgctgaaccgc tttccggaaacccgctttgtgacccagctggcgaaaaaacgcgcgagctggattgtgttt ggccattatctgaccccggcgcagtttgaagatatggatttttttaccaaccgctttaac gcgattctggatatgtggaaagtgggccgctatgaagtggcgctgatggatggcgaactg accagcgaacatgaaaccattctgaaagcgctggaactggattatgcgcgcattcaggat gtgccggatctgaccaaaccgggcctgattgtgctggatatggatagcaccgcgattcag attgaatgcattgatgaaattgcgaaactggcgggcgtgggcgaagaagtggcggaagtg accgaacgcgcgatgcagggcgaactggattttgaacagagcctgcgcctgcgcgtgagc aaactgaaagatgcgccggaacagattctgagccaggtgcgcgaaaccctgccgctgatg ccggaactgccggaactggtggcgaccctgcatgcgtttggctggaaagtggcgattgcg agcggcggctttacctattttagcgattatctgaaagaacagctgagcctggattatgcg cagagcaacaccctggaaattgtgagcggcaaactgaccggccaggtgctgggcgaagtg gtgagcgcgcagaccaaagcggatattctgctgaccctggcgcagcagtatgatgtggaa attcataacaccgtggcggtgggcgatggcgcgaacgatctggtgatgatggcggcggcg ggcctgggcgtggcgtatcatgcgaaaccgaaagtggaagcgaaagcgcagaccgcggtg cgctttgcgggcctgggcggcgtggtgtgcattctgagcgcggcgctggtggcgcagcag aaactgagctggaaaagcaaagaaggccatcatcatcatcatcat
 * length of your protein in Amino Acids: 335 (** Julia C.: 326 **)**
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website:** 36981.5 Da
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 34045
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * CDS Gene Sequence (paste as text only):**

__Julia C.__ The gene sequence I found: 1 ATGGACGCGCTGACCACGCTGCCGATCAAAAAACACACCGCGCTCCTGAACCGTTTCCCG 61 GAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCTGGATCGTTTTCGGTCAC 121 TATCTGACCCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATC 181 CTCGATATGTGGAAAGTGGGTCGTTATGAGGTTGCGCTCATGGACGGTGAACTCACCTCT 241 GAACACGAAACCATTCTGAAGGCGCTGGAACTCGATTACGCACGTATCCAGGACGTTCCG 301 GACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCAGATCGAA 361 TGCATCGACGAAATCGCGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAA 421 CGTGCTATGCAGGGCGAACTGGATTTCGAACAGTCTCTGCGTCTCCGTGTTTCTAAACTG 481 AAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAAACCCTGCCGCTGATGCCGGAA 541 CTGCCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGT 601 GGTTTTACCTACTTCAGCGACTACCTGAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCT 661 AACACCCTCGAAATTGTTTCTGGTAAACTCACTGGTCAGGTTCTCGGTGAAGTTGTCTCC 721 GCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATGACGTTGAAATTCAC 781 AACACCGTTGCGGTTGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTG 841 GGTGTGGCCTACCACGCGAAACCGAAAGTCGAAGCAAAGGCGCAAACGGCGGTTCGTTTT 901 GCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCTGGTGGCGCAGCAAAAACTG 961 TCTTGGAAATCTAAACCGTAA

Dax: This is the gene sequence I found, and what was used for primer tail design and ordering. It has a 100% query coverage and 88% match to Julia's sequence, but when put in an amino acid sequence, the two match at 100% for both query coverage and identity.

ATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGCTGCTGAACCGTTTC CCGGAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC TCTTGGAAATCTAAACCGTAA

Oligo sequence:

1 ATGGACGCGCTGACCAC 17

2 CCGGGAAACGGTTCAGGAGCGCGGTGTGTTTTTTGATCGGCAGCGTGGTCAGCGCGTCCA 60

3 TCCTGAACCGTTTCCCGGAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCT 60

4 TCTTCAAACTGTGCCGGGGTCAGATAGTGACCGAAAACGATCCAGCTCGCGCGTTTCTTC 60

5 CCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATCCTCGATAT 60

6 CGTCCATGAGCGCAACCTCATAACGACCCACTTTCCACATATCGAGGATGGCATTAAAGC 60

7 GGTTGCGCTCATGGACGGTGAACTCACCTCTGAACACGAAACCATTCTGAAGGCGCTGGA 60

8 TTGGTGAGGTCCGGAACGTCCTGGATACGTGCGTAATCGAGTTCCAGCGCCTTCAGAATG 60

9 CGTTCCGGACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCA 60

10 ACACCCGCCAGCTTCGCGATTTCGTCGATGCATTCGATCTGAATCGCGGTAGAATCCATG 60

11 CGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAACGTGCTATGCAGGGCG 60

12 CAGTTTAGAAACACGGAGACGCAGAGACTGTTCGAAATCCAGTTCGCCCTGCATAGCACG 60

13 CGTCTCCGTGTTTCTAAACTGAAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAA 60

14 GTCGCAACGAGCTCTGGCAGTTCCGGCATCAGCGGCAGGGTTTCACGAACTTGGCTCAGG 60

15 CCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGTGGT 60

16 CCAGAGAGAGCTGCTCTTTCAGGTAGTCGCTGAAGTAGGTAAAACCACCGGAGGCGATTG 60

17 GAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCTAACACCCTCGAAATTGTTTCTGGTAA 60

18 GCGCGGAGACAACTTCACCGAGAACCTGACCAGTGAGTTTACCAGAAACAATTTCGAGGG 60

19 GTGAAGTTGTCTCCGCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATG 60

20 TTCGCGCCATCGCCAACCGCAACGGTGTTGTGAATTTCAACGTCATACTGCTGGGCGAGC 60

21 TGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTGGGTGTGGCCTACCA 60

22 ACGAACCGCCGTTTGCGCCTTTGCTTCGACTTTCGGTTTCGCGTGGTAGGCCACACCCAG 60

23 GCAAACGGCGGTTCGTTTTGCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCT 60

24 TTACGGTTTAGATTTCCAAGACAGTTTTTGCTGCGCCACCAGCGCGGCAGACAGAAT 57


 * GC% Content for gene: 56.3%**
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**
 * GC% Content for gene (codon optimized):**

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): **
 * ( link to DNA Works output text file - ** that should be saved in your Google Docs folder after you did the primer design protocol)

__ Upstream: __ TACTTCCAATCCATGGACGCGCTGACCA __ Downstream (reverse complemented): __ TATCCACCTTTACTGTTACGGTTTAGATTTCCAAGAGAG *Note that this insert contains code that codes for a cut by BsaI.
 * Primer design results for 'tail' primers (this is just 2 sequences): **

We will be ordering from Shelby-Dax Garibay.

Resources:
See ** ProtocolTargetDiscoveryVDS.docx ** for more Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334

@http://www.niaid.nih.gov/Pages/default.aspx @http://eupathdb.org/eupathdb/ @https://patricbrc.vbi.vt.edu/portal/portal/patric/Home @http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes @http://tubic.tju.edu.cn/deg/ @http://csgid.org/csgid/cake/pages/community_request_gateway @http://tdrtargets.org/ @http://gsc.jcvi.org/status.shtml Possible inhibitors: [] Comparison of phosphoserine and amino acid binding in another bacteria: http://www.rcsb.org/pdb/explore/explore.do?structureId=1L7P Comparison of protein to other bacteria with cation in active site: []
 * Databases of genes/organisms: **

@http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
 * Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations) **

NCBI GENE Page: @http://www.ncbi.nlm.nih.gov/gene BLAST Page: @http://blast.ncbi.nlm.nih.gov/
 * Gene Information: **

NCBI Protein Page: @http://www.ncbi.nlm.nih.gov/protein Protein Expression Website Protein Expression Paper:
 * Protein Information: **


 * Primer Overlap PCR Articles **


 * Is my target good for Virtual Screening programs? **