Sajan+S.

worked on enzyme assays and inhibition assays
 * Week 14: **

This week I worked on FPLC and we concentrated the protein. We also worked on an enzyme assay on J.C. enzyme. I redid the homology model and I will start the virtual screening. Protein concentrated after FPLC to 1mL
 * Week 13: **
 * FTHAP FPLC- Tubes 30-38 are contaminated**

nanodrop results before concentrating

Lane 1: n/a Lane 2: ladder Lane 3: sample 1 Lane 4: n/a Lane 5: sample 3 Lane 6: sample 4 Lane 7: n/a Lane 8: elution 1 Lane 9: elution 2 Lane 10: n/a

112612 - for Week 13 - do you have some virtual results. Dr B


 * Week 12: **

** this week I worked on characterization and purification protocol. **
Ok - Dr. B 11/19/12

I Continued working on Homology Model and starting the virtual Screening. Finished Protein Expression of FTHAP worked on Material and Methods for cloning
 * Week 11: **

This week I worked on making PNIC BSA-4 for cloning. The concentration however was not large enough. I then started to work on the surrogate protein expression FTHAP on thursday and friday. Nanodrop for PNIC BSA4 for cloning
 * Week 10: **

This week I worked on running the gel for my PCR and also redoing PCR since the band is low. I also worked on the research paper Lane 1- skip Lane 2- 1 Kb ladder Lane 3- PCR 1 Lane 4- Secondary PCR with annealing 55 degrees Lane 5- Secondary PCR with annealing 57 degrees 102112 - Sajan, show some results here. - Dr. B
 * Week 9: **
 * Week 8: **


 * This week I worked on virtual screening refresher. I also worked on PCR secondary.**
 * Week 7: **

=
This week I worked on making the primer dilution which tom ordered. I made a oligo mix which was used to do primary and secondary PCR for my target. I ran a gel for the primary and secondary PCR. I also did PCR Squared for my target. All of the gels looked good and I will proceed to PCR cleanup. I also worked on starting my virtual screening of my target by doing a homology model and then proceeding to screen from that, ======

Lane 1: skip
Lane 2: 100 bp Ladder Lane 3: PCR 1 Lane 4: PCR 2

Lane 1: skip Lane 2: 100 bp ladder Lane 3: PCR squared 1 Lane 4: PCR Squared 2 Lane 5 :PCR squared 3 Lane 6: PCR squared 4

100912 - Sajan, ok good. Show your gels for primary and secondary PCR. - DR. B This week I worked on running the gel from the primary and secondary PCR. The gels unfortunately failed. I also worked on doing my primer design. I started the PCR primer overlap. I worked on the primer dilution and PCR one and secondary PCR for the Primer Overlap.
 * Week 6: **

Figure 1: agarose gel of pGBR22 with M13 primer.

Lane 2:100bp DNA ladder

Lane 7:1:1000 dilution

Lane 8:1:1000 dilution

Lane 9:1:100 dilution

Lane 10: no DNA control

= Week 5: = 093012 - sajan, missing Tail Primer Design. Good analysis on your gel. Should include results of your PCR's. Also, use the standard format for labeling your gel with each lane on a separate row. - Dr. B This week I worked on running the gel for restriction enzyme digest from the week 3 which I had not done. I also did PCR and PCR two this week. I have also finished doing Pymol Refresher this week. This is the gel run from the re digest. The Ladder is located on Lane two and the uncut plasmid is in lane three. Lane four through six contains the plasmid cut with ECO

RI, PVU II and both ECO RI and PVU II. The plasmid however could not be seen. This could be due to the length between the preparation and running the gel. The DNA

could have degraded while in the -20 freezer or there could be loss of the plasmid through not properly capping the tubes. = Week 4: = sajan - looks good. Dr. B This week I worked on Midi prep. I also did a nanodrop to find the concentration of sample to be 53.8 ng/uL.

the sample was also sent to DNA sequencing to determine the sequence so that we can proceed to cloning. Here is the forward sequencing

code NNNNNNNNNNNNNCTTTAGNNGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAG AACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATG ATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAA ACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAG ACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGC AAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGT AACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGA TGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCG TTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAAT CCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAG GCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTT GCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGA CAGCTGGAAAACGCTGGCNGCGTCTTTNAAGACAGCGACAAATTCNATGCAAATGATNCTATCCTAAAAGACCNAACNCA NAATGGNCNNNNANCNNNTTTACNTCTGANGAAAATCCGTTATNCTANNNTGATTNNCNNANNNTNNGNANCAANNCTGN NANTGNNNNANTANNNNNCANCATNNNNNNNNNTGNANNTNNNCNGNNNANNNNNNNNAATNNNNNTNACGNNNANNNNN NNNNNNNNANNNNNNNNNNNNNNNNNNANTCNNNNNNNNGGNNNNNCNNANNNNNNNNNNNNNNNNNNNNNN

code

It was determine that it was the pNIC vector through a nucleotide blast. = Week 3: = Sajan - can you include an image of your RE digest with analysis? -- DR. B 091812

This week I worked on the RE digest along with doing transformation that will be later used for the midi prep next week.

= Week 2: = This week I did primer dilution and the DNA sequencing. I did a forward primer dilution which was then used with a reverse primer for the submission to the DNA sequencing facility for pNIC-Bsa4 vector. On Friday I did a DNA sequence analysis protocol where we used the purple protein and did a protein blast. We also looked at restriction enzyme cuts on the computer. = = = = = = = Week 1: =

This week I worked on finding a target for this semester. Here is my info from the target page.


 * *Target (protein/gene name): **phospholipase C


 * *NCBI Gene # or RefSeq#: **NCTC8237

GenBank: CAA31943.1

[]

>gi|194293816|gb|EU839779.1| Clostridium perfringens strain S01 phospholipase C (plc) gene, complete cds


 * *Organism: Clostridium Perfrigens **


 * *Background/Disease Information: **//C. perfringens // is a bacteria that leads to necrosis, bacteremia and gas gangrene. It is the most common bacterial reagent leading to gas gangrene. It is the third most common cause of food poisoning in the United Kingdom and the United States.

**Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens. **

[]


 * Complex of enzymes: **


 * *EC#: 3.1.4.3 **


 * Link to BRENDA EC# page: **[]


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




 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **

[]


 * Structure Available (PDB or Homology model) **


 * -- PDB # or closest PDB entry if using homology model: 2WXT **


 * Inhibitors **


 * || Inhibitors || Commentary || Organism || Structure ||
 * EDTA || enzyme activity against 1-myristoyl-lysophosphatidylcholine, glycerophosphorylcholine, and p-nitrophenyl phosphorylcholine are readily inhibited by EDTA. Indicates that a divalent cation is essential for catalytic activity || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">Homo sapiens || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">[[image:http://vdsstream.wikispaces.com/site/embedthumbnail/placeholder?w=200&h=51 width="200" height="51" caption="Description: http://brenda-enzymes.org/images/structure.gif"]] ||  ||
 * <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">EGTA || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">enzyme activity against 1-myristoyl-lysophosphatidylcholine, glycerophosphorylcholine, and p-nitrophenyl phosphorylcholine are readily inhibited by EGTA. Indicates that a divalent cation is essential for catalytic activity || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">Homo sapiens || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">[[image:http://vdsstream.wikispaces.com/site/embedthumbnail/placeholder?w=200&h=51 width="200" height="51" caption="Description: http://brenda-enzymes.org/images/structure.gif"]] ||  ||
 * <span style="color: #669933; font-family: 'Times New Roman',serif; font-size: 16.5pt;">additional information || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">the catalytic activity of NPP6 toward p-nitrophenyl phosphorylcholine is partially inhibited by the divalent cations. Attempts are unsuccessful to recover the activity of EDTA treated NPP6 by adding various divalent cations || <span style="font-family: 'Times New Roman',serif; font-size: 16.5pt;">Homo sapiens || <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16.5pt; text-align: center;">- ||  ||


 * Expression Information (has it been expressed in bacterial cells): **<span style="font-family: Arial,sans-serif; font-size: 13.5pt;">homo sapiens, E. Coli and mus musculus. Yes it was expressed in E. Coli


 * Purification Method: **

<span style="font-family: Arial,sans-serif; font-size: 13.5pt;">mono Q ion exchange chromatography column and eluted with a linear gradient of NaCl (0-2 M) using the deltaKTA system in homo sapiens and mus musculus




 * *Amino Acid Sequence: ****<span style="font-family: Arial,sans-serif; font-size: 13.5pt;">* **

1 MSRLVVVSNRIAPPDEHAASAGGLAVGILGALKAAGGLWFGWSGETGNEDQPLKKVKKGN

61 ITWASFNLSEQDLDEYYNQFSNAVLWPAFHYRLDLVQFQRPAWDGYLRVNALLADKLLPL

121 LQDDDIIWIHDYHLLPFAHELRKRGVNNRIGFFLHIPFPTPEIFNALPTYDTLLEQLCDY

181 DLLGFQTENDRLAFLDCLSNLTRVTTRSAKSHTAWGKAFRTEVYPIGIEPKEIAKQAAGP

241 LPPKLAQLKAELKNVQNIFSVERLDYSKGLPERFLAYEALLEKYPQHHGKIRYTQIAPTS

301 RGDVQAYQDIRHQLENEAGRINGKYGQLGWTPLYYLNQHFDRKLLMKIFRYSDVGLVTPL

361 RDGMNLVAKEYVAAQDPANPGVLVLSQFAGAANELTSALIVNPYDRDEVAAALDRALTMS

421 LAERISRHAEMLDVIVKNDINHWQECFISDLKQIVPRSAESQQRDKVATFPKLA


 * Primer design **

1 ATGTCTCGTCTCGTTGTTGTTTCTAATCGTATCGCGCCTCCAGACGAACACGCGGCGTC 59

2 GCCGCCTTGAGGGCACCGAGGATACCCACCGCGAGGCCACCAGCAGACGCCGCGTGTTCG 60

3 TGCCCTCAAGGCGGCAGGCGGTCTGTGGTTCGGTTGGTCCGGTGAGACCGGTAACGAGGA 60

4 CCCAGGTGATGTTACCTTTCTTAACTTTTTTGAGTGGCTGATCCTCGTTACCGGTCTCAC 60

5 AGAAAGGTAACATCACCTGGGCGTCTTTCAACCTGTCTGAACAAGACCTGGACGAATACT 60

6 GGAACGCAGGCCAGAGAACGGCGTTAGAGAACTGGTTGTAGTATTCGTCCAGGTCTTGTT 60

7 TCTCTGGCCTGCGTTCCATTACCGTCTCGACCTGGTGCAATTTCAGCGTCCAGCGTGGGA 60

8 GGGAGGAGTTTATCCGCCAGCAGCGCGTTAACACGCAGATAGCCGTCCCACGCTGGACGC 60

9 TGGCGGATAAACTCCTCCCGCTCCTCCAGGACGACGATATCATCTGGATTCACGACTACC 60

10 CGCCACGCTTACGCAGCTCGTGCGCGAACGGCAGGAGGTGGTAGTCGTGAATCCAGATGA 60

11 CTGCGTAAGCGTGGCGTAAATAACCGTATCGGTTTCTTCCTGCACATCCCGTTCCCAACC 60

12 CAGCAGGGTGTCGTAGGTCGGGAGTGCGTTAAAGATTTCCGGGGTTGGGAACGGGATGTG 60

13 ACCTACGACACCCTGCTGGAACAGCTCTGTGACTACGACCTGCTCGGCTTCCAGACCGAG 60

14 GGGTCAGATTAGAGAGGCAGTCCAGAAACGCCAGACGGTCATTCTCGGTCTGGAAGCCGA 60

15 CTGCCTCTCTAATCTGACCCGTGTTACCACTCGTAGCGCGAAATCTCATACTGCGTGGGG 60

16 TTCGATACCGATAGGGTAAACTTCGGTGCGGAACGCTTTACCCCACGCAGTATGAGATTT 60

17 AAGTTTACCCTATCGGTATCGAACCGAAAGAAATCGCGAAACAAGCAGCCGGTCCGCTGC 60

18 TTCTGAACGTTTTTCAGTTCCGCTTTGAGCTGGGCCAGCTTTGGTGGCAGCGGACCGGCT 60

19 CGGAACTGAAAAACGTTCAGAACATCTTCTCTGTTGAACGCCTGGACTACTCTAAAGGTC 60

20 TTTCCAGGAGCGCTTCGTACGCCAGGAAACGCTCCGGCAGACCTTTAGAGTAGTCCAGGC 60

21 ACGAAGCGCTCCTGGAAAAATACCCGCAACATCATGGTAAAATTCGTTACACCCAGATTG 60

22 CTTGGTACGCCTGCACGTCGCCACGAGAGGTCGGCGCAATCTGGGTGTAACGAATTTTAC 60

23 CGTGCAGGCGTACCAAGACATCCGTCACCAACTGGAGAACGAAGCAGGCCGCATCAACGG 60

24 GGTTCAGGTAGTACAGCGGAGTCCAACCCAGCTGACCGTACTTACCGTTGATGCGGCCTG 60

25 CCGCTGTACTACCTGAACCAACACTTCGACCGTAAACTGCTGATGAAAATCTTCCGTTAC 60

26 TACCGTCGCGCAGTGGGGTCACCAGACCAACGTCAGAGTAACGGAAGATTTTCATCAGCA 60