Divya+G.

__**Week 13:**__ This week I did concentration after FPLC

Results of the protein characterization are shown below (from a long time ago) ​

Lane 1: n/a Lane 2: ladder (smo671) 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

__**Week 12:**__

This Week I worked on FPLC and concentrated my protein here are the results form FPLC



__**Week 11:**__ Divya - show the alignment as a screen shot to preserve the formatting. An alignement doesn't mean much when it is out of alignment. Dr. B 11/19/12

I worked on my homology model and prepared it for virtual screening. Additionally I characterized FTHAP and concentrated the protein to be used for FPLC

The results from the Homology model are shown below:

Score E Sequences producing significant alignments: (bits) Value

ExPDB|1auiA|2.1| HUMAN CALCINEURIN HETERODIMER 336 2e-92 ExPDB|1m63A|2.8| Crystal structure of calcineurin cyclophilin cy... 335 2e-92 ExPDB|1tcoA|2.5| TERNARY COMPLEX OF A CALCINEURIN A FRAGMENT CA... 335 4e-92 ExPDB|1wao1|2.9| PP5 STRUCTURE 193 2e-49 ExPDB|1s95B|1.6| Structure of serine threonine protein phosphata... 191 8e-49 ExPDB|3v4yG|2.098| Crystal Structure of the first Nuclear PP1 ho... 188 6e-48 ExPDB|1fjmB|2.1| Protein serine threonine phosphatase 1 alpha i... 187 8e-48 ExPDB|3p71C|2.7| Crystal structure of the complex of LCMT 1 and ... 187 1e-47 ExPDB|3v4yC|2.098| Crystal Structure of the first Nuclear PP1 ho... 187 1e-47 ExPDB|3c5wC|2.8| Complex between PP2A specific methylesterase PM... 186 2e-47 ExPDB|2o8gA|2.5| Rat pp1c gamma complexed with mouse inhibitor 2 186 3e-47 ExPDB|2nppF|3.3| Structure of the Protein Phosphatase 2A Holoenzyme 185 4e-47 ExPDB|1u32A|2| Crystal structure of a Protein Phosphatase 1 Cal... 184 1e-46 ExPDB|3fgaC|2.7| Structural Basis of PP2A and Sgo interaction 184 1e-46 ExPDB|2iaeC|3.5| Crystal structure of a protein phosphatase 2A ... 183 2e-46 ExPDB|1s70A|2.7| Complex between protein ser thr phosphatase 1 ... 180 1e-45 ExPDB|3icfA|2.3| Structure of Protein serine threonine phosphata... 159 3e-39

>[Template]|1auiA|2.1| HUMAN CALCINEURIN HETERODIMER Length = 378 [|[Display Alignment in DeepView]] Score = 336 bits (861), Expect = 2e-92, Method: Composition-based stats. Identities = 165/372 (44%), Positives = 234/372 (62%), Gaps = 26/372 (6%) Query: 17 PPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTEPNVLRI 76 PP AK + D GK ++++ H +++GRL + AL II +S+LR E N+L ISbjct: 11 PPSHRLTAKE--VFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILRQEKNLLDI 68 Query: 77 ADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKT-YLFLGNYIDSCFFSTECILLLLAAKL 135 D V V GD+ GQF+DL K+ +G S A T YLFLG+Y+D +FS EC+L L A K+Sbjct: 69 -DAPVTVCGDIHGQFFDLMKLFEVGG--SPANTRYLFLGDYVDRGYFSIECVLYLWALKI 125 Query: 136 THPSCVFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCV 195 +P +FLLRGNHECR ++ F F+ EC KY++ VY+A M AFDCLPLAA++N Q+ CVSbjct: 126 LYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCV 185 Query: 196 HGGLSPDVTSVDNIRLIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGN 255 HGGLSP++ ++D+IR + RF+EPP+ G MCD+LWSDP D N Sbjct: 186 HGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEK--- 230 Query: 256 GPSYGTAPCFLDNEQRGCSYLFNYHSVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSN 315 T F N RGCSY ++Y +V FL N LL ++R+HE QD GY++YR + + Sbjct: 231 -TQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTG 285 Query: 316 FPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQFCCSPHPYVLPKHLNAFEWSFSYLL 375 FP ++++FSAPNY D +NK AVL E + I+QF CSPHPY LP ++ F WS ++ Sbjct: 286 FPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVG 345 Query: 376 DSVRDIFASIIS 387 + V ++ ++++Sbjct: 346 EKVTEMLVNVLN 357

__Better alignment:__ code Query: 17 PPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTEPNVLRI 76 PP    AK   + D  GK  ++++  H +++GRL +  AL II   +S+LR E N+L I Sbjct: 11  PPSHRLTAKE--VFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILRQEKNLLDI 68

Query: 77 ADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKT-YLFLGNYIDSCFFSTECILLLLAAKL 135 D V V GD+ GQF+DL K+  +G   S A T YLFLG+Y+D  +FS EC+L L A K+ Sbjct: 69  -DAPVTVCGDIHGQFFDLMKLFEVGG--SPANTRYLFLGDYVDRGYFSIECVLYLWALKI 125

Query: 136 THPSCVFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCV 195 +P +FLLRGNHECR ++  F F+ EC  KY++ VY+A M AFDCLPLAA++N Q+ CV Sbjct: 126 LYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCV 185

Query: 196 HGGLSPDVTSVDNIRLIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGN 255 HGGLSP++ ++D+IR + RF+EPP+ G MCD+LWSDP D  N Sbjct: 186 HGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEK--- 230

Query: 256 GPSYGTAPCFLDNEQRGCSYLFNYHSVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSN 315 T  F  N  RGCSY ++Y +V  FL  N LL ++R+HE QD GY++YR +  + Sbjct: 231 -TQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTG 285

Query: 316 FPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQFCCSPHPYVLPKHLNAFEWSFSYLL 375 FP ++++FSAPNY D +NK AVL  E   + I+QF CSPHPY LP  ++ F WS  ++ Sbjct: 286 FPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVG 345

Query: 376 DSVRDIFASIIS 387 + V ++ ++++ Sbjct: 346 EKVTEMLVNVLN 357 code

__**Week 10:**__ Since the PCR failed yet again, A surrogate protein was used form now on. FTHAP was experssed and purified this week. Next week, FPLC will be run. I also started my homology model.

__**Week 9:**__ This week I redid my primary and secondary PCR at both 56 and 57 degrees. Results to be posted soon.

__**Week 8:**__ 102112 - Divya, I would also do another primary at 57 degrees - then do a couple of different secondary's (from your original primary and from this new primary). -- DR. B I Gel checked my primary and secondary PCR and got the following results

Lane 1: Skip Lane 2: 100 bp ladder Lane 3: Primary PCR Land 4:Secondary PCR

Results: The secondary PCR did not show on the gel, so I think I need to change the annealing temperature. I will have 2 samples one will be 56 degrees with from the old and new PCR and I will do the same for 57 degrees.

__**Week 7:**__ 101612 - Divya, show some of your results/gels. -- Dr. B

This week I diluted my primers, made an oligomix, and ran my primary PCR. I will gel check it with secondary PCR. The results from both primary and secondary PCR will be posted next week.

__**Week 6:**__ 100912 - Divya - ok, not a great gel - but move on to cloning PCRs so taht you can catch up with the others. - Dr. B

This week I did my second PCR practice this week and next week I will dilute my primers and begin PCR^2 on my target (Finally). I need to make an oligomix as well. The results of my second PCR are shown below. there is a small crack at the bottom but it does not affect the results obtained.




 * 1) Skip
 * 2) DNA Ladder 100kp
 * 3) Sample A Lowest concentration
 * 4) Sample B Medium concentration
 * 5) Sample C Highest concentration
 * 6) Sample D control No DNA

__**Week 5:**__ 093012- Divya. Good but show some results here. Dr. B This week I did a PyMol refresher and I completed the primer design for pNIC-Bsa4 cloning. The results were posted to google docs. I had a lot of tests this week so I could not get too much done. Next week I will do PCR2

Results from the Pymol Refresher are shown here:

__**Week 4:**__

Divya - in midi-prep you are purifying plasmid DNA instead of protein. Also, your PCR is ~ok. I don't think you have contamination but rather some smearing of the DNA. The sharp bands are actually about the right size (the GBR22 gene is about 1,000 bp). So - good job. You can move on to PCR#2. Also, eventually you might need to re-do the Midiprep if you need more for your cloning. -- Dr. B - good labeling of your marker too next to the PCR gel. Could specify exactly what it is you are amplifying in the PCR caption.

This week I did midi-prep and I did my first PCR practice. The protein concentration from midi-prep yielded low protein concentration, which was most likely due to the fact that too much elution buffer was added initially.






 * 1) Skip
 * 2) Ladder 100bp
 * 3) Sample A
 * 4) Sample **B**
 * 5) Sample C
 * 6) Sample D (No DNA)

__**Week 3:**__ Divya - ok - good image. Include brief analysis of image. Are the bands correct? Any anomalies? Also, you get some back credit for last week. -- Dr. B 091812 This week I Did transformation of E-coli and I completed the RE Digest. I was not successful in transforming my bacteria so I borrowed bacteria form another person in lab (initials SB). I then I grew the bacteria in LB Broth and spun down my sample to obtain a pellet, which I then froze in -20 C fridge. In the RE Digest I ran a gel electrophoresis which is pictured below: The 100bp ladder is shown on the left. gel Results for pGBR22 DNA (above) (Fixed the lane numbers) Lane 1; Skip Lane 2; 100 bp NEB DNA Ladder Lane 3; Plasmid Lane 4; 25ul control Lane 5; 25ul EcoRI Lane 6; 25ul PvuII Lane 7; 25ul EcoRI and PvuII

__**Week 2:**__ I diluted Primer for pNIC-Bsa4 and prepared it for sequencing. I also analyzed the DNA sequence for PGBR-22.

__**Week 1:**__ I created my target page as shown below

Disease: Trypansoma brucei Enzyme: [|Protein Phosphatase 2B]

Essentiality: Essential, used for [|cytokinesis]

Not complex EC#: 3.1.3.16 Drugability: 0.8 (yes) Assay: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16 crystal structure:

[|Amino Acid sequence:]

MLPILKAGEDEGGPRIPPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTE PNVLRIADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKTYLFLGNYIDSCFFSTECILLLLAAKLTHPSC VFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCVHGGLSPDVTSVDNIR LIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGNGPSYGTAPCFLDNEQRGCSYLFNYH SVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSNFPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQ FCCSPHPYVLPKHLNAFEWSFSYLLDSVRDIFASIISCEGA

[|Gene Sequence:] ATGCTGCCTATACTAAAGGCCGGGGAAGACGAGGGGGGTCCCCGGATCCCCCCTCCTCTATGGTGTGAGG CGAAGCGTGAGGCGTTAATCGATGGCAAAGGAAAAGTTAATCTTGAGGTAATGCTTGTTCATTTTCTTCG GCAAGGACGTTTGAGCAAGCACGATGCACTCAACATAATTCAAAATGCATCAAGTGTACTTCGTACGGAA CCCAACGTCCTGAGGATTGCTGATCCTTCGGTTGTAGTCGTCGGCGACGTTCGGGGACAGTTCTACGATT TAGCAAAAATTATATTTATAGGTAATATGTTCAGTAGGGCAAAAACGTATTTGTTTCTAGGTAATTATAT TGACTCATGTTTTTTTAGCACCGAGTGTATACTCCTTTTGCTTGCGGCGAAACTAACCCACCCGAGTTGT GTATTTTTGCTTCGTGGAAACCATGAATGCCGTTTTATGTCGAATATTTTCGATTTTCGGGGAGAGTGTC TGAAGAAATATAATGACGATGTTTACGAAGCCATTATGACAGCGTTTGATTGTCTTCCACTGGCAGCTGT TGTAAACAATCAGTATTTTTGTGTACATGGAGGTCTTTCACCTGACGTTACGAGTGTTGACAATATTCGC TTAATTTATCGATTTCGGGAACCCCCGTCGAAAGGGGCTATGTGTGATTTATTATGGTCTGATCCGTTTT GGGATGTAGAGAATCCGTCGTCAGTTTGCGAAGGTTCTAGAGATGATTATTACACCCCTGGAAATGGCCC ATCGTACGGTACGGCGCCATGCTTTCTTGACAACGAGCAGAGAGGTTGCAGTTATTTGTTTAACTATCAT AGTGTAAAGCACTTTTTGTTGACGAATGGTCTTCTTTGCGTCGTACGTAGCCATGAGGTTCAAGATGATG GTTATAAGTTGTATAGGTTCAACAGTGTTTCCAATTTTCCGTGCATGATGTCCGTTTTTTCTGCCCCTAA TTACTGTGATAACTTGCACAACAAGGGTGCAGTCCTCATTCTTGAGGGGAAACAAATAGGTATAAAGCAG TTTTGTTGCTCGCCTCATCCTTATGTTCTCCCAAAGCATCTTAACGCATTTGAATGGTCATTCTCTTACC TGTTGGATAGTGTTCGCGACATATTTGCTTCCATCATTTCGTGTGAGGGAGCATGA