Kevin+E.

Please make distinction for weeks more clear. Also include data/pics from virtual -UM 12/6/13 __Enzyme Assay and Inhibition Assay__

Inhibition Assay Absorbance Data:

Fig 79. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library

Fig 78. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library (run 1)

Enzyme Assay Data:

Fig 77. Enzyme Assay results graph of enzyme concentrations vs. absorbance measured by the spectrophotometer.

12/5/13 Fig 76. Characterization poly-acrylamide page gel of YopH. Lane 1: DNA ladder, Lane 2: Cell lysate before induction, Lane 3: cell lysate after induction, Lane 4: soluble fraction, Lane 5: flow through, Lane 6: wash, Lane 7: Elution 1, Lane 8: Elution 2

FIG 75. Elution 2 of YopH after purification. Fig 74. Elution 1 of YopH after purification. 11/20/13

Expression of YopH

Fig 73. YopH, LB, and Kan solution after expression resuspended pellet in lysis buffer.

weight of pellet: 1.47g

Measuring OD values - 23uL of total culture Time Abs at 600nm 9:53 .086 10:29 .075 11:00 .099 12:09 .28 12:34 .36 12:46 .385 1:07 .47 1:12 .47 1:20 .52

Ligand Prep protocol was completed on 11/14/13, in order to make the ligands ready to be screened. On 11/1/5/13, virtual screening of our target was begun and is currently being run through GOLD.

11/15/13

Fig 72. DNA sequencing results from clone 2 of PP2A in T. brucei. Obviously, no cloning whatsoever happened. The vector may have simply closed up. No results were close enough for any match for comparison because the sequence was simply: NNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNGGTGGTGGTGGNGNNNCTCTANGNNNNNNNNTGNNGANNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNGNNNNNNGAANNNNNNNNNNNNNNNNNNNNANACNNNTTNTNNNNNANNNGT Fig 71. DNA sequencing results from clone 1 of PP2A in T. brucei. As evident, only the ends of the gene were cloned with nothing in the center. I have to talk to Dr. B about this as to why still. Fig 70. Master plate of single colony picked from the original transformation of 2ul pNIC-bsa4 and 4ul PP2A.

Fig 69. Regrown transformation plate of 2ul vector, pNIC-bsa4, and 4ul insert, PP2A, resulting in the same as below of streaking with no true colonies.

Fig 68. Regrown transformation plate of 3ul vector, pNIC-bsa4, and 6ul insert, PP2A. This was regrown by streaking a pipette tip from the overgrown plate onto this plate. Obviously, it continued to grow just on the areas streaked, resulting in no solo colonies.

Fig 67. Miniprep concentration of trial 2 of pNIC-bsa4 and PP2A. Trial 2 is from the same colony as trial 1, however, it is just a regrown colony since trial 1 was left in the shaking incubator for an extra day.

Fig 66. Miniprep concentration of trial 1 of pNIC-bsa4 and PP2A. I believe the reason why the concentration was so high is due to the fact that it was left in the shaking incubator for an extra day. However, the nanodrop reading looks to be normal, just highly concentrated.

Fig 65. PCR cleanup concentration of sample 4 of pNIC-bsa4 and PP2A

Fig 64. PCR cleanup concentration of sample 3 of pNIC-bsa4 and PP2A

Fig 63. PCR clean up concentration of sample 2 of pNIC-bsa4 and PP2A

Fig 62. PCR cleanup concentration of sample 1 of pNIC-bsa4 and PP2A

10/29/13 Fig 62. Chart of all the postive and negative control ligands for virtual screening for PP2A.
 * = Where ligand is from? ||= Organism ||= Protein ||= Name ||= Control ||=  ||= XLogP ||= Molecular weight ||= H-donor ||= H-acceptor ||
 * = PDB -> pubChem ||= Homo sapiens ||= 4LAC ||= adenosine 5'-O-(3-thiotriphosphate) ||= positive ||= CID_440317 ||=  ||=   ||=   ||=   ||
 * = PubChem ||=  ||=   ||= 4-aminophenylphosphate (pNPP) ||= positive ||= CID_77949 ||=   ||=   ||=   ||=   ||
 * = PubChem ||= Homo Sapiens ||= 2NPP ||= calyculin A ||= positive ||= CID_5311365 ||= 1.8 ||= 1009.169702 g/mol ||= 8 ||= 18 ||
 * = PubChem ||= Homo Sapiens ||= 2NPP ||= fostriecin ||= positive ||= CID_6436285 ||= -0.5 ||= 430.386042 g/mol ||= 5 ||= 9 ||
 * = PubChem ||= Homo Sapiens ||= 2NPP ||= microcystin ||= positive ||= CID_445434 ||= 2.3 ||= 995.17166 g/mol ||= 10 ||= 13 ||
 * = PubChem ||= Homo Sapiens ||= 2NPP ||= okadaic acid ||= positive ||= CID_446512 ||= 3.4 ||= 805.00292 g/mol ||= 5 ||= 13 ||
 * = PubChem ||= Homo Sapiens ||= 2NPP ||= nodularin ||= positive ||= CID_6437077 ||= 1.7 ||= 824.9627 g/mol ||= 8 ||= 11 ||
 * = PuChem ||= Homo Sapiens ||=  ||= caffeic acid phenethyl ester ||= positive ||= CID_5281787 ||= 4.2 ||= 284.30654 g/mol ||= 2 ||= 4 ||
 * = PubChem ||= Homo Sapiens ||=  ||= glautamate ||= positive ||= CID_33032 ||= -3.7 ||= 47.12926 g/mol ||= 3 ||= 5 ||
 * = PubChem ||= Homo Sapiens ||=  ||= endothall ||= positive ||= CID_3225 ||= -0.5 ||= 186.162 g/mol ||= 2 ||= 5 ||
 * =  ||=   ||=   ||= Mesalazine ||= positive ||= CID_4075 ||= 1.3 ||= 153.13538 g/mol ||= 3 ||= 4 ||
 * = PubChem ||=  ||= Asprin ||= ACETYLSALICYLIC ACID ||= negative ||= CID_2244 ||= 1.2 ||= 180.15742 g/mol ||= 1 ||= 4 ||
 * = Chembridge ||=  ||=   ||=   ||= negative ||= 5150705 ||= 3.43 ||= 516 g/mol ||= 6 ||= 10 ||
 * = Chembridge ||=  ||=   ||=   ||= negative ||= 5224197 ||= 3.14 ||= 486 g/mol ||= 6 ||= 10 ||
 * = Chembridge ||=  ||=   ||=   ||= negative ||= 5127723 ||= 2.74 ||= 401 g/mol ||= 5 ||= 10 ||
 * = Chembridge ||=  ||=   ||=   ||= negative ||= 7845952 ||= 3.41 ||= 493 g/mol ||= 5 ||= 8 ||

As one can see, the plates did not grow as clean of colonies as intended. In an effort to get around this, I decided to swipe some of the bacteria from each plate and spread it out on new plates (respectively), in an effort to regrow the bacteria in better looking colonies. However, one colony from the 2ul/4ul ratio was picked and chosen to make a master plate out of.

Fig 61. Transformation plate of 3ul vector (pNIC-Bsa4) and 6ul insert (protein phosphatase 2A gene).

Fig 60. Transformation plate of 2ul vector (pNIC-Bsa4) and 4ul insert (protein phosphatase 2A gene).

10/24/13

After some pretty low concentrations resulting from PCR cleanup, I decided to only go ahead and use sample 4 for transformation.

Fig 59. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 4.

Fig 58. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 3.

Fig 57. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 2.

Fig 56. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 1.

Fig 55. Virtually cut vector of pNIC-Bsa4, emphasizing where bands should be seen if cut correctly. These bands represent where in the pNic-Bsa4 that the genome is cut, allowing tail primers of the insert (gene) attach on, where the cut out sequence would be.

Fig 54. Cut accepting vector of pNIC-Bsa4 run on a gel to ensure the cuts happened and happened in the right location.

PCR squared was completely again, following up with an unsuccessful gel extraction. More accepting vector was created for our next rounds of cloning.

Virtual screening with control ligands was run. More ligands must be found and ran on GOLD.
 * < Where ligand is from ||< Species ||< Protein ||< Name ||< Control ||  ||<   ||<   ||
 * < PDB -> pubChem ||< Homo sapien ||< 4LAC ||< adenosine 5'-O-(3-thiotriphosphate) ||< positive ||< CID_440317 ||  ||   ||
 * < PubChem ||  ||   ||< 4-aminophenylphosphate (pNPP) ||< positive ||< CID_77949 ||   ||   ||
 * < PubChem ||  ||< Asprin ||< ACETYLSALICYLIC ACID ||< negative ||< CID_2244 ||   ||   ||

__**Week 5 &****6**__
 * Nice Job keeping your work up to date. Good captions but please try to add a brief analysis after new data. Thank you. -Max 10/07/2013**
 * __Homology Model__:**
 * A homology model was created since our protein, protein phosphatase 2A, does not have one.**


 * Fig 53. Molprobity Multi-criterion chart of the newly created homology model for PP2A created using 2nppF.**


 * Fig 52. Molprobity Multi-criterion chart of chain F of 2npp**


 * Chain F of 2npp has a 71% Identity and 84% Positives**
 * Top hit from SWISS-MODEL test is chain F of 2NPP. The amino acid sequence for 2nppF is:**
 * MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNY **
 * LFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVD **
 * GQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRA **
 * HQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRTPDYFL **


 * Fig 51. SWISS-MODEL homology test top results.**


 * Fig 50. BLASTp results to find a homology model for PP2A**


 * __Homology Model__:**
 * A homology model was created since our protein, protein phosphatase 2A, does not have one.**


 * Nanodrop results of the second round of cloning look much more promising than the first round. Results should be in in a day or two.**
 * Fig 49. Nanodrop concentration of tube 8 after miniprep**


 * Fig 48. Nanodrop concentration of tube 7 after miniprep**


 * Fig 47. Nanodrop concentration of tube 6 after miniprep**


 * Fig 46. Nanodrop concentration of tube 5 after miniprep.**


 * Fig 45. Nanodrop concentration of tube 4 after miniprep.**


 * Fig 44. Nanodrop concentration of tube 3 after miniprep.**


 * Fig 43. Nanodrop concentration of tube 2 after miniprep.**


 * Fig 42. Nanodrop concentration of tube 1 after miniprep.**


 * Fig 41. Master plate used in second round of cloning, 2 ul of accepting vector and 4 ul of insert**


 * Fig 40. Secondary PCR of T. brucei - Lane 1-4: Renee and Tony's samples, Lane 5: blank, Lane 6: 100 base pair ladder, Lane 7: primary PCR, Lane 8: secondary PCR sample A, Lane 9: secondary PCR sample B.**


 * None of the results received were completely successful. Tube 3 was the closest but there was one deletion which will not let us precede with this sample. A second round of cloning will soon begin.**
 * Fig. Tube 3 of master plate B cloning results, Query cover 99%, Identity 98%, closest sample we had, yet there was a deletion in the second row.**


 * Fig. Tube 7 of master plate tube B cloning results, Query cover 93%, Identity 96%**


 * Fig. Tube 5 cloning results, Query cover 89%, Identity 99%**

__Week 3 & 4__
 * Kevin - ok good work on the colonies. Show if these sequences were positive or not from Sequencing. - DR. B 101013**
 * Fig 39. Nanodrop concentration of tube 1 after miniprep**


 * Fig 38. Nanodrop concentration of tube 2 after miniprep.**
 * Fig 37. Nanodrop concentration of tube 3 after miniprep.**
 * Fig 36. Nanodrop concentration of tube 4 after miniprep.**
 * Fig 35. Nanodrop concentration of tube 5 after miniprep.**
 * Fig 34. Nanodrop concentration of tube 6 after miniprep.**
 * Fig 33. Nanodrop concentration of tube 7 after miniprep.**
 * Fig 32. Nanodrop concentration of tube 8 after miniprep.**
 * Fig 31. Master plate with transformation mixture from tube B (3ul of accepting vector and 6 ul of insert) **


 * Fig 30. Master plate with transformation mixture from tube A (2ul of accepting vector and 4 ul of insert) **


 * Master plates were grown with 8 colonies from the transformation plate in 5mL LB and 5 uL kanamacyn. We also started a back up master plate in case these results were not successful.**


 * Fig 29. Successful cut vector of pNIC-BSA4 gel. After taking a picture without the rig, the strange imaging at the bottom of the gel disappeared.**




 * Fig 28. NEBcutter, virtual gel of the cut vector pNIC-BSA4.**




 * Fig 27. PCR clean up for the 8 samples run off of secondary PCR**

__Fall 2013 - Week 1 & 2__
 * Kevin, need some more results here (PCR's etc.) Also include an image of PyMol refresher. Each week should have some image ideally. - Dr. B 090913**


 * This week in VDS, I completed a PyMol refresher.**
 * Also, I completed the preparation of pNIC-Bsa4 as accepting vector. This RE Digest is necessary for cutting the pNIC so that my gene can insert into the DNA before cloning.**
 * Fig 26. PyMol of 2H2Q PDB. Selected substrates NAP and DU shown as red. Polar contacts between atoms shown as black. Hydrophobic residues shown as yellow; ionic residues shown as blue; polar residues shown as orange. All other atoms colored by element with carbon as green. **


 * Fig 25. Alignment of 1U72 and 3CL9 on PyMol. Active sites around MTX (red) are pink for 3CL9 and navy for 1U72. In all, 1U72 is colored by element with carbon as green and 3CL9 with carbon as cyan. **
 * Week 6**
 * Kevin - good conc of pNIC. SHow some more of your Overlap and Secondary PCR.s - Dr. B 071713**


 * Ordered new correct primers:**
 * Fig 24. Successful Secondary PCR of T. Brucei tail primers**
 * Lane 1: Skip, Lane 2: 100 bp ladder, Lane 3: Sample 1, Lane 4:**
 * Sample 2, Lane 5: Sample 3, Lane 6: Sample 4, Lane 7: Sample**
 * 5, Lane 8: Sample 6, Lane 9: Sample 7, Lane 10: Sample 8**

Cycles, 72 for 2 minutes, 4 for forever 4: 68.3°, Sample 5: 66°, Sample 6: 64.3°, Sample 7: 63°, Sample 8: 62°
 * Sample cycle: 98 for 30s, [98 for 10s,** for 30s, 72 for 30s] 25
 * Sample 1: 72°, Sample 2: 71.4°, Sample 3: 70.1°, Sample

Fig 22 and 23. Measurement 1 and 2 of pUC19 from the nanodrop after midiprep

Fig 21. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 12, Lane 3: Sample 13, Lane 4: Sample 14, Lane 5: Sample 15, Lane 6: Sample 16, Lane 7: Sample 17, Lane 8: Sample 18, Lane 9: Sample 19, Lane 10: Not our sample Sample 12: 98 degrees for 30s, [98 for 10s, 72 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 13: 98 degrees for 30s, [98 for 10s, 70.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 14: 98 degrees for 30s, [98 for 10s, 68.1 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 15: 98 degrees for 30s, [98 for 10s, 64.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 16: 98 degrees for 30s, [98 for 10s, 59.9 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 17: 98 degrees for 30s, [98 for 10s, 56.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 18: 98 degrees for 30s, [98 for 10s, 53.8 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever. Sample 19: 98 degrees for 30s, [98 for 10s, 52 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.

Fig 20. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 6, Lane 3: Sample 7, Lane 4: Sample 8, Lane 5: Sample 9, Lane 6: Sample 10, Lane 7: Sample 11 Sample 11: 98 degrees for 30 s, [98 for 15s, 66.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever. Sample 10: 98 degrees for 30 s, [98 for 15s, 67 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever. Sample 9: 98 degrees for 30 s, [98 for 15s, 68.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever. Sample 8: 98 degrees for 30 s, [98 for 15s, 69.1 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever. Sample 7: 98 degrees for 30 s, [98 for 15s, 69.7 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever. Sample 6: 98 degrees for 30 s, [98 for 15s, 70 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.

Fig 19. Secondary PCR gel of T. Brucei Lane 1: blank, Lane 2: 100 bp ladder, Lane 3: Sample 3, Lane 4: Sample 4.

Sample 4: 98 degrees for 30 s, [98 for 15s, 60 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever. Sample 3: 98 degrees for 30 s, [98 for 15s, 60 for 10s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.

Fig 18. PCR gel of T. Brucei; Lane 1 & 2 blank, Lane 3: 100 bp ladder, Lane 4: Primary PCR, Lane 5-8: Jackie's sample, Lane 9: Sample 1, Lane 10: Sample 2. Sample 2: 98 degrees for 30 s, [98 for 30s, 65 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever. Sample 1: 98 degrees for 30 s, [98 for 10s, 65 for 20s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.

Fig 17. Nanodrop trial 1 and 2 after midiprep of pNIC-BSA4. Determination of concentration is 72.2 ng/uL.

Fig 16. Graph of FtHap FPLC of ptp1b

Fig 15. Gel of PCR overlap of T. Bruci Lane 1: KB ladder Lane 2: Primary PCR Lane 3: Secondary PCR Failure due to using the ladder instead of the blue dye in samples. Will run using same samples next.

Fig 14. PCR gel of pNIC-Bsa4. Lane 1 – 1 KB ladder Lane 2 – lowest concentration Lane 3 – medium concentration Lane 4 – highest concentration Lane 5 – control, no template

Fig 13. Gel of PCR of pGBR22 purple protein. Lane 1: 1 KB ladder, Lane 2: Sample A (1:1000 dilution of DNA template, 1 microliter), Lane 3: Sample B (1:1000 dilution of template, 10 microliters), Lane 4: Sample C (1:100 dilution of template, 10 microliters), Lane 5: Sample D (control, no DNA template). Template seen through gel in lane 5, the control, may be due to leakage of the wells.

Fig 12. RE Digest of pGBR22 plasmid with restriction enzymes EcoRI and PvuII. Lane 1: skipped, Lane 2: 1 KB ladder, Lane 3: Uncut plasmid, Lane 4: EcoRI digestion, Lane 5: PvuII digestion, Lane 6: EcoRI and PvuII digestion, Lane 7: 1 KB ladder, Lane 8: EcoRI digestion, Lane 9: PvuII digestion, Lane 10: EcoRI and PvuII digestion.

pNIC-Bsa4 FASTA sequence with vector in it: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAA CCTG TACTTCCAATCC __ATGCTGTCCCTGACCCGCATCCTCCGTAAGAAGATCCCTGTTCACGAACTGGCCGGCAAGATTTCTCGTCCACCACTCCGTCCGTTTTCCGTCGTCGTTGCCAGCGATGAAAAGGGT __ __GGTATTGGCGACGGTGGTACTATTCCTTGGGAAATCCCAGAAGACATGCAATACTTCCGCCGTGTGACGACCAACCTGCGTGGCAAAAATGTTAAGCCGAGCCCATCTAAGCGTAACGCGGTCGTGATGGGT __ __CGCAAAACCTGGGATAGCCTGCCACCTAAGTTCCGTCCACTCTCTAATCGTCTGAATGTGGTTCTGTCCCGTTCTGCGACTAAAGAGCAACTGCTCGCGGGTATTCCGGACCCAATCAAGCGTGCGGAAGCT __ __GCTAACGACGTGGTGGCTGTTAACGGTGGCCTGGAAGATGCACTGCGTATGCTCGTAAGCAAGGAACATACCTCTAGCATCGAGACCGTGTTTTGCATCGGTGGCGGTACGATCTACAAACAGGCGCTCTGT __ __GCCCCTTGCGTTAATGTTCTGCAAGCGATCCACCGTACCGTTGTACGTCCGGCGTCTAACTCTTGCTCTGTTTTCTTCGACATCCCAGCGGCTGGCACCAAGACCCTCGAAGGCCTCGAACTCGTACGTGAGT __ __CTATTACCGATGAGCGTGTTTCTACGGGCGCTGGTGGTAAAAAGTATCAATTTGAAAAGCTCGTGCCGCGCAATAGCGAAGAAGAGCAGTATCTCAACCTCGTGGGCCGCATTATCGACGAGGGTTGCACCA __ __AATGTGACCGCACTGGTGTAGGTACGCGTTCCCTCTTCGGTGCTCAGATGCGTTTCTCTCTGCGTAACAACCGTCTGCCTCTGCTGACTACTAAACGCGTCTTTTGGCGTGGTGTATGTGAAGAGCTCCTCTG __ __GTTCCTCCGCGGTGAAACCAACGCAAAGCTCCTGTCTGATAAGGGCATCCATATTTGGGACGGTAATGGTTCCCGTGCTTTTCTCGACTCCCGTGGTCTGACCGACTACGACGAGATGGACCTCGGCCCAGT __ __CTATGGCTTCCAATGGCGTCACTTTGGCGCGGACTACATCTCTTGCAAAGAGGATTCCGAGGGTAAGGGCGTAGACCAAATCGCGAATATCGTCAAATCTCTCATCGAAAACCCTGATGACCGCCGTATGATTT __ __GCACCGCGTGGAACCCGGCTGCCCTCCCTCGCATGGCACTCCCGCCTTGCCACATGATGGCGCAATTTTACGTGTCTAATGGCGAGCTGTCCTGCATGCTCTATCAGCGCTCCTGCGATATGGGCCTGGGTG __ __TGCCGTTCAATATCGCGTCCTATGCGCTGCTCACGTTTCTGATGGCTAAAGCGTCTGGTCTGCGTCCGGGTGAGCTGGTACATACTCTGGGCGATGCGCACGTATACTCCAACCACGTTGAACCATGCCGTAA __ __ACAGCTGAAGCGTGTCCCGCGTCCATTCCCGTTTATCGTCTTCAAGCAAGATAAGGAGTTTCTGGAGGACTTTCAAGAAAGCGACATCGAGGTGATCGACTATTCTCCATACCCAGTAATCTCTATGGAAATGG __ __CAGTGCTCGGCGACAGCCCTGGCTACGTTATGTCCAACATCGAATTCCGTCAACTGACGCGTGGTCATAGCCCGTCCGACGAACGCGAGGCACGCCGCGTCGAGGAGGCGGGTGGTCAACTCTTCGTGAT __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">TGGCGGCGAACTCCGTGTAAACGGTGTCCTGAATCTCACGCGCGCACTCGGTGACGTGCCTGGTCGCCCGATGATTTCTAATGAACCGGAGACCTGTCAGGTACCGATCGAATCCAGCGACTATCTGGTCCT __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">GCTGGCATGTGATGGCATTTCCGATGTGTTTAATGAGCGCGATCTGTATCAACTGGTCGAAGCGTTCGCCAACGATTACCCGGTTGAAGATTATGCCGAACTCTCTCGTTTTATCTGCACGAAAGCCATTGAAG __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">CGGGCTCTGCCGACAACGTTAGCGTTGTTATTGGTTTTCTCCGCCCACCGCAGGACGTCTGGAAACTGATGAAACATGAATCTGATGATGAAGATAGCGATGTTACGGACGAAGAATGCCAAGTCCCGATTG __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">AATCTAGCGATTATCTCGTACTCCTCGCCTGCGACGGCATCAGCGACGTATTCAATGAACGTGACCTGTACCAACTCGTGGAAGCATTCGCAAATGACTACCCTGTAGAAGACTACGCTGAACTGAGCCGCTTT __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">ATTTGTACGAAGGCTATTGAGGCCGGTAGCGCGGATAATGTCTCTGTCGTCATCGGCTTCCTGCGCCCTCCGCAAGATGTATGGAAGCTCATGAAGCATGAGAGCGACGATGAGGACTCCGACGTGACTGAC __ __<span style="font-family: 'Calibri','sans-serif'; font-size: 13.3333px;">GAGGAATGA __ CAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTG GCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCT TCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACC CTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTC AGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAAC TGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCA TTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTC CCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACA TCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATAC CCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG GTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTAC AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGC TTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCT CATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGG GCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAA CATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGT TCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTT GTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAA CGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGC GTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGC ATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAA TGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTG CGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGC CCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACT CGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCAC TCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCG CGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCC GGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAG GCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTG GAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGC AGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCT CTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGG TTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCT CATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTA GAGGATCGAGATCTCGATCCCGCGAAAT

Fig 11. Elution 1 of FtHAP after purification.



Fig 10. Elution 2 of FtHAP after purification

Fig 9. Agarose gel PCR of pmCherry into pGBR22 plasmid Lane 1 - 1KB ladder, Lane 2 - sample 1, Lane 3 - sample 2, Lane 4 - sample 3, Lane 5 - sample 4, Lane 6 - sample 5, Lane 7 - sample 6, Lane 8 - sample 7, Lane 9 - sample 8
 * Agarose gel**

Fig 8. 1KB DNA ladder used from NEB used in running gels.

Fig 7. Agarose gel of pGBR22 plasmid, primers M13 forward and reverse: Lane 1 – 1KB latter Lane 2 – Sample A Lane 3 – Sample B Lane 4 – Sample C Lane 5 – Sample D

Fig 6. Nanodrop measurement 2 of pNIC-Bsa4 plasmid at 230 nm.

Fig 5. Nanodrop measurement 1 of pNIC-Bsa4 plasmid + FABI at 230 nm. Fig 4. Transformation of E. coli with 5ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.

Fig. 3 Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.

Fig. 2 Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.

DNA Sequencing of pGBR22 plasmid: NNNNNNNNNNNNNGGGCGATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGG TGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTAC ATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCA GTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATT GTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCG TATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTC CCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCG ACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCA TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANCTGTC NTGNCAGCTGCATTAATGAATCGGCNACNNNCGGGGNANNNGNNGTTTGCGTANTGNNNCTCNTCCGNTNCNCGCTCANT GANTCNNNNNNCTCNNNCNNNCGGCNNCNGNNNNNNNGNNTCANCNNNNNNNNNNGNNNGNNNNNNNNNTNNNCCNNNNN NNNGNNNNNACNNNGAANANNNNNNNNNNNNNNCNAANGCNNNNNNNNNAAAGNCNNNNNNNNNNNNNNNNNNTNNNNNN


 * Nanodrop Spectrophotometer**

Fig 1. Nanodrop spectrophotometer measurements 1 and 2 of pGBR22 plasmid blanked using TE buffer.

Kevin - update your page and properly annotate everything. All of your results for the last 2 weeks shoudl be here. Also repeat this PCR. What was the PCR of? - Dr. B

Kevin - good reverse chronological order, but segment your results by each week (We are now on Week 6). Also put dates for each image (roughly) - Dr. B 070913