Gabe+H.

= Week 9 =

8/1/13 ICM1 Results
Reran 20 ligands from VS1 (see below) but using ICM (Maestro ligprep was used for pH-ing) against 1U72. The following rank table was compiled:

For reference, here is the VS1 results table using GOLD:

Here is the histogram of the scores ICM provided for me:

7/30/13 VS4 Results
Ran verification library against NDM-1 Beta-Lactamase sample in GOLD using original protocol and redo protocol (specific protonation of histidines and removal of metal CONNECT records). Inserted results onto table. Do note Maestro ligprep was used to pH the library. Figure 10: VS4 Results Table (4HL2 vs. Verification Library) = Week 8 =
 * Name || Protein Code (PDB) || POS1 || Faropenem || Penicillin || Clavulanic Acid || Aspirin || Original Ligand || REDO Pos1 || REDO Faropenem || REDO Penicillin || REDO Clavulanic Acid || REDO Aspirin || REDO Original Ligand ||
 * Gabe H. || 4HL2 || 115.04 || 80.56 || 76.95 || 65.54 || 66.96 || 86.35 || 113.25 || 85.00 || 68.53 || 70.53 || 69.24 || 91.88 ||

7/26/13 VS3 Results
Ran VS3 Protocol selecting 3QY7 ( Tyrosine-protein phosphatase of Bacillus subtilis) using database CB1k_42. Controls were the original phosphate ligand, aspirin, pNPP, and two positive controls (known inhibitors). Table ranked by GOLD fitness score. 1st positive control has highest score in its three forms, however it does not fit Lipinki's rules. The top novel inhibitors all follow Lipinski's rules. Here are images for the dockings of the top performing control along with the 2 best novel inhibitors:

**7/22/13 - 7/26/13 Protein Expression of FabI with Modified Expression Temperature and IPTG Levels** Continued work on expressing Daniel's target. This time, the incubation for expression conditions and IPTG levels were altered: 125uL IPTG @ 30degrees C (4-5 hours) 250uL IPTG @ 30degrees C (4-5 hours) 125uL IPTG @ 22degrees C (room temp) (overnight) 250uL IPTG @ 22degrees C (room temp) (overnight)

The room temperature incubation showed significant levels of the target protein in the solluble fraction of the cell following sonication. Work is now being done on purifying the protein.

7/23/13 VS2 Results
Ran VS 2 protocol. The following table shows results for docking onto 3DAT (bacterial):

The following shows results for docking into 1u72 (human): = Week 7 =

7/17/13 - 7/19/13 Protein Expression of FabI with Modified Lysis Buffers
Began work on helping Daniel express his target. The issue we are dealing with is the protein not appearing in the soluble fraction of the cell after lysing and sonicating the culture. The following additives were applied to the lysis buffers in hopes of increasing the protein's solubility: 1. KCl 1M 2.MgCl2 .2M 3. Tween 80 .2% w/v 4.Tween 20 120uM 5.Sucrose 1M 6. Glycine 2% 7. Glycerol 40% v/v Gel results showed no significant amount of protein in soluble fraction.

7/17/13 GFP + T7 Sequencing Results and Gel of GFP Plasmid
Resequenced Cloned GFP with T7 Primer:

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNCTTTCNTCNNGACGTTNCACACCNNTANGGTACGATCCAG

ATCNGGCTAGGCAGNCATTTTGAGNNAAGCGAGAACGCTAAACGANGGCAGAGCGNNAATCNCCCGNNNCCCATGCCCAC

ATCANGTTNNNTATTCANCATACCNNNNTNANTCAATGTTCCNNCCCACCAACGCATGCATTGACGATGTGGTTCTNAGA

NACTGCTTCATGGTCCANCTCATGCTATGNNNGGNACACAAGACNAAATCNNCTTTGANTGATCGAANAACTACCATCAC

TTANGTAAGCAGGGCGGGAAACCCACATTTTGCTTACACGATACAGGCCANAACAGATAACCGAAGANGNATGCCAGGTG

GAAGTTGTTGCGCTAGCTGGGACCCAACCACNCGNGAGANCNNNAAACNACTCNNNNNNNNTTTNNNNNNNNTCNANNNT

TNNNTNGGNNNGGGNTGGGGCGTNCTNCNTCCNNNNNNNAGGANTGNNNNNNNNCNNNNNNGTTGNNNGCCTGCATACAN

NNANACCNCNNCNACCNNACCCNNNTANNGGNNNNNNNNTNATCGNCNNCNNNGACNNCNNNNANNTCGAAGGACNNGAA

NNANCGNGCTTNNNNTNTACGACCNGNNNNNNNNNNACNTGANNCNACNNNCNNNANNNNGTNTNNANANTNNNGAGNGA

TNGACTNGNATNCTTNNTATNCNNNNNNTTNTNNCNNTATGNNNAAATNCNNCNTNANNNNNNTGNTCATGCNAGNGCTA

NGGGTTNTGGCCNTNNNNNNCNCCTTNNAGGNNAGNTCNCNGANNGNNGNNGCGGNNNTANNNGCTGGNANCCCGGNNTC

GGNGNNCGNGGANTGNTGAGCACNNNNNNNTGANGNTNGNNCGNTCNNNNGCTTGNGGCNTANNATCCANNNNATNAANC

TCNTTTNNNNCATTTNNNTTNNNNNNNNNNNACNGNNN BLAST Search also failed on this sequence.

A gel was run on the whole plasmid to help understand results. It appears to contain contamination:

7/16/13 VS1 Results
Followed VS1 Lab Protocol. The following is a table of the ligand docking results ranked by GOLD Fitness Score.

= Week 6 = Gabe - good results. Post some virtual results (table) when you get some. - Dr. B 071713

DNA Sequencing Results for Purified GFP Plasmid after Transformation 7/12/13
GFP + M13Reverse: NNNNN It appears no DNA was found

GFP + M13Forward: NNNNNNNNNNNNNNNNNNANNNNNNTNCNTNNNANNAATANNNCNNNNTTNTTCNNNNNAANNCGGCNTTCTCNNNNNAGNNNTT NNNNNCNTTNNCGGGANNGNNTGNNNNNANTTTCNNNNCNGNCNNNNNANNNNTGNNNNNNNNNNCNNNANNNCCNCTCTNNNCN NTNGNNNNNCNNTGNTCNGNNNGGN The sequencing facility notified me that this sample is being rerun.

BLAST search was attempted on the GFP + M13 Forwards sample, but no results were found.

Post-Transformation GFP Plasmid Midi Prep Purification 7/10/13
Performed transformation on cells with GFP plasmid. Performed midi prep to purify the DNA from the cell sample. Nanodrop results demonstrated the following: Avg. Results: ng/ul: 79.35 Volume ul: 1200 260/280: 1.91 260/ 230: 4.15 yield (ng): 95220 yield (ug): 95

= Week 5 =

PCR 7/3/13
Conducted PCR cloning on pgbr22 = Week 4 = **RE Digest 6/25/13** RE Digest was done on pgbr22 plasmid using EcoRI, PvuII, and a combination of both. A gel was run with the following results (gel shared with Anita V.): Lane 1: Blank Lane 2: DNA Ladder Lane 3: Uncut Plasmid (Anita V.) Lane 4: EcoRI HF + NE2 Buffer (Anita V.) Lane 5: PvuII + NE2 Buffer (Anita V.) Lane 6: EcoRI + PvuII + NE2 Buffer (Anita V.) Lane 7: EcoRI + NEB Buffer (Anita V.) Lane 8: PvuII + NE2 Buffer (Gabe H.) Lane 9: PvuII + EcoRI HF + NE2 Buffer (Gabe H.) Lane 10: EcoRI HF + NE2 Buffer (Gabe H.)