Brandon+N.

This week, I completed my virtual screening of the cb_306_3d library and the CB-kin_UT library. The top 10 ligands were kept from each of the libraries. I also completed an inhibition assay with PSTP. Ligand 5153298 was chosen as the inhibitory compound. All absorbance values taken at 410 nm were 0. This means the enzyme was not functional to begin with because the controls, those tubes with no compound, did not yield any absorbance. cb_306_3d was considered the better library because of the higher scores in GOLD and the values for Lipinski's Rule. The top 3 ligands of this library were viewed in the active site of STP1 using PyMOL.
 * __Week 15 __**

Figure 1: Spectrophotomer reading of different concentrations of ligand 5153298 with PSTP enzyme.

Table 1: Top 10 GOLD scoring ligands in the CB-kin_UT ligand library. Lipinski's info also included.

Table 2: Top 10 GOLD scoring ligands in the cb_306_3d ligands library. Lipinski's info also included.

Figure 2: Top scoring ligand from the cb_306_3d library in the active site of STP1. Enzyme is shown as lines colored by element with carbon being green. Ligand 5192853 is shown as sticks with colored by element with carbon being blue. Polar contacts are shown as black dashes. Figure 3:Second top scoring ligand from the cb_306_3d library in the active site of STP1. Enzyme is shown as lines colored by element with carbon being green. Ligand 5175515 is shown as sticks with colored by element with carbon being blue. Polar contacts are shown as black dashes.

Figure 4: Third top scoring ligand from the cb_306_3d library in the active site of STP1. Enzyme is shown as lines colored by element with carbon being green. Ligand 7842136 is shown as sticks with colored by element with carbon being blue. Polar contacts are shown as black dashes.

This week, I performed two enzyme assays. The assays were not performed on our surrogate protein PSTP but rather PTP1b. This selection was made by Dr. B. The first assay showed fluctuation instead of a positive linear behavior. Therefore, a second assay felt necessary. Also, it was advised that I increase the concentration of the enzyme 4 times. The second assay displayed a constant increasing of absorbance with increasing concentration of enzyme, which is ideal. Therefore the enzyme proves to be functional.
 * __Week 14: __**

Figure 1: Spectrophotometer absorbance reading at 410 nm of a series of dilutions of the PTP1b enzyme. Concentration of enzyme before any dilution is 2 ng/ul.

Figure 2: Second spectrophotometer absorbance reading at 410 nm of a series of dilutions of the PTP1b enzyme. Concentration of enzyme before any dilution is 8 ng/ul.

112612 - Good but include some 'data'. Dr B This week, I started virtual screening of my target enzyme, STP1. In the process, an active site had to be defined because of the missing ligands with the crystal structure.
 * __We __****__ek 13: __**

Brandon - ok let's try it with someone's FtHAP enzyme instead ( or another groups PSTP that works). - Dr. B 11/19/12
 * __Week 12: __**

This week, I performed an enzyme assay with the surrogate enzyme PSTP. There were no values other than zero at 410 nm so I believe there was an error on my behalf.

Figure 1: Spectrophotometer reading of different dilutions of enzyme PSTP with pnPP substrate. This week, my team finished expression of our protein surrogate, PSTP. After they finished the protein purification step, I started protein characterization which involved using samples they took during different steps in the purification step and performing gel electrophoresis. I then put the gel in imperial stain. My team then finished the characterization step. Also, Suman completed the FPLC step which further purified the protein. Results from FPLC indicate low yield but no contamination.
 * __Week 11: __**

Figure 1: Dry gel of PSTP samples. Soaked in imperial protein stain. Lane 1: Skip Lane 2: Protein Ladder Lane 3: Sample 3 - flow through Lane 4: Sample 4 - wash Lane 5: Sample 5 - elution 1 Lane 6: Sample 6 - elution 2 Lane 7: Sample 3 (2) - flow through Lane 8: Sample 4 (2) - wash Lane 9: Sample 5 (2) - elution 1 Lane 10: Sample 6 (2) - elution 2

Figure 2: Nanodrop reading of concentrated protein PSTP. Figure 3: Second Nanodrop reading of protein solution PSTP.

Figure 4: Nanodrop reading of protein concentration after FPLC.

Figure 5: Second Nanodrop reading of protein concentration after FPLC.

This week, I performed secondary PCR with tail designed, first and last primers of the oligo mix, and the third and the sixteenth primer of the oligo mix. I then did a gel check of the secondary PCR product. There were no obvious bands for the designed primers but there are strong bands for the oligo mix primers. The bands correctly correlate with the primers. Also this week, I performed a portion of the expression of PSTP which is Sadhana's target and also our surrogate target. This portion was day 2 of the expression and included bacteria with target and measuring OD values with a spectrophotometer.
 * __Week 10: __**

Figure 1: Agarose gel check with secondary PCR product of STP1 with designed forward and reverse tail primers, 1st and 18th primer of oligo mix, and 3rd and 16th primer of oligo mix. Lane 1: 1kb DNA ladder Lane 2: Secondary PCR product with designed tail primers at 57 degrees Celsius Lane 3: Secondary PCR product with 1st and 18th primer of oligo mix at 57 degrees Celsius Lane 4: Secondary PCR product with 3rd and 16th primer of oligo mix at 57 degrees Celsius

This week, I performed secondary PCR with 55 degrees Celsius annealing temperature with all other parameters being the same. Then, I gel checked my products for 53,55, and 57 degrees Celsius. As you can see below, there was no visible bands, smears, or anything which implies unsuccessful PCR. This week, I also completed the lab report detailing the research target STP1 and the experiment proposal. Later in the week, I went to a target meeting with my other target members and Dr. B to discuss the experiment and troubleshoot any problems. After the meeting, it was apparent that all members were having the same problem as me. We set a plan to do 3 secondary PCRs: one with tail designed primers, one with the first and last primer of the oligo mix, and one with the third primer and sixteenth primer of the 18-primer oligo mix.
 * __Week 9: __**

Figure 1: Agarose gel check with secondary PCR product of STP1 with primary PCR template and designed forward and reverse tail primers. Lane 1: 100bp DNA ladder Lane 2: Secondary PCR product at 53 degrees Celsius Lane 3: Secondary PCR product at 55 degrees Celsius Lane 4: Secondary PCR product at 57 degrees Celsius

102112- Brandon, ok - also try the trick of doing a new primary PCR at a lower temp. This may give you a better 'seed' for the secondary. -- DR. B
 * __Week 8: __**

This week, I gel checked my PCR product from last week with annealing temperatures of 48 and 60 degrees Celsius. I chose these parameters because Ruifei had done PCR with several temperatures around the standard of 58 degrees Celsius. **Ruifei and I** are collaborating to successfully get secondary PCR product. If you look very closely, there are a few bands near the bottom of the 100bp DNA ladder. You said this was incorrect because of the length and caused by probably contamination. I performed secondary PCR with temperatures of 53 and 57 degrees celsius because these temperatures have not yet been tested. I also completed the Virtual Screening Refresher. Figure 1: Agarose gel check with secondary PCR product of STP1 with primary PCR template and forward and reverse tail primers. Lane 1: 100bp DNA ladder Lane 2: Secondary PCR product at 48 degrees Celsius Lane 3: Secondary PCR product at 60 degrees Celsius

Figure 1.Number 1 ligand(highest GOLD score), 9039798, in DHFR active site. Ligand is shown as blue sticks. Protein is shown as lines and colored by element with carbon being green. Polar contacts are shown as black dashes. Ligand has 1 polar contact. Figure 2. Original ligand, MTX, in DHFR active site. Ligand is shown as gray sticks. Protein is shown as lines and colored by element with carbon being green. Polar contacts are shown as black dashes. Ligand has 6 polar contacts. 101612 - So Brandon - state your result here since it is not totally clear which band is which. I think that your Primary worked but secondary PCR did not ?? - Dr. B
 * __Week 7: __**

This week, I performed secondary PCR for my target and did a gel check on both the PCR product of my first and secondary PCR. I have a smear for my primary PCR results and nothing visible for my secondary results. I then performed PCR for secondary with 48 and 60 degrees Celsius annealing temperature. Froze PCR product in fridge. Figure 1: Agarose Gel Check of primary and secondary PCR product of STP1 with oligo mix. Lane 1: 100bp DNA ladder Lane 2-6: skip Lane 7: Primary PCR product Lane 8: Secondary PCR product Lane 9-12: Not mine

100912 - Brandon, ok good. I am eager to see if your PCR worked. - Dr. B
 * __Week 6: __**

This week, I finished the PyMol refresher and I prepared my oligo mix containing the necessary 18 primers for STP1 and PCR primer overlap. Then later this week, I used the oligo mix to perform the first primary PCR to piece together the DNA for STP1. I froze my PCR product awaiting to start my secondary PCR with tail primers.



Figure 1. //T. cruzi// DHFR aligned with human 1U72 DHFR. Active site is defined 5 angstroms from MTX inhibitor. MTX from the 1U72 is shown as sticks and is colored by element with carbon being orange. MTX from the 3CL9 is shown as sticks and is colored by element with carbon being forest green. Active site of 1U72 is shown as sticks and is colored by element with carbon being silver and active site of 3CL9 is shown as sticks and is colored by element with carbon being green. NAP from the 1U72 is shown as sticks and colored by element with carbon being pink. NAP from the 3CL9 is shown as sticks and is colored by element with carbon being pink. Chlorine ions of 3CL9 are shown as green spheres.

100112 - Brandon, missing your Tail primers. Did you do PyMol too? -. B
 * __Week 5: __**

This week, I performed an agarose gel check of my PCR product from last week. I found out there was a issue with the cycling program that I ran for the PCR so it was expected that my PCR product came out incorrectly. As a result, I performed another PCR practice with the pGBR22 and this one was a bust as well. This week I also completed the PCR primer design for pNIC-Bsa4 cloning in regards to // Staphylococcus agalactiae. //

Forward Primer: TACTTCCAATCCATGGAAATCTCTCTCCTCACCG

Reverse Primer: TATCCACCTTTACTGTTATTCTTCGTCCGTCACG



Figure 1: Agarose gel check of fail pGBR22 PCR product with M13 forward and reverse primers. Lane 1:100bp DNA ladder, Lane 2:1:1000 dilution Lane 3:1:1000 dilution Lane 4:1:100 dilution Lane 5:No DNA control.

Figure 2: Another fail agarose gel of pGBR22 with M13 primer. Lane 2:100bp DNA ladder Lane 3:1:1000 dilution Lane 4:1:1000 dilution Lane 5:1:100 dilution Lane 6: no DNA control Other lanes belong to Sajan. Brandon - ok you may need to do Midi-prep again if you need more pNIC for your cloning. -- Dr. B
 * __Week 4: __**

This week, I completed the midiprep protocol to extract the DNA from the transformed bacterial cells with pNIC-Bsa4 and sent the DNA to DNA sequencing. I also finalized the restriction enzyme digest with the agarose gel check. The gel matched my prediction but it was more faded than expected probably due to error such as incorrect pipetting. I will fix the contrast next time to better see the quality of the bands. I began the first part of the practice PCR which consists of the physical PCR with the thermal cycler. I then stored my PCR product in -20 degrees Celsius for later gel check.

Figure 2: Nanodrop results from DNA extraction of bacterial cells with pNIC-Bsa4. The concentration is 39.4 ng/ul.

Forward Sequence: NNNNNNNNNNNNNNACTTTAGNNGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCG AGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTA TGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAG AAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAAT AGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAG GCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGC GTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAAC GATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAG CGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAA ATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAA AGGNCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCT TTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATT GACAGCTGGAAAAACGCTGGGCNNCGTCTTTNAAGACAGCGACAANTCGATGCAAATGATTCTATCCTAAANANNNANCA NANANNGNCNNNNAGCNNCNTTTNCNTCTGANNNAAANCNNNATNCTANNCTGATTNNNNCNNANNNTNNGNNANCNANN CTGANNNTNNNNNNNNNNNNTCANCATNNNNNNNNNNTNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNGNNNNNNN NNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNN

Reverse Sequence: NNNNNNNNNNNCCGGNNCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCG GATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTG TTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCA AACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGG TACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCG GCTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATT TTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTAC TGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGT TTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCA TAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTT GTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACT GCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTG TCTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTTGTTTGCCGTANGNTTACNGGANAAATCANTGNANANTAAA CGNATTTTNCGTCNNATGTAAATGNNNTGNNNGACNNNCNTNNGNTTNNCNTTTNGNNNNNANCNNTNGCATCNATTNTC NCTNNNNTTNNNNNNNNNGNNNNTTTCNNCNNNCANTANANTTNNNCNANTTNNNNNNANNNNNNNNNNANNNNNNCNNN NNNNNNCNNNNNNNNNNNCANNNNNNNNNNNNNNNTNNNNNN

Figure 1: Agarose Gel of pGBR22 plasmid. Lane 2 is 1kb DNA ladder, lane 3 is uncut plasmid, lane 4 is plasmid with EcoR1 enzyme buffer, lane 5 is with PvuII enzyme buffer, and lastly lane 6 is with both the enzyme buffers.

Brandon - just say EcoRI enzyme vs. 'EcoRI enzyme buffer'. The buffer actually isn't doing the cutting. -- Dr. B

Brandon - ok good. Be sure to include your gel image when you get it - Dr. B 091812
 * __Week ____ 3 __**__: __

This week, I continued from Day 1 and completed the transformation of competent cells for plasmid prep of pNIC-Bsa4. Kept pellets from centrifugation in -20 degrees Celsius refrigerator. Completed first part of the restriction enzyme digest protocol. Froze samples in -20 degrees Celsius.

This week, I completed my target discovery and updated my wikispace target page. I finished PCR primer design for Primer Overlap Assembly PCR for my target, inositol-phosphate phosphatase for //S. aureus.// I completed primer dilution from stock solutions of primers. I made an order and sent tubes of diluted forward and reverse primers to MBB 1.426 for DNA sequencing. I finished Day 1 for transformation of competent cells for plasmid prep of pNIC-Bsa4 which involves growing up DH5alpha //E.Coli// bacteria with plasmid. I started analyzing DNA sequence protocol. Below is a link to the target page and the sequences for the oligonucleotide primers.
 * __<span style="font-family: Arial,Helvetica,sans-serif; font-size: 20px; line-height: 29px;">Week 2: __**

http://vdsstream.wikispaces.com/Target-+Inositol-phosphate+phosphatase+%28S.+aureus%29................................Brandon+N.

1 ATGACCGACAAAACCCTGCAGCAGATCGATAAACTCATTTGTTCTTGGCTCAAGC 55 2 TCATTTCCATAATCAGTTGAGGGATCACATTGTCGATTTGCTTGAGCCAAGAACAAATGA 60 3 CCCTCAACTGATTATGGAAATGACGACCGAAACGAAGCGTCACCGTTTCGACCTCGTTAC 60 4 GGAATTGCTGGAACTGCTGTTGGATCTGTTTGTCAACATTGGTAACGAGGTCGAAACGGT 60 5 ACAGCAGTTCCAGCAATTCCTCGCGACCCACTTTCCTGAGCACCAGCTGCTGGCGGAAGA 60 6 TCCAGAGGTGGTTGATTTCGTTGGTAATCATTTCATTAGATTTTTCTTCCGCCAGCAGCT 60 7 CGAAATCAACCACCTCTGGATCATGGACCCGATCGACGGTACGGCGAACCTGGTTAAACA 60 8 CTTCGTAGAAGTACGCCAGGATGATGCAGTAGTCTTCCTGTTGTTTAACCAGGTTCGCCG 60 9 CCTGGCGTACTTCTACGAAGGTAAACCTATGCTGTCTTACGTTTATGACTATCCTCACAA 60 10 ATGCACCCTCACCGCGGATAGCTTTATACAGTTTTTTGTGAGGATAGTCATAAACGTAAG 60 11 CGCGGTGAGGGTGCATTTTGCAACGGTATCAAAATGGAAGAACCGCCTTCTCTCAAACTG 60 12 GGTTCATAACCTGCGCGTTAAAAGAGATGATGGCGTCTTCCAGTTTGAGAGAAGGCGGTT 60 13 ACGCGCAGGTTATGAACCTCGACACTGTTCAGGACCTGTTCGATGCGTCCTTTTCTTATC 60 14 AACACGCATAGAGTCCAGACCGCACGCACCAACCAGGCGATAAGAAAAGGACGCATCGAA 60 15 GTCTGGACTCTATGCGTGTTGCTAAAGGTCAGTTCGGTGCCCACATCAACACCAATCCTA 60 16 GTTCCGCGAAGAGAAATTGCGCTGCGATGTCCCAAGGCTTAGGATTGGTGTTGATGTGGG 60 17 GCAATTTCTCTTCGCGGAACTCCTCAATCTCAAGATGACCACGCTGGACGGTAAAGCCAT 60 18 GCTTTGTTAGAAATAATGAACGGAGCGCCTTTCAGGTGATCGATGGCTTTACCGTCCAGC 60 19 CTCCGTTCATTATTTCTAACAAAGCGTGCCACGAAACCGTTCTGAAAATTCTCAATGCGA 60 20 GGAGAGTCACCCAGGCGGTATTTCTGGTAACCACCGTTCGCATTGAGAATTTTCAGAACG 60 21 CGCCTGGGTGACTCTCCGGGTTACGTTATGTCTAACATCGAATTCCGTCAGCTCACCCGT 60 22 TCTTCGACACGGCGAGCTTCACGTTCGTCAGATGGAGAATGGCCACGGGTGAGCTGACGG 60 23 GCTCGCCGTGTCGAAGAAGCCGGTGGTCAGCTGTTCGTTATTGGTGGTGAACTCCGTGTA 60 24 GGAACGTCGCCCAGGGCACGCGTCAGGTTCAGCACACCATTTACACGGAGTTCACCACCA 60 25 CCCTGGGCGACGTTCCGGGTCGTCCGATGATCAGCAACGAACCGGAAACCTGTCAAGTTC 60 26 TCGCATGCCAGGAGTACCAGATAGTCGCTGCTCTCAATCGGAACTTGACAGGTTTCCGGT 60 27 GGTACTCCTGGCATGCGACGGCATCTCTGATGTGTTCAACGAGCGTGATCTGTACCAACT 60 28 ATAATCCTCAACCGGGTAGTCGTTTGCGAAGGCTTCAACCAGTTGGTACAGATCACGCTC 60 29 GACTACCCGGTTGAGGATTATGCAGAACTGTCTCGTTTCATCTGCACCAAGGCGATCGAG 60 30 GCAGGAAACCGATCACAACAGAGACGTTGTCCGCAGAACCAGCCTCGATCGCCTTGGTGC 60 31 TGTTGTGATCGGTTTCCTGCGTCCGCCGCAAGACGTTTGGAAACTGATGAAGCACGAGTC 60 32 TTATTCTTCGTCGGTAACGTCAGAGTCCTCGTCATCAGACTCGTGCTTCATCAGTTTC 58

This week, I started target discovery which involves searching through databases of proteins of many different organisms. I'm having troubles on finding a organism/disease pair that has essential phosphatase enzymes.
 * __<span style="font-family: Arial,Helvetica,sans-serif; font-size: 20px; line-height: 29px;">Week 1: __**