Jason+P.

=**FALL 2013**=

== The top 5 compounds from libraries HF9PlatesPlates5_9.sdf and cb_306_3d.sdf. An inhibition assay was done on the surrogate target YopH. The results of the assay were varied, and there was no real pattern. The failure to show any patterns is most likely due to errors setting up the assay. Several of the samples were actually prepared incorrectly, so they had to be remade. It's possible that the discrepancy in incubation time at room temperature between the samples that were made correctly and the samples that had to be remade is responsible for some of the variation. Additionally, the samples were all put in the water bath before any of the substrate was added, so that may have influenced the results as well.
 * Week 13, 14. 15 18-Nov - 8-Dec **



An enzyme assay was done on FabG. The blue line measured absorbance at 340nm (NADPH) and the red 260nm (NADP+). The decrease in the blue line shows that the protein has some sort of activity going on. More runs will be necessary to check for consistency of the protein, and also to see how change in substrate affects the progression of the reaction.


 * Week 11 & 12 4-Nov - 17-Nov **

Screening against two libraries has been started, **HF9PlatesPlates5_9.sdf** and **HF9_180_Plates_lum3D_catnum.sdf**.
 * Library Screening:**

For HF9Plates..., all 400 compounds were run at an autoscale of 2 on one blade. For the HF9_180... library, compounds were divided up amoung 6 blades, with 2400 compounds per blade, for a total of 14400 compounds. The autoscale was set to 0.1 for this library so that it would run faster.

Future errors to avoid: make sure the file path is correct, don't accidentally change the conf file for a run.

//The gold.conf file for HF9PlatesPlates5_9.sdf.//

//The gold.conf file for HF9_180_Plates_lum3D_catnum.sdf//

Results from control docking were quite different from what I expected. Some of the negative controls performed better than the positive controls, and the scores of the positive controls were relatively low, given that they had good IC50 values based off previous studies.
 * Images of Control Docking Results: **

Something that should be noted is that the second substrate, NADPH, was not present in the control docking procedure. Some of the positive and negative controls appear to clash with the NADPH site, so it may be wise to rerun the entire control procedure with NADPH in the site (after docking the NADPH) to ensure that these compounds are binding in the correct sites. Even though previous papers have noted that NADPH is not completely necessary in the inhibition of FabG in //Plasmodium Falciparum//, it would be better to double check.

The listing of the pymol images is in the same order of the compounds in the chart below. Physio-chemical properties displayed in the chart below were taken from Zinc and PubChem.




 * Week 9 & 10 21-Oct - 3-Nov **

Good work! Continue with virtual screening of control ligands and protein expression. - Suman 11/5/13

The next step in virtual would be to start screening against my control ligands after consulting with a mentor/Dr.B. For wet lab, protein expression sometime this week.

Jason do you have any data for weeks 7 & 8? Please upload what data you do have. Thank you. -Max 10/21/13 Week 5 & 6 23-Sep - 6-Oct
 * Jason nice job on adding your data but try to include captions and an analyses for the data. Thank you. -Max 10/07/2013**

//DNA sequencing of miniprep product//
 * 2013.10.03**

Clone 1 (primer: pLIC-forward) code NNNNNNNNNNNNNNNNNNNTTANNNGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTAC CGAGAACCTGTACTTCCAATCCATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCTTATCGACGGTGGTCTGTCTCCGT AACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACC ACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCC TGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG GGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCC TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTT AACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTANGTGGCACTTTTCNGGGAAATGTGCGCGGAA CCCCTATTTGTTTATTTTTCTAAATACNNTCAAATATGTATCCGCTCATGAANTAATTCTTAGAAAAACTCATCGAGCAT CAANNGAAACTGCNATTTATTCNTATCNNNANNATCAATANCATATTTTTGAAAANCCGTTTCTGTAATGNANGGANNAA AACTCACCNANGCNNNTNCATNNGNNGGCNNGATNCTGGNATCNGNNNGNNNANNNCNANNNNNNNNATCNATNCNNCNN NTNANTTNCCNNNNNNNAAAATANNNNATNANNNNNNNANNNNCNNGNNNNNNNNACNNNNNNCNNNNNNNNANNNNNNN NNNNNNNNNNNNNNNNTTNNNNNNNNNNNNNANNNNNNNNNNNNNNNN code  Range 1: 103 to 137

Alignment statistics for match #1 ||~ Score  code Query 1    ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  35 ||||||||||||||||||||||||||||||||||| Sbjct 103  ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  137
 * ~ Expect ||~ Identities ||~ Gaps ||~ Strand ||
 * 65.8 bits(35) || 2e-14 || 35/35(100%) || 0/35(0%) || Plus/Plus ||

code

Clone 2 (primer: pLIC-forward) code NNNNNNNNNNNNNNCTTNNGNNGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA GAACCTGTACTTCCAATCCATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCTTATCGACGGTGGTCTGTCTCCGTAAC AGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACT GAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTT GGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGT AGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCC TTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGT TCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA TAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAA CCCTATCTCGGTCTATTCTTTTGATTTATAAGGNATTTTGCCNATTTCGGCCTATTGGNTAAAAATGANCTGATTTAACA AAANTTAACGCGAATTTTNANNAAATATTAANNNTTACANTTNNNGGNACTTTTNNNNAATGNNGNNNNGNNCCCTNNTT NNTTNNTTTTTNNAAATNNNTTCAANNNNGNNNCNNCNNCANGNNNNNNNNTNNAAAANNNNNNNNNNNGCNTNNANNNA NCNGNNNTNNTNNNNNNNNTNNNNNNNNNNNNNTTTNANAANNNNTNNNNNNNNNNNANNNNNNNCNNNNNNNNNNNNNN NNNGNNNNNNNGNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNN code  Range 1: 100 to 134

Alignment statistics for match #1 ||~ Score  code Query 1    ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  35 ||||||||||||||||||||||||||||||||||| Sbjct 100  ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  134 code
 * ~ Expect ||~ Identities ||~ Gaps ||~ Strand ||
 * 65.8 bits(35) || 2e-14 || 35/35(100%) || 0/35(0%) || Plus/Plus ||

Clone 3 (primer: pLIC-forward) code NNNNNNNNNNNNNNNTTNAGNNGAGNNNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA GAACCTGTACTTCCAATCCATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCTTATCGACGGTGGTCTGTCTCCGTAAC AGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACT GAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTT GGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGT AGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCC TTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGT TCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA TAGACGGNTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCNA CCCTATCTCGGTCTATTCTTTTGATTTATANNNNTTTGCCNATTTNNNCTATTGGTTAAAAAATGAGCTGANTTANAAAN TTANGCGANTTTNNCAAANNNTANNNCTTANANTNNGNGNNNTTTNGGNAANNNNNNNNGNNCCCNANTNNNTTNNTTTT CNNNNNCNTNNNNNNGNNNCCGCTCATGNANNNNNNTANNNNANNNNNNCNNNNCNTNNNNGANNGNNNNNNNTTNNNTN NNNTNNNNNNNNNNNNNTNNAANNNNNNNNNNNNNNGNNNNNNNNNNNNNNGNNNNNNCCNNNGNNNNNANN code  Range 1: 100 to 134

Alignment statistics for match #1 ||~ Score  code Query 1    ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  35 ||||||||||||||||||||||||||||||||||| Sbjct 100  ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCT  134
 * ~ Expect ||~ Identities ||~ Gaps ||~ Strand ||
 * 65.8 bits(35) || 1e-14 || 35/35(100%) || 0/35(0%) || Plus/Plus ||

code Will have to retry cloning.

// Miniprep //
 * 2013.10.02 **

The resulting plasmids were sent off to DNA sequencing to determine whether or not the proper sequence was accepted by the plasmid

Clone 1

Clone 2 Clone 3

Jason - resutls of your cloning? Also do you have some virtual work yet? - Dr. B 100113 //Cohesive End Generation// //-nothing to show yet-//
 * Week 3 & 4 9-Sep - 22-Sep **
 * 2013.09.18 **

//Vector Digest (pNIC-Bsa4)// Two samples were made, one containing three tubes of digested pNIC, and one with five tubes of digested pNIC. The results of the sample containing three volumes of digested pNIC is on the left, and the results of the one with five volumes is on the right. For cloning, the one on the right will be used first. -Bands on the gels to make sure they were the right lengths (5.3kB and 1.9kB)
 * 2013.09.13 **

//PCR Squared// Another PCR squared after the previous one had poor yields. This time, 400uL of PCR product was used compared to 200uL time, and the extracted DNA was collected into only one column in 40uL of elution buffer. Each lane in the pictures below contained 50uL PCR product with 16.667uL of dye. The leftmost lanes on the two gels is the 1kB ladder, which helps make sure that the correct gene sequence was amplified (903bp long).
 * 2013.09.10 **

//Gel Extraction// This extraction was much more successful than the previous attempt. This product will be viable for cloning.

//Gel Extraction// The first gel extraction resulted in a poor final concentration, mainly due to a low amount of PCR squared and the use of two final columns instead of one. -Column 1 -Column 2
 * 2013.09.08 **

Jason - good. Don't put link to WORD docs here. I just want embedded images. Thanks. Dr. B 090913 //03.09.2013// PymolRefresher
 * Week 1, 2:** ** 26.08.2013 - 06.09.2013 **

Secondary PCR:
 * Lane 1: 100 bp ladder
 * Lane 2: 1kb ladder
 * Lane 3: Amplified full length gene

//05.09.2013// PCR Squared:
 * Lane 1: 1kb ladder
 * Lane 2: 100 bp ladder
 * Lane 3: Full length gene + primer dimer

=**SUMMER 2013**=


 * Week 5: **

Forward: TACTTCCAATCCATGAAAAAAACCTTCCTGATCGCG Reverse: ­TATCCACCTTTACTGTTAACGGGTCTTTGCAGAAGA Full Document:
 * Primers for HpPAP_FOR, HpPAP_REV (07.01.2013) **
 * || ** Forward **  ||  ** Reverse **  ||
 * || TACTTCCAATCCATGAAAAAAACCTTCCTGATCGCG || TATCCACCTTTACTGTTAACGGGTCTTTGCAGAAGA ||
 * [Mg2+](mM) || Tm (oC) || Tm (oC) ||
 * 0.0 || 63.1 || 63.2 ||
 * 1.5 || 70.7 || 70.7 ||
 * 2.0 || 71.2 || 71.2 ||
 * 4.0 || 72.1 || 72.2 ||
 * 6.0 || 72.6 || 72.6 ||

=Week 4:=


 * Analyzing a DNA sequence (pGBR22)** -- work only:


 * Nanodrops of Purified Protein (FtHap)**


 * Elution 1:**


 * Elution 2:**

**Week 3:**
 Lane 1: 100 bp DNA ladder Lane 2: Sample A [1:1000 template dilution, 1uL] Lane 3: Sample B [1:1000 template dilution, 10uL] Lane 4: Sample C [1:100 template dilution, 10uL] Lane 5: Sample D [control]
 * PCR pGBR22 (06.18.2013) **

**PCR pmCherry (06.18.2013)**
Lane 1: 100 bp ladder Lane 2: VDSR1/2, 1:1000 dilution Lane 2: VDSR1/2, 1:100 dilution Lane 2: VDSR1/2, 1:10 dilution Lane 2: VDSR1/2, no DNA Lane 6: 100 bp ladder Lane 7: M13 Forward/Reverse, 1:1000 dilution Lane 8: M13 Forward/Reverse, 1:100 dilution Lane 9: M13 Forward/Reverse, 1:10 dilution Lane 10: M13 Forward/Reverse, no DNA


 * pGBR22 Sequence Verification**

=Week 2:=

**pGBR22 Colonies: **
== == == = =

** pGBR22 Nanodrop Spectrophotometer Results: **
Trial 1: == Trial 2: == The average concentration of the sample was 45.25 ng/uL.

**pGBR22 DNA Sequencing Results:**
code NNNNNNNNNNNNNGGGCGATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGG TGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTAC ATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCA GTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATT GTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCG TATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTC CCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCG ACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCA TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTANTTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAANCTGT CNTGCCAGCTGCATTAATGNATCGNNCAACNNNNGGGGNANNNNNGTTTGCGTATTGGGNNGCTCTTNCGNNNCNNNNNN NACTGACTCNCNNNNNNNNNNNNNNGNNGCGGNNNNNNNNNNNNNNNNNNNNNNNANNNNNANNNNNNNNNCNNNNNNAA NCNGGGNANNNNCNNNNNANNNNNNNNNNAANNNNNNNNNNNNNNNNNCNNNNAAAGNCCCNNNNNNNNNNN

code

Lane 1-5: Vicki's Lane 6: 1 kb DNA ladder Lane 7: Sample A [1:1000 template dilution, 1uL] Lane 8: Sample B [ 1:1000 template dilution, 10uL] Lane 9: Sample C [1:100 template dilution, 10uL] Lane 10: Sample D [control]
 * PCR pGBR22 (06.14.2013)**

=Week 1= = =

Run 1:
== Run 2: == = = The results from the two trials provided similar data for the purity of the sample in comparison to protein (260/280) and in comparison with to other contaminants (260/230). The average purity in relation to protein was 1.855, and the average purity in relation to other contaminants was 2.315. Although purity measures were similar, there was a rather large difference between the two measures of concentration, about a 330 ng/uL discrepancy. This could possibly indicate that the plasmid concentration was not consistent throughout the sample. = =

**pGFP DNA Sequencing Results:**
code NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNTTTCTTNNNTTACNNNGCNNTTNCNGAGCATTANTNNTTTNNNANCNN GANCCNTTANNCANCNTGNAAACTCTTGTTCNACATNGTCCNNANNNNACTCCCATTNNGGTGCTTGTGGGACGTTTAAC NNANCATGTCCNNNTNNNTAANANTTCNTTNNNNNTANGTACACTTTCTCTTTTNNNNNNTCCACCCCTTGCTTGTTTTN NNTCCCCANATGAAATTCCGAATGAANNNNGGTTGNATTCACCNATTTTTGCNNNNNANCNNGNTCTANNGCNGGATTAA CCCCATTTTNCATGTGTCCGATCCATTTACTTACNTTTGAAGGACATTTAANCGTTCNTTTTCNNAATTACANTGNAAAA AGGAATACCCCCNTGNATNCNATATCNATATGGGGNAAANAANGTTCAAAAATNTNNAANTAAAACTTTCATTTGGNAAC TTTATTAANNANGAANAANAATGGAGGTCCTACNTTTNNTTCANATAAANTATATNAAAATGNACNAANANGTCAANATC TNATNCTACCCTTTCTTTCCATTNCNNAATTTTGGCAATNCCAACACTTCNTTGTATCCTTATGANTCNGATAANACNNT GTTATTCCCNNTACTNATATNAATTCCNATNTCNTTAGNNCCGTTNNATTCTAACGANTTCCTCTCCAANCNNNATGNG code

**PCR pGBR22 (06.07.2013)**
Lane 1: 1 kb DNA ladder Lane 2: Sample A [1:1000 template dilution, 1uL] Lane 3: Sample B [1:1000 template dilution, 10uL] Lane 4: Sample C [1:100 template dilution, 10uL] Lane 5: Sample D [control]


 * Jason - good job. Segment your stuff by Week 1, Week 2, etc.. put weeks in reverse chronological order. -- Dr. B **