Research+Page-+Krishna

Krishna's Research

=**__Fall 2011 RESEARCH__**=

=Week 14= I nanodropped the VDS staff sample of the protein.

I ran another control enzyme assay with the new protein sample. The Sal CA was not active so I could not continue with research ): = = I cleaned out my box and completed the one hour clean up. = = =Week 13= I ran a control enzyme assay. However, the assay did not show significant differences at various enzyme concentrations. This means that the enzyme might not be active.

==

Started using Dr. B's expressed protein. Concentrated it and added 20% glycerol. I ran out of time to nanodrop so I will do that next week.

=Week 12= Only did virtual screening stuff this week for lab. Ran and completed CBkinUT library (4000) ligands after third try. Ran second run and got a best ranking list with 55 compounds. Ordered two compounds from it

I added 20% glycerol to my sample.
 * ID || Mol Weight || Mol Formula || Mol Name || cLogP || RB || hDon || hAcc ||
 * 7541339 || 444 || C21 H22 Cl N5 O2 S || 2-({5-[4-(acetylamino)phenyl]-4-methyl-4H-1,2,4-triazol-3-yl}thio)-N-(4-chlorophenyl)butanamide || 3.311 || 6 || 2 || 5 ||
 * 7551780 || 439.5 || C21 H21 N5 O4 S || methyl 4-{[({5-[4-(acetylamino)phenyl]-4-methyl-4H-1,2,4-triazol-3-yl}thio)acetyl]amino}benzoate || 1.941 || 7 || 2 || 7 ||

I will run a control assay on Monday. =Week 11= I finished drying my page gel but could not take a photo of it because I could not find the USB cable. I will upload it sometime next week. I concentrated my protein after FPLC

I ran three virtual screens on CBkinUT library. The first two failed but everything was corrected an as of Friday 11-11-11 the first virtual screen seems to be running properly. I will run the second run on Monday. =Week 10= I ran protein characterization on Monday. Let it wash for a few days until Thursday. Took a preliminary photo before drying as seen below.



Lane 1: Skip Lane 2: Protein Ladder (Fermentas Page Ruler Prestained Protein Ladder) Lane 3: Sample 0 (Cell Lysate Before Induction) Lane 4: Sample 1 (Cell Lysate After Induction) Lane 5: Sample 2 (Soluble Fraction) Lane 6: Sample 3 (Flow Through) Lane 7: Sample 4 (Wash) Lane 8: Sample 5 (Elution #1) Lane 9: Sample 6 (Elution #2) Lane 10: Skip

I concentrated the protein in Elution #1 before FPLC.

I ran FPLC as well on Thursday.

=__Week 9__= Prepared SDS page gel to run next week. Gel leaked the first first time so I had to redo it. Began running virtual screening for ChemBridge library but the Best Ranking List file showed in the wrong folder so I had to run it again. Ran ChemBridge library first run again on Friday.

Nanodrop of Elution #1 and #2 from protein expression. - **that's a good yield! - Dr. B**

=__Week 8__= Virtual screening refresher failed again. The job has been run too many times so the files all have new names and its rather confusing. I moved on to virtual screening of my protein but this time I will make sure my file names are in order and organized. I attempted to run the first virtual screen but I could not get gold to run on two different processors to search 400 compound in the library. I will try again with the new tips next week.

I finished day 4 of expression and saved the supernanant.

While prepping for protein purification i slipped and dropped my .3 OD expression samples in the sink and lost it! I had to dispose of that one as barely any remained in the tube to be used for protein purification. I can only use the .6 OD expression sample to continue with my research. I ran protein purification but did not have time to nanodrop the elutions. The elutions are in the 4 degree fridge and I will nanodrop them next week as well as run protein characterization hopefully. = = = = =__Week 7__= I had to rerun the second job of virtual drug screening refresher because of a fatal error. I created a pymol image for the pymol contest and finished the research report. I was not able to get much lab work done this week because of tests and the research assignments. I will run protein purification.

=__Week 6__=

I ran midi prep on the two 80 ml samples of DH5alpha cells will pNIC and my accepting vector. The samples were combined in the midi prep. The nanodrop of the concentration is below. I did not have a chance to send these results to sequencing on Wednesday but I did on Friday so the results should come in sometime next week.

On Wednesday I made LB media and prepped for expression for Day 1 on wednesday. I grew transformation plates from the plasmid that went through midi prep. I transformed it into BL21 bacteria cells.

On Thursday I transferred two colonies to the shaker in 50 mL.

On Friday I ran the 8 to 10 hour long process of expression and will finish Day 4 of the protocol next week. I also sent in the plasmid after midi prep to DNA sequencing.

Krishna - good deal. I am interested to see how it turns out. -- Dr. B

=__**Week 5**__= Successfully inserted Sal CA gene into pNIC-BSA 4!!!!!! Krishna, yeah! Good job on getting it to work. - Dr. B

DNA sequencing Forward Primer Results >filtered DNA sequence consisting of 1213 bases. NNNNNNNNNNNNNNTTTANANGAGANNTACATATGCACCATCATCATCATCATTCTTCTG GTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGAAAGACATCGACACCCTGA TCTCTAACAACGCGCTCTGGTCTAAGATGCTGGTTGAAGAAGACCCGGGCTTCTTCGAGA AACTGGCGCAGGCGCAGAAACCGCGTTTCCTGTGGATCGGTTGCTCTGACTCTCGTGTTC CGGCGGAACGTCTGACTGGTCTGGAACCGGGTGAACTGTTCGTTCACCGTAATGTTGCGA ACCTGGTTATCCACACCGACCTGAACTGCCTGTCTGTTGTTCAGTACGCAGTTGATGTTC TGGAAGTTGAACACATCATCATCTGCGGTCACTCTGGTTGCGGTGGTATCAAAGCGGCGG TTGAAAACCCGGAACTGGGCCTGATTAACAACTGGCTGCTGCACATCCGTGACATCTGGC TGAAACACTCTTCTCTGCTGGGTAAAATGCCGGAAGAACAGCGTCTGGACGCGCTGTACG AGCTGAACGTAATGGAGCAGGTTTACAACCTGGGCCACTCTACCATCATGCAGTCTGCGT

TGCGTGACCTGGACGTTACTGCGACCAATCGTGAAACCCTGGAAAACGGTTACCACAAAG GTATCTCTGCGCTGTCTCTGAAATACATCCCGCACCAGTAACAGTAAAGGTGGATACGGA TCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCA CTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGA NCAATAACTAGCATAACCCCNTGGGGCCTCTNAACGGGTCTTGAGGGGTTTTTTGCTGAA AGGAGGAACTATATCCGGANTGNNGAATGGGNNNNGCCCTGTAGCGGCGCATTAAGCGCG GCGGNNGNNGNGGNNACNCNCAGCNNGACCNCNNCACTTGCCAGCGCCNNNNCNNCCNNC TCTTTNNCTTNNNTNNNNNNTTNNNCGNCNNNNNNCGNNTTNCCNNNANNNNNNAANTCG GGGGNNNNNTNNNNCNANTNNNNGNTTNNNNNNNNNNNCNAANNNNGNNNNGNNNNNNNN NNNNNGGGNNNNN



DNA sequencing Reverse Primer Results >filtered DNA sequence consisting of 1729 bases. ATGAAAGACATCGACACCCTGATCTCTAACAACGCGCTCTGGTCTAAGATGCTGGTTGAA GAAGACCCGGGCTTCTTCGAGAAACTGGCGCAGGCGCAGAAACCGCGTTTCCTGTGGATC GGTTGCTCTGACTCTCGTGTTCCGGCGGAACGTCTGACTGGTCTGGAACCGGGTGAACTG TTCGTTCACCGTAATGTTGCGAACCTGGTTATCCACACCGACCTGAACTGCCTGTCTGTT GTTCAGTACGCAGTTGATGTTCTGGAAGTTGAACACATCATCATCTGCGGTCACTCTGGT TGCGGTGGTATCAAAGCGGCGGTTGAAAACCCGGAACTGGGCCTGATTAACAACTGGCTG CTGCACATCCGTGACATCTGGCTGAAACACTCTTCTCTGCTGGGTAAAATGCCGGAAGAA CAGCGTCTGGACGCGCTGTACGAGCTGAACGTAATGGAGCAGGTTTACAACCTGGGCCAC NNNNNNNNNNNNNNNNNNNNGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTC GACGGAGCTCGAATTCGGATCCGTATCCACCTTTACTGTTACTGGTGCGGGATGTATTTC AGAGACAGCGCAGAGATACCTTTGTGGTAACCGTTTTCCAGGGTTTCACGATTGGTCGCA GTAACGTCCAGGTCACGCAGCAGACCGTCGTTGATAGAGTACGCCCAACCGTGGATGGTA ACGTTCTGACCACGTTTCCACGCAGACTGCATGATGGTAGAGTGGCCCAGGTTGTAAACC TGCTCCATTACGTTCAGCTCGTACAGCGCGTCCAGACGCTGTTCTTCCGGCATTTTACCC AGCAGAGAAGAGTGTTTCAGCCAGATGTCACGGATGTGCAGCAGCCAGTTGTTAATCAGG CCCAGTTCCGGGTTTTCAACCGCCGCTTTGATACCACCGCAACCAGAGTGACCGCAGATG ATGATGTGTTCAACTTCCAGAACATCAACTGCGTACTGAACAACAGACAGGCAGTTCAGG TCGGTGTGGATAACCAGGTTCGCAACATTACGGTGAACGAACAGTTCACCCGGTTCCAGA CCAGTCAGACGTTCCGCCGGAACACGAGAGTCAGAGCAACCGATCCACAGGAAACGCGGT TTCTGCGCCTGCGCCAGTTTCTCGAAGAAGCCCGGGTCTTCTTCAACCAGCATCTTAGAC CAGAGCGCGTTGTTAGAGATCAGGGTGTCGATGTCTTTCATGGATTGGAAGTACAGGTTC TCGGTACCCAGATCTACACCAGAAGAATGATGATGATGATGGTGCATATGTATATCTCCT TCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGNTATCCGCTCNCAATNNCCCTA TAGTGAGTCGTANTNATTTCGCGGNATCGAGATCTCGATCCNCTACNNCGGACGCATCGT GGCCGGGCNTCACCGGCGCCACAGGTGNNNNTTGCTGGCGNNTNNANNGNCGACNTCANC GANNGGGGNAGATCGGGCNTCGCNNCTTNCNGGNNNATGANNNCTNGNTTNNNCNNNGGN NATNGNNGNNNNGNCCNNNNCNNNNNNNNNNNNNNNNNNNNATNTCNNNNNNNNNNNCTN TNCNNNNNNNNNNNNGGNNNNTNANNGGNCNNNNNCNNNNNNNNNNGNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNN



I ran miniprep on the transformation pellet and have stored the sample in the fridge. I grew up more bacteria in the shaker in two tubes of 80mL LB. I spun them down and stored the pellets in the freezer.

I ran virtual screening refresher last week but the second run did not work properly. I ran the second run again on Thursday 9/29/11 so I will complete the analysis portion on Monday.

__**Week 4**__
I ran PCR clean up on my 4 PCR^2 samples and combined them into one tube.



I ran annealing and transformation again on Thursday. Hopefully I will see results and send it off to sequencing this weekend or the beginning of next week.

=**__Week 3__**= I ran the restriction enzyme digest for pNIC-Bsa4 and ran it on a gel along with my 4 PCR^2 tubes. The pNIC-Bsa4 did not cut properly as there is only one band and it matches that of the uncut pNIC-Bsa4 plasmid. Lane 1: Skip Lane 2: 100 bp Ladder Lane 3: Sample A PCR2 Lane 4: Sample B PCR2 Lane 5: Sample C PCR2 Lane 6: Sample D PCR2 Lane 7: Skip Lane 8: PNIC Bsa-4 uncut plasmid Lane 9: PNIC Bsa-4 cut plasmid

Joey had made extra restriction enzyme digest of pNIC-Bsa4 and I ran a gel to confirm if his two samples had cut properly. The gels in fact did cut properly as there are two distinct bands in each lane. Lane 1: Skip Lane 2: 100 bp ladder Lane 3: “My Box” pNIC-Bsa4 Accepting Vector Lane 4: “My Personal” pNIC-Bsa4 Accepting Vector

I completed the pyMol refresher lab that involved studying four protein molecules (2H2Q, 3CL9, 1U72, and 3HBB). The file for pyMol refresher is attached below.

=__**Week 2**__= Ran annealing and transformation protocol into PNICBsa4 but there was no growth on the plates. It was said that the restriction enzyme digest did not work properly and the PNICBsa4 was not cut properly.

I ran PCR^2 after this to amplify my gene concentration. The two largest concentrations are show in the nano drop below





=__**Week 1**__=



Lane 1: Skip Lane 2: 100 bp Ladder Lane 3: Sample A Secondary PCR (74.3 ng/ul) Lane 4: Sample B Secondary PCR (134.2 ng/ul)

Sample B was used for the next step of research as it had the highest concentration.

=__**SUMMER RESEARCH**__= = = __**Transformation Efficiency Lab (6/8/11)**__





__**RE Digest Lab (6/10/11)**__ = Lane 2: 1kb DNA Ladder Lane 5: EcoR I Lane7: Pvu II Lane 8: EcoR I + Pvu II Lane 9: Uncut Plasmid

__**PCR for pGBR22 (6/17/11)**__

Ruoyi Pu Lane 2: Ladder Lane 3: A 0.3 ng Lane 4: B 3 ng Lane 5: C 30 ng Lane 6: D 0 ng (Control)

Krishna Patel Lane 7: Ladder Lane 8: A 0.3 ng Lane 9: B 3 ng Lane 10: C 30 ng Lane 11: D 0 ng (Control)

__**PCR for pGFP (6/20/11)**__ Lane 2: 100 bp Ladder Lane 11: 100 bp Ladder

Primers: VDS 1 and 2 Forward and Reverse Lane 3: A 1.58 ng Lane 4: B 0.158 ng Lane 5: C 0.0158 ng Lane 6: D 0 ng (Control)

Primers: M13 Forward and Reverse Lane 7: E 1.58 ng Lane 8: F 0.158 ng Lane 9: G 0.0158 ng Lane 10: H 0 ng (Control)

Lane 2: 100 bp Ladder

Primers: VDS 1 and 2 Forward and Reverse Lane 3: A 1.58 ng Lane 4: B 0.158 ng Lane 5: C 0.0158 ng Lane 6: D 0 ng (Control)

Primers: M13 Forward and Reverse Lane 7: E 1.58 ng Lane 8: F 0.158 ng Lane 9: G 0.0158 ng Lane 10: H 0 ng (Control)

__**PCR protocol for pLIC sequencing vectors of pNIC-Bsa4**__



Ruoyi Pu Lane 2: Ladder Lane 3: A Lane 4: B Lane 5: C Lane 6: D

Krishna Patel Lane 7: Ladder Lane 8: A 3.14 ng Lane 9: B 0.314 ng Lane 10: C 0.0314 ng Lane 11: D 0 ng (Control)

__**PCR for pNIC-Bsa4 cloning (Zhang Protein)**__

Lane 2: Ladder Lane 3: A 0 mM MgCl 2 Lane 4: B 1.5 mM MgCl 2 Lane 5: C 2 mM MgCl 2 Lane 6: D 4 mM MgCl 2 Lane 7: E 6 mM MgCl 2 Lane 8: F Control

__**PCR_PrimerOverlap (Carbonic Anhydrase for Salmonella**__

Lane 2: Ladder Lane 3: Primary PCR (Primer Mix) Lane 4: Secondary PCR (DNA Strand for Forward and Reverse Primer) Lane 5: Ladder

Lane 2: Ladder Lane 3: Sample A Lane 4: Sample B Lane 5: Sample C Lane 6: Sample D