Priya+P.

=** Week 15 & 16 (Dec 2 - Dec 8) **=

Cation Enchange
Cation Exchange was completed. Nanodrop results show that it was unsuccessful. A protein gel will be run to verify these results.

Elution 4: 0% Buffer A: 100% Buffer B (1M NaCL, 20mM HEPES)







Elution 3: 25% Buffer A: 75% Buffer B









Elution 2: 50% Buffer A: 50% Buffer B



Elution 1: 75% Buffer A: 25% Buffer B





The goal concentration was to get as close to .1 mg/ml as possible. The -0.00 concentration indicates that all the contaminants were positively charged, and stuck to the negatively charged resin. This is a highly unlikely occurrence so the rest of the procedure was completed as a safety precaution.

=** Week 13 & 14 (Nov 18 - Dec 1) **=

The enzyme Assay was unsuccessful. This could be due to contaminants in the sample. Cation exchange will be used to further purify the sample since Nickel column purification was not effective. = =
 * Enzyme Assay **
 * Protein Expression **

Small cultures went in at 10:30AM, after 8 hours (6:30PM) no growth was apparent. The cultures were left in the shaking incubator over night to see if they needed more time to grow, or if the bacterial colonies were dead. At 7:07, small culture tubes were made for the second plate of bacterial colonies with the RpFabG insert to determine if the plate was still viable. All samples grew up overnight, expression protocol was continued. Two 500mL flasks of LB were used. Flask A failed to grow while Flask B was successful. Sample B was sonicated then sun down, sterile filtered, and purified with the Nickel column. A protein gel was run to determine the purity. Elution 1 had a yield of 17.31mg in 2mL of buffer and Elution 2 had a yield of 11.2mg in 4mL of buffer. Elutions were concentrated into 1 mL and then .5mL was stored with glycerol and .5mL was snap frozen.

=** Week 11 & 12 (Nov 4 - Nov 17) **=

Great work Priya, nice assays. -UM
 * Enzyme Assays **

Conditions in each of the enzyme assays (1-6) had different conditions. Enzyme assay 1 (Green) began at the same conditions as the assays conducted in figure 64, but was unsuccessful. Since the assay was unsuccessful, calculations were adjusted to account for more enzyme. In assay 2 (Blue) 30 uL of enzyme was used at the start, enzyme was added multiple times in 5 uL increments. Enzyme assay 3 (purple) was adjusted to being with 50 uL of enzyme. Assay 4 (orange) Started with 70 uL of enzyme and more was added multiple times in 10 uL increments. Assay 5 (pink) started with 100uL of enzyme and additional enzyme (50 uL) was added once. Enzyme assay 6 (red) had enzyme added in 50 uL increments for a total of 500 uL. Assay 1-4 were with sample stored in the -80C freezer while assay 5r and 6 were using sample stored with glycerol. All enzyme assays were unsuccessful. This could be attributed to having a contaminated sample or samples with low concentrations. There is a possibility that the enzyme can not be stored for long periods of time and still be functional.





Enzyme assay 1 (Orange) began at the same conditions as the assays conducted in figure 64, but enzyme was added multiple times in 5uL increments. In assay 2 (Pink) 20 uL of enzyme was used at the start. Enzyme assay 3 (green) had the same starting conditions as assay 2 but enzyme (5uL) was added one additional time. Assay 4 (blue) Tris buffer was used instead of HEPES since the assays were not successful. This assay showed that NADPH was being oxidized by the enzyme (RpFabG) since absorbance was decreasing.
 * Enzyme Assays**




 * Virtual Screening**

The top 5 ligands from the CB_306 library with a logP below 3 were ordered.



=** Week 9 & 10 (Oct 21 - 3- Nov) **=

Great work! It would be good to include the results table from your ligand virtual screening run, too! -Suman 11/5/13
 * Virtual Screening**









The orange line is the end of the third assay. The green line is NADPH, buffer and water, the blue is Buffer, water, NADPH, and enzyme. The red line is enzyme,ACC, NADPH, water, and buffer. These results show that NADPH is the smaller hill at about 340nm. The next steps will be to attempt to replicate this data and then to vary the amount of NADPH while keeping the amount of AAC constant. Then vary the amount of AAC while keeping the amount of NADPH constant in several enzyme assays.
 * Enzyme Assay**

The absorbance of NADPH decreased as the reaction progressed. NADPH was oxidized to NADP. The graph indicated the assay worked. = =

Dilutions for NADPH and AAC were calculated and prepared. Enzyme assay calculations were completed and three assays were conducted. Five negative controls and 10 positive controls were obtained using PDB. The SDF files were concatenated into a control library. The library was then lig-prepped using ICM since Maestro was still down and docked using GOLD. The control ligand docking was analyzed and confirmed as accurate. Script files were made to run screening jobs on the Hit Finder library, NIH Clinical, Cb 306, and In House libraries.
 * Virtual Screening**



The pdb structure of RpFabG was obtained (3F9I). No ligands were docked, so NADPH was extracted from FabG from another organism. An attempt to ligand prep NADPH was made but ICM and Maestro were down. After numerous attempts at different times on different days, NADPH was prepared using ICM since Maestro was still down. NADPH was then docked multiple times using ICM with each attempt resulting in various errors or a low docking score. GOLD was then used to dock NADPH into the active site, generating 30 poses.

= =

=** Week 7 & 8 (Oct 7 - Oct 20) **= Good captions and brief analysis of your data. Where are you with virtual work? Thank you. -Max 10/21/13 RpFabG Sonication, Spin Down, Purification, Characterization, Concentration, and Storage **

= =



The two samples saved from the previous week were sonicated, spun down, filtered, and then purified. The concentrations of these elutions are relatively high when compared to past elution samples for RpFabG. This can be attributed to using the custom made HEPES buffer with a high concentration of imidazole instead of the elution buffer. A characterization protein gel was run containing elution 1 and 2 samples. The purified samples were stored for 2 days.The samples were then concentrated to about 2mL, split into two 1mL samples that were stored by snap freezing with liquid nitrogen and glycerol respectively. There is still contamination in sample, but FPLC was skipped this time since all previous attempts failed.





==

=** Week 5 & 6 (Sept 23 - Oct 6) **= Good Captions but try to add an analysis of your data. Thank you. -Max 10/07/2013 10/1/13 Virtual Screening
 * Positive and Negative controls were found for target. Since NADPH was not docked in the original crystal structure, similar structures of FabG were used to dock NADPH and define the active site after completing the ligand prep protocol. **

RpFabG Expression
 * RpFabG was expressed using the new expression protocol, Option A. The sample was stored to be sonicated.**

9/27-28/13 Protein Expression** RpFabG sample that was stored after sonication was spun down, purified using the nickel column, and then purified through FPLC. FPLC result show a minimal amount of protein, but not enough to be purified. RpFabG will be expressed again and samples will be purified in the room temperature FPLC machine using a nickel column.

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= =

= =

=** Week 3 & 4 (Sept 9 - Sept 22) **=
 * 9/16/13 **
 * Priya - Good work on purifications. what does your gel look like for these? There doesn't seem to be much of a peak at 280nm on Nanodrop - more of a shoulder of the 260nm peak. - Dr. B 100113 **

TEV was expressed, and spun down. TEV will be used to purify RpFabG since FPLC combine with the Nickel column is not working.

Samples saved from summer (samples 2 a&b) were sonicated, spun down, and purified. The results of the new experimental purification method used instead of FPLC are indicated in the nanodrop measurements. Another round of RpFabG expression was also started, grown up over night, spun down and stored for later usage. The pellet weights were 1.13 g and 1.67 g respectively.

Protein characterization was completed on 9/10/13. The protein sample was concentrated down to 1 mL and run through the FPLC machine. Another round of protein purification via FPLC was completed on 9/10/13. The FPLC machine on both these two rounds and the previous round skipped collection tubes and only half filled others. The graphs also indicated that there was no protein in the sample. RpFabG is 700bp ~ 300AA long, so it should have shown up towards the end of the graph. Since this method has failed three times, another purification method will be used. The protein will be purified using a nickel column multiple times and the use of protein TEV will also be involved.



=Week 1 & 2 (of Aug. 28- Sept 6)= Priya - good work. Hopefully the next FPLC will be good. Dr. B 090913

FPLC
The graph indicates that the custom buffer did not enter the purifying column until the brown line peaked. The brown line indicates conductivity which would increase when a significant concentration of salt has entered the system. The custom buffer has a NaCL concentration of 500mM. The protein Rp Fab G should be at a peak in the solid blue line, since there is no peak, but instead a drop where the custom buffer entered the system, it is likely that the peak is hidden. It should be around the red fraction number 24- 50. The protein is 700bp wich translates to 300 AA. FPLC will be re run with the correct buffer already running through the column with the remaining .8mL of concentrated elution 1 Rp Fab G protein sample.

Spinning Down and Concentrating Samples




After 12 hours of storage, Sample 1 and 2 of Elutions 1 had precipitated, so the samples were combined, spun down, nano dropped, concentrated to 1 mL, and then nano dropped again to get concentration readings. Elutions 2 were combined and concentrated together to .5 mL and then combined with .3mL of concentrated Elution 1. The .8mL sample was run through the FPLC machine.

Protein Purification


== Protein expression, purification, and FPLC were complete this week. Elution 1 of the protein had precipitated after 12 hours of storage in the 4C fridge. =Fall 2013=

= = =Week 8=

Protein Characterization
Protein characterization was started from OEV's positive clones. Pellets were spun down, re-suspended, and stored for long term use in the -80 C freezer.

Mini Prep




The samples in wells 1-3 were loaded, but the positive and negative cables were connected incorrectly so that the sample ran up the gel instead of down. The cables were fixed and then wells 5-7 were loaded when the original samples were thought to have reached the wells again. Wells 5-7 served as a back up just in case the DNA had already run off the gel from lanes 1-3. =

= =Week 7= Cloning was attempted twice, the second attempt being somewhat successful with one colony present after one 24 hr period. The one colony was then used to continue on with the master plate protocol.

PCR Clean Up




RE Digest
==

=Week 6= Priya - include your PCR results of squared - DR. B 071713 7/13/13 Cloning plates were checked. No colonies were apparent.

7/12/13

PCR Clean Up
Since the previous cut plasmid had a concentration that was too low, 4 new samples (50uL each) were made and the combined into one 200uL sample in PCR clean up to increase the concentration.



DNA Sequencing Results for PNiC-BSa4
NNNNNNNNGNNNACTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTG GTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATAT ACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATT AAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAA ACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTAT CAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTC GAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAG GGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGT AACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACAT AAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAAC CTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCA AAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAAT CCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAA AAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGA CGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAA AAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGA CAGCTGGAAAACGCTGGNCNNCGTCTTTNAAGACAGCGACAANTCGATGCNNTGATNCTA TCCTAAAANACCAAACNNANAANGGNCAGGNTCANCNNCATTTACATCTGANNNAANNNN NTNNTTCTNNNNTGATTTNNNCNGNNNNNNNNGNNANNNANNNCTNNNNNNNNNNNNNNN CNNATCNNNATCNNANNNNNNTTNNANNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNN NNNNANNNNNNNANNNNNNGNANNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNN
 * Filtered DNA Sequence**:


 * Nucleotide Blast Results for PNiC-BSa4**

7/11/13

PNiC-BSa4 Cloning
Two samples (50uL each) were prepared for PNiC-BSa4 in case one failed or had a concentration too low to be used. The concentrations of both samples would ideally be around 50ng/uL. Both samples indicate high purity through the 260/280 and 260/230 values. The RE Digest Gel shows that the cutting of plasmid PNiC-BSa4 was successful. With the current concentrations of the samples, cloning might be successful and will be carried out.











7/10/13

PCR Clean Up Re-Do(s)
After completing PCR Clean Up correctly the concentration of the sample was over 100 ng/uL. This supports the conclusion as to why the precious sample had low concentrations.



In this PCR clean up, step 1 was skipped. In step 1, the column filter is prepared for maximum DNA binding to the membrane. Since this step was overlooked, the concentration of the sample suffered. PCR^2 was redone as well as PCR Clean Up.



PCR Clean Up
The concentrations of these samples could be low due to PCR^2 not being successful or because the primary and secondary PCR samples were too weak for PCR^2 to have a significant impact on the amplification of DNA present in the sample.



7/8/13
Elution samples for PfDXR were spun down and concentrated into a 1mL sample. =
 * FPLC**

= =Week 5=

Tail Primer Design
__ Forward Primer: __ 5’ **__ TACTTCCAATCC __****__ ATG __****__ ATTGACCTCACGGGCAA __** 3’ __ 32 __ bp  Content __ 46.9 __% 0 mM Mg2+ Tm __ 64.4 __oC 1.5 mM Mg2+ Tm __ 71.5 __ oC 2 mM Mg2+ Tm __ 72.0 __ oC 4 mM Mg2+ Tm __ 72.9 __ oC 6 mM Mg2+ Tm __ 73.4 __ oC

__ Reverse Primer __ : 5’ **__ GTGGTATGCTGATGGTT __****__ TAA __****__ CAGTAAAGGTGGATA __** 3’ 5’ TATCCACCTTTACTGTTAAACCATCAGCATACCAC 3’ __ 35 __ bp GC Content __ 40.0 __% 0 mM Mg2+ Tm __ 61.2 __ oC 1.5 mM Mg2+ Tm __ 68.8 __ oC 2 mM Mg2+ Tm __ 69.3 __ oC 4 mM Mg2+ Tm __ 70.3 __ oC 6 mM Mg2+ Tm __ 70.8 __ oC
 * Reverse complement it: **



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= =Week 4=

[[image:vdsstream/vlg_pmp_elution2a_pfdxr.jpg width="552" caption="Figure 14: MEasurement of PfDXR; max abs at 280 nm; Elution 2"]]
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= =**Week 3**=



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= =**Week 2**=









Midi Prep DNA Sequencing Results:

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= = Week 1 =

DNA sequence results: NNNNN

The DNA Sequencing results indicated that there were nucleic acids present in the sample but the specific bases were not able to be determined. pNiC Plasmid.