Max+M.


 * Research Page - Max M. Virtual Drug Screening Summer/Fall 2012**
 * 12/07/2012**
 * On the final day of lab work for the semester, I finished up my Final report and revised the data in my notebook.**
 * 12/06/2012**
 * Inhibition assay was performed on the FabV enzyme using the compounds obtained from Robertus's Lab. Recal these volume were very low. When trying to perform the assay, I started having trouble with creating a control with the substrate alone. Rather than producing a constant line of absorbance at wavelength 340nm, the graph was created with many fluctuations. This should not be the case. Ultimately, the enzyme was introduced to the third ranking compound from the screening of the HF9 library, but no inhibition was measured.**
 * 12/05/2012**
 * Time was spend working on the final report.**
 * 12/04/2012**
 * Attempted to determine the concentrations for the enzyme inhibition assay. Very small volumes of potential inhibitors were acquired from the stock held in Robertus's lab. Since there are such low volumes and the LogP values are relatively high, the entire volume of compound obtaind will be placed into the assay. Aside from determining the concentrations for the assay, today was spent working on the report.**
 * 12/03/2012**
 * The final organization of the compounds screened from the HF9library. Inhibition assays will maybe be performed with the lop few ligands achieved through the virtual screening of this particular library. The ligands which have their plate location listed have been placed through Lipinski's rule and had satisfactory results.**


 * 11/30/2012**
 * Today was spent organizing the data that has been collected thus far. Also I analyzed enzyme assays performed by my research partner and compared them to the assays performed through my enzyme. It appears as if both of our enzymes are functional, however I have reason to believe that I have miscalculated the concentration of my enzyme. Due to the reaction rate associated with my enzyme, the assay itself might be being performed at higher concentrations then what I am under the impression of. This can be quickly checked through Spectrophotometry of my enzyme sample. I will nanodrop the enzyme in order to determine its absorbance and maybe calculated its actual concentration. Also I looked over the HF library to organize the virtual screening of that library. **


 * 11/29/2012**
 * More enzyme assays were performed on the FabV enzyme.**
 * The results from this enzyme assay are much more promising. In this assay we can see that the slopes of the reactions are starting to become much more linear. This suggests that we are going in the right direction with the alterations of the concentrations. The more dilute enzyme appears to be working much more efficiently. This must be a result of how the reaction can be displaced over a longer period of time once the ratio is determined. Recall this ratio is trying to be discovered in order to obtain kinetic value of the enzyme. Once we have found a satisfactory ratio, potential inhibitors can be introduced into the reaction. If these inhibitors have any binding to the enzyme then the substrate absorbance will either be constant or vary a bit more. **


 * 11/28/2012**
 * Studying for a Genetics Exam.**
 * 11/27/2012**

Ligand with the highest ranking fitness score implemented into the mol2 version of 3ZU4, which is the crystallized structure of //Yersinia pestis’// FabV enzyme. The mol2 structure is shown as cartoon in complete blue. The ligand docked by the GOLD virtual screening software is shown with cyan carbon, red oxygen, blue nitrogen, orange phosphate, and white hydrogen molecules. Polar contacts are shown as black dashes. However, since these virtually docked ligands were implemented into the mol2 enzyme structure rather than the original PDB structure, the number of polar contacts may not be reliable. Ligand with the second highest ranking fitness score implemented into the mol2 version of 3ZU4, which is the crystallized structure of //Yersinia pestis’// FabV enzyme. The mol2 structure is shown as cartoon in complete blue. The ligand docked by the GOLD virtual screening software is shown with cyan carbon, red oxygen, blue nitrogen, orange phosphate, and white hydrogen molecules. Polar contacts are shown as black dashes. However, since these virtually docked ligands were implemented into the mol2 enzyme structure rather than the original PDB structure, the number of polar contacts may not be reliable.
 * the same problem persists in these reactions. As can be seen, the reaction is still not going to completion and the substrate is kept at a constrant absorbance. This means that the reaction is still plateauing and that a satisfactory slope is yet to be determined. These assays were performed at varying concentrations of the enzymes. These concentrations can be found in yesterday's posting.**
 * 11/26/2012**
 * Concentration were calculated for the next enzyme assays. We have decided to test varying concentrations the enzyme in the reaction. As has been seen by the old reaction the ratio of enzyme to substate to cofactor has not been determined yet. We are attempting to reach an optimal ratio that provides a slope of satisfactory nature. So far the reactions have been plateauing too quickly. For the following enzymes we decided to use 30ug/mL, 40ug/ml, 60ug/mL, 65ug/mL, and 70ug/mL of the enzyme along with varying volumes of substrate and cofactor. These concentration were also determined using the spread sheet found on google docs.**
 * 11/21/2012**
 * Left home for the Thanksgiving Holiday.**
 * 11/20/2012**
 * An enzyme assay was performed to gather data of how quickly the substrate is cleaved with the cofactor in solution.**
 * This enzyme assay showcases the potential for the reaction to be performed as a constant slope. As suggested by Dr. B. the concentration of the enzyme should be further reduce. This particular reaction shows that the volume of cofactor placed into the reaction should be held at around 2uL. Higher concentration of the cofactor causes the reaction to occur at faster rates. This data could be helpful in the future analysis of this enzyme if the reaction ever needs to be made quicker.**
 * 11/19/12**
 * Concentrations were determined for the upcoming enzyme assays. Based off the data collected from the first enzyme assay, the concentration must be diluted further because the reaction is occuring much too quickly. concentrations were determined using the spread sheet on Google docs. There will be 100ug/mL of the enzyme reacted with varying amounts of substrate and cofactor.**
 * 11/16/2012**
 * Started the virtual screening of the "inhousecompound" library. This was the third library that was screened against the enzyme.**
 * 11/15/2012**
 * Images were generated to determine whether the screened ligands generated from the virtual screening data were placed into the proper location in the**
 * active site of the enzyme.**


 * 11/14/2012**
 * Results from virtual screening of the enzyme against the chembridge library cb306. This library contained 306ligands and was reduced after one screening of the library. Along with being ranked by fitness scores, the compounds were placed through Lipinski's rule( data not shown) in order to determine whether it would be a candidate for a drug. As can be seen, just because a compound has a high fitness score does not that it would be emerge as a good candidate from Lipinski's rule.Some of the top results matched those my partner achieved from his screening, but the third compound on this list had good enough fitness score and emerged from Lipinski's rule therefore it was retrieved from the stock in Robertus's lab.**
 * 11/13/2012**
 * Virtual screening results were anazyled and ranked from the chemical compound library CBKin. I found the data needed to apply Lipinski's rule. This data showcased that the top ligands from the screening did not have strong LogP values. These high LogP values are associated with compounds that are not very good drug candidates. I was mistaken to have not collected more data for the subsequent compounds. I did this only for the top ten with the highest fitness scores, but since they are not goood drug candidates i must collect data for the rest of the compounds as well.**


 * 11/12/2012**
 * Results from virtual screening of the enzyme against the chembridge library cbkin. This library contained 4000 ligands and was reduced after two screening of the library. Along with being ranked by fitness scores, the compounds were placed through Lipinski's rule in order to determine whether it would be a candidate for a drug. As can be seen, just because a compound has a high fitness score does not that it would be emerge as a good candidate from Lipinski's rule.**


 * 11/10/2012**
 * -Molprobity analysis of our FabV enzyme. This table shows the clashscore, all atoms data, and upon which percentile the enzyme would fall into. The data from Molprobity will help is determine whether the virtual virtual enzyme will provide reliable results when it is screened against the ligand libraries.**

The first thing done in the enzyme assay was creating the base of absorbance for NADH without any enzyme or crotonyl in the sample. This was done to create a control for comparison for the activity occurring on the substrate once the cofactor was introduced into the sample. As expected, there was a steady absorbance of about 0.3 without enzyme of cofactor. Next a baseline for comparison of crotonyl was created and this can be found with an absorbance of zero since it has none at this wavelength. Next the first test was conducted with 8uL of substrate NADH and 8uL of cofactor crotonyl. As can be seen the enzyme if very active as it quickly used up the substrate and produced NAD+. This is a problem since we must find an optimal ratio of the substrate and cofactor. Once the substrate was used it was spiked again with NADH, hence the spike. From the second trial, I found that even with 12uL of NADH and 6uL of crotonyl, the substrate was quickly used up again, however is 8uL more of NADH was added during the assay, a steady slope was created. This is interesting because it implies that an optimal ratio can be found with enough trial and error. 102112- Max, ok good work - looking forward to getting some more FabV soon. - Dr. B Absorbance readings for the CPR clean up procedure that the PCR Gel Extraction product was run through This is the result of placing the PCR gel extraction final product through another PCr clean procedure. As can be seen the previous image, the nucleic acid concentration of the PCR gel extract product was high but there seemed to be large contamination. Therefore it was placed through a second clean up procedure. This product is low in concentration, but if a gel check shows that only a band of 1200 bp is present, it will be very pure and may still be used in annealing and transformation. Absorbance readings for PCR Gel Extraction Procedure ran on the prior gel image posted Nanodrop reading #1: PCR gel extraction was performed as previously mentioned. The absorbance of the sample was then taken at 260 nm in order to determine the concentration of Nucleic acids within the solution. The absorbance reading of 248.9 in the first reading was expected to be high considering that this elution was performed in 25uL of elute where as the following elution was done into 50uL of elution solution. The ratios were off in thes sample, so it was run through s secondary PCR clean up procedure in order to purify the sample. Nanodrop Reading #2: PCR gel extraction was performed as previously mentioned. The absorbance of the sample was then taken at 260 nm in order to determine the concentration of Nucleic acids within the solution. The absorbance reading of 133.7 in the second reading was expected to be lower considering that this elution was performed in 50uL of elute where as the previous elution was done into 25uL of elution solution. This sample was not placed through a secondary PCR clean up procedure in hopes of preserving as least one sample retrieved from the PCR gel extraction.
 * 11/08/2012**
 * Enzyme assay**
 * Final stock solution of FabV stored in 20% glycerol.**
 * Spectrophotometry results for the final FabV stock stored in 20% glycerol. The absorbency of 6.564 was used in the Google docs spreadsheet to determine what concentration must be used during enzyme assay.**
 * 11/01/2012**
 * The samples were concentrated using centrifugation and placed through FPLC to achieve satisfactory concentration.**
 * FPLC results shown protein of the FabV enzyme size were expressed and isolated. The peak is located after the 40 kilodalton mark, which is acceptable since the FabV enzyme is about 43 kilodaltons large. Tubes 36 through 44 were kept, concentrated and stored in 20% glycerol in the -20 degree Celsius freezer.**
 * 10/30/2012**
 * The soluble fraction was placed through a series of protein characterization and purification protocols, starting with purification. This purification was achieved by utilizing the his tag found on the FabV enzyme by placing it through Ni-resin affinity purification. samples were taken at each elution for protein characterization. Then the concentration of elution 1 and 2 from both sources was determined using spectrophotometry.**
 * Elution 1A**
 * Elution 1 from source A was placed through spectrophotometry analysis to determine its absorbency __prior__ to FPLC. This was done to have a comparison of the spectrophotometry results of the elution after placed through the purification step of FPLC.**
 * Elution 1B**
 * Elution 1B shows similar results to that found in the results for elution 1A from the other source. This was done to determine the absorbency of the sample prior to the purification of FPLC.**
 * Both of these elutions were combined to placed through FPLC. Note elutions 2 from both sources were excluded from the FPLC machine due to such low concentration and and absorbency.**
 * 10/29/2012**
 * Sonication was performed on Monday afternoon. The soluble fraction was kept and the pellet of cell wall waste was discarded. By this time, the soluble fraction was believed to contain the our gene of interest. To validate this, the soluble fraction would be placed through a series of characterization and purification steps, including FPLC.**
 * 10/27/2012**
 * Sonication was to be performed on the pellets formed the previous night, however due to complication with the sonicator, lysozyme was added to both samples along with 12mL of sonication buffer. The lysozyme was added to slowly break down the cell membrane until Monday when sonication will be attempted once again. Re suspended pellets, sonication buffer, and lysozyme were frozen for the next couple of days.**
 * 10/26/2012**
 * FabV expression was conducted to its completion. OD600 values were measured from both sources every 30 mins. Source A and source B showed parallel growth during the expression. 2 hours and 45 mins after large scale expression was started, IPTG was added to induce transcription of the FabV gene sequence and eventual translation of the enzyme. Both sources were incubated for 4 hours of protein synthesis. Then both sources were centrifuged to produce pellets of about three grams. These pellets were frozen.**
 * 10/25/2012**
 * Starter cultures have been started. If these two starter cultures have bacterial growth and become cloudy after eight hours of incubation, then protein expression will be continued tomorrow.**
 * 10/19/2012**
 * Transformation of the pnic/FabV plasmid into competent BL21 bacterial cells was a success. Well defined colonies grew. We can be assured that these colonies contain out gene of interest because the media was selective. It was selective against //kan^(-)// bacteria. Furthermore, there was sucrose present in the media, only cells not containing the SacB gene would have grown. Protein expression can now be performed.**
 * 10/18/2013**
 * The starter cultures initiated yesterday were checked. It appears my research partner's colonies have lost their viability. This most likely resulted from their storage in the fridge since July. This means I will now attempt to transform BL21 cells with the Pnic-BSa4/FabV plasmid today in order to have a new collection of colonies from which we could express more protein from. The concentration of the annealed plasmid was 33.1 ng/ul and 1.5ul so therefore this volume of the plasmid will be introduced into the BL21 bacteria cells. The agar plates will be assessed tomorrow to determine if the transformation was a success.**
 * 10/17/2012**
 * As instructed by the research educator, I have moved forward onto the project my lab-mate, who has successfully annealed, transformed, and isolated a pNIC-Bsa4/FabV plasmid. I have had difficulty performing this task, and it may be beneficial to the research study as a whole to express the protein and create a stock of the enzyme. With a stock of the enzyme, and data from virtual screening, the chances of discovering an inhibitor will be amplified. Today I started the protein expression protocol by creating starter cultures from the colonies my lab-mate had saved. Three individual colonies were picked and placed into 80ml of Lb media (from two separate sources) alone with 80uL of kanamycin antibiotic. If the colonies still have viable bacteria, the Erlenmeyer flask will be cloudy tomorrow.**
 * 101612 -** Max, ok good deal. Shift over to protein work. Then you can come back to this when you have some down time. -- Dr. B
 * 10/11/2012**
 * 10/10/2012**
 * Nanodrop Reading #1**
 * Nanodrop Reading #2**
 * 10/09/2012**
 * 10/08/2012**


 * 10/07/2012**
 * Annealing of the FabV gene into the pNIC-BSa4 accepting vector, and attemt to transformation into DH5 alpha bacterial cells procedure was not successful**
 * A pNIC-BSA4 accepting vector was created and treated with T4 polymerase. The FabV gene of interest was also treated with T4 polymerase to create cohesive ends for the two nucleic acid molecule in order to anneal the them. Theoretically, the successfully annealed and transformed nucleic molecules should have bestowed kanamycin resistance. So if any colonies did grow, they would hace been expected to contain our gene of interest. As can be seen, no colines grew. This could have occured due to a number of problems including poor reagents and poor quality FabV genes. New stock of reagents will be used in the next trial. First a stronger FabV gene will be created to ensure annealing.**

Nanodrop reading number one: although a higher concentration would have inclined towards a more reliable vector, a concentration of 35.3 ng/ul is comfortable enough to attempt a transformation of the FabV gene into the cut pNICBsa vector. This low concentration can most likely be related to having a low initial concentration of the plasmid o begin with. The next step is to do a gel check of the vector to make assure it was cut correctly.
 * 09/29/2012**
 * A new accepting vector was created, cleaned through a PCR cleen up procedure, and then nanodropped to determined its concentration. A gel check is still to proceed.**

Nanodrop reading number two: although a higher concentration would have inclined towards a more reliable vector, a concentration of 34.7 ng/ul is comfortable enough to attempt a transformation of the FabV gene into the cut pNICBsa vector. This low concentration can most likely be related to having a low initial concentration of the plasmid o begin with. The next step is to do a gel check of the vector to make assure it was cut correctly.

A new attempt will be done at making a more reliable vector. 09/27/2-012 The accepting pNIC-BSa vector was created using the BSaI restriction enzyme. Gel check to proceed shortly. 09/25/2012 Part of Tuesday was spent reading over academic articles in order to increase my awareness of recent advancements in the scientific community.
 * 09/282012**
 * a pNIC-BSa plasmid was treated with the restriction enzyme BSAI and was not allowed sufficient time to run its reaction. Results implied that a reliable vector was not created due to the mismatching of band size.**

0921/2012 Nanodrop results showed a concentration of about 65ng/ul. Although this concentratoin could have been higher in a more ideal experiment, it is comfortable enough to move onto the insertion of the FabV gene into an accepting vector and attempt to clone it. So the next step to do is to create am accepting pNIC-BSa vector. 09/21/2012 09/17/2012
 * Trial 1 for Nanodrop Concentration Measurement of FabV gene isolated through PCR clean up**
 * Trial 2 for Nanodrop Concentration Measurement of FabV gene isolated through PCR clean up**

09/15/2012

09/13/2012
 * Nanodrop1**
 * Nanodrop2**

Nanodrop results 1 and 2 are those for the concentration measurement of my FabV gene following PCR cleaup of the Squared samples previously performed. As can be seen, the concentration of my sample was 16.9ng/ul for one sample and 14.0ng/ul for the second sample. These are not acceptable concentrations as they are much too low to give positive results during gene transformation and cloning. The low concentration may have occured due to poor PCR cleanup technique. This work must be performed again to achieve a higher concentration. 9/12/2012

09/10/2012
 * Week Three:**


 * Week two: Since I ran out of FabV gene stock and the last successful cut pNIC-BSA4 plasmid, this week will be dedicated to rebuilding that stock.**

​09/06/2012


 * 07-18-2012 pNIC-BSA plasmid cutting with Restriction Enzyme BSAI (attempt 2)**


 * 07-16-2012 pNIC-BSA plasmid cutting with Restriction Enzyme BSAI**

Trial 1: Nanodrop results aquired from PCR Clean Up procedure. The concentration of the plasmid was 78.9 ng/uL which is an acceptable concentration for this plasmid at a wavelength of 260 nm**.** The 260/280 ratio was 1.95 and the 260/230 ratio was 2.93 and these are once again acceptable ratios.
 * 07-09-2012 Nanodrop results for FabV Plasmid trial 1**

Trial 2: Nanodrop results aquired from Midi-prep clean up. The concentration of the plasmid was 80.3 ng/uL which is an acceptable concentration for this plasmid at a wavelength of 260 nm**.** The 260/280 ratio was 1.97 and the 260/230 ratio was 2.96 and these are once again acceptable ratios.
 * 07-09-2012 Nanodrop results for FabV Plasmid trial 2**

Note: "??" after each 50 signifies uL
 * 07-09-2012 PCR re-do squared results**

**6-26-2012 PCR squared results**

Lane 2: 100 bp ladder Lane 7: Primary mix Lane 8: Secondary mix
 * 6-26-2012 Primary Secondary Gene PCR results **


 * 6-29-2012** Failed Primary Secondary Gene PCR Results

Trial 1: Nanodrop results aquired from Midi-prep clean up. The concentration of the plasmid was 128.7 ng/uL which is an acceptable concentration for this plasmid at a wavelength of 260 nm**.** The 260/280 ratio was 1.89 and the 260/230 ratio was 2.63 and these are once again acceptable ratios. Trial 2: Nanodrop results aquired from Midi-prep clean up. The concentration of the plasmid was 128.1 ng/uL which is an acceptable concentration for this plasmid at a wavelength of 260 nm**.** The 260/280 ratio was 1.90 and the 260/230 ratio was 2.65 and these are once again acceptable ratios.
 * 07-02-2012 Midi-Prep pGBR22 Plasmid Nanodrop**

**6-22-2012 pNIC PCR Results**


 * -Target PCR Primary Results **

PCR results for target FabV from Yersninia Pestis. Although better results for lane 6 were expected, the procedure will be redone with a newly created olgio mix, and this may result in clearer marks to determine the size of the DNA.


 * -First for pGFP PCR 6-08-2012**

PCR for pGFP.


 * -PCR for pGBR22**

Figure 1: 1 ng of pGRR22 into DH5 alpha
 * -Culture images of increasing Concentrations for efficiency calculations**

Figure 2: 5 ng of pGRR22 into DH5 alpha

Figure 3: 25 ng of pGRR22 into DH5 alpha

Lane 1: Skip Lane 2: Ladder Lane 3: PvuII cut Lane 4: PvuII & EcoRI cut Lane 5: EcoRI cut
 * -Restriction Enzyme Digest**

Nanodrop Trial 1: concentration determination of pGBR22 using nanodrop. Cencentratoin was 410.2 ng/ml. Concentration at different wavelengths is also shown.
 * -06-05-2012 Nanodrop exercise to determine concentration of pBGR22 Plasmid**

Nanodrop Trial 2: concentration determination of pGBR22 using nanodrop. Cencentratoin was 524.9 ng/ml. Concentration at different wavelengths is also shown

Trial 1: Nanodrop results aquired from Midi-prep clean up. The concentration of the plasmid was 128.7 ng/uL which is an acceptable concentration for this plasmid at a wavelength of 260 nm**.** The 260/280 ratio was 1.89 and the 260/230 ratio was 2.63 and these are once again acceptable ratios.