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Michael T.'s Research Page Extravaganza
Fall: Week 15

Virtual screening was performed previously for both the CB306 and the HF9 libraries. The top scoring ligands were then analyzed using Lipinski's Rule. There was not time to test any of these compounds yet, but we should already have them in lab. After the break, or perhaps during the break, these compounds will be tested in an inhibition assay. They seem very promising, as a lot of them follow Lipinski's Rule, and the their structures are somewhat similar to the 2-pyridone ring structure of known inhibitors. My protein level is also running kind of low, but Max has some. Maybe an expression can be done during the break if anything. A lot of the HitFinder compounds scored very high and they followed Lipinski's Rule. I am curious to see whether or not they are able to inhibit the function of FabV. We already have compounds from both these libraries, so after the break or during the break inhibition assays can be performed to determine whether or not I have found a potential inhibitor.

Fall: Week 14

The inhibition assay was performed for FabV enoyl-ACP reductase. None of the inhibitors proved to be very effective. The drop in absorbance readings for the last two inhibition assays is probably a result of bad NADH. However, the slopes remain consistent. None of the slopes are zero, meaning that none of the inhibitors are significantly effective. It was no surprise that triclosan was ineffective, as seen in literature, triclosan is not effective against FabV like it is against other Fab family enzymes. Since these inhibitors were not effective, more screening work needs to be done, or perhaps compounds can be tested from the readily available CB306 library.

Fall: Week 13



After the library was screened, the top compounds from CBUT were docked in the structure and checked for polar contacts. These were the two compounds that were ordered and that would be tested for inhibitive properties. These two compounds scored relatively high and followed Lipinski's rule, so hopefully they have inhibitive properties. When the compounds arrive, they will be tested using the newly discovered optimal concentration for FabV (240 uM in 400 uL).

Fall: Week 12

The best scoring ligands from the CB-kin UT library. A lot of the compounds scored within the hundreds, which was peculiar. It is not something I am used to seeing based on previous experiences with GOLD. However, this is an updated GOLD application and the scoring function is a little different. A possibility could be that the gold.conf file was created incorrectly, but the same one that was used for CB306 was used, but modified for different libraries. These high scoring compounds will be tested for Lipinski's Rule and the ones that are eligible will be ordered and tested in an inhibition assay.

Fall: Week 11

This is the multiple assays performed in order to determine the optimal concentration of enzyme needed in the final volume of 400 uL for the enzyme assay to run the desired five minutes. After many failed attempts, and the enzyme almost immediately consuming all of the NADH within a matter of seconds, an ideal mixture was determined for the solution. 240 uM of enzyme (100 uL volume), 400 uM NADH (32 uL), 300 uM Crotonyl-CoA (10 uL), and the remaining volume was Tris Buffer to give a final volume of 400 uL. Now that the enzyme is known to be functional, and an ideal mixture has been discovered, an inhibition assay can be performed in order to find potential inhibitory compounds.

Ok - Dr. B 11/19/12 Fall: Week 10

This week, the a lot of virtual screening work was performed. The CB306 library finished screening, however the same cannot be said about the diversity library. It is around 50,000 ligands and the first run has been running for several days now. Also this week more protein was expressed using plasmid that I had stored in the -20 degree freezer. I helped Max with the characterization process and some other expression work. We should have enough protein to last us the rest of the semester now.



Fall: Week 9

Some more protein expression work was performed, however the sonicator was broken. So the protein was placed in a lysis reagent. Also during the weekend, more libraries were screened. A gold script was created for 3ZU5 and screening work is currently being performed for both 3ZU4 and 3ZU5. The same libraries will be screened for both to determine if the results are similar in order to find the best possible inhibitors. After all of the libraries have been screened, the top results from each will be concatenated and analyzed and ordered to perform more assay work. An optimal mixture of protein, NADPH, and Crotonyl has still not been discovered. Concentrations of each have been altered, but nothing seems to be working. This week, I will try once more to make the assay run the complete 5 minutes. The NADPH continues to be disappearing before or at the 3 minute mark.



Fall: Week 8

102112- Michael, ok good. Show some type of data or image file here as your results for the week. Even some enzyme assay values. - Dr. B

Some new possible inhibitors have been discovered from the Platesplates library. Next week, I will scan more libraries and hopefully express more protein with Max. Then perhaps we can crank out more assays to find the Km values of our specific protein. While the screening continues to progress, I may and try to decrease the enzyme concentration for the assay. Nothing else seems to be working with regards to changing up the amounts of NADH, Crotonyl, or buffer. So perhaps, decreasing my concentration of enzyme may help the assay run a bit longer. I also want to try and look at order inhibitors this coming week.

Fall: Week 7

101612 - Michael - ok good. Hopefully you guys will be able to crank out some assays and get the Km and kinetics vaules with the program and then do some inhibition test soon. Dr. B

There was a lack of progress this week due to an abundance of tests. The only thing new that was discovered this week was the Km values of FabV and Cortonyl CoA. However, these values varied a bit organism to organism. So a remote desktop will be used in order to run a graphing program that will allow a more reasonable Km value for my own FabV. I messed around with it a little bit over the weekend, but I am still not quite familiar with it as I hope to be, so I will spend some more time with this program. Also, the virtual screening will be performed, so that I can go ahead and order my inhibitors, also some assays will be run next week while GOLD is screening my target. My target does not need a homology model since it has already been crystalized with a different substrate.

100912 - ok I think the Shackleton is useable for now. Just be sure you are getting a baseline signal if you use it. The revised VS protocols are online - I think you will just need to add in MolProbity. -- Dr. B Fall: Week Six

It was confirmed that both snap frozen samples and glycerol samples were active enzymes. Shackleton was determined to have read low, and Henry reads a little high. The Km value for FabV and Crotonyl CoA was determined to be around 0.2. There has not been much progress on the delaying of enzymatic activity to run for the full five minute course. So this week, I instead focused a little bit on the dry lab portion and worked a little bit on setting up the screening of my target on GOLD.

100112 - Michael - ok, I am interested to see how the assay goes this week and if you can get it 'tamed' -- Dr. B

Fall: Week Five

Another Enzyme Assay was performed this week. This was done using various ratios of NADH to Crotonyl CoA to determine an optimal mixture to perform an inhibition assay. The data was inclusive as there was no optimal mixture; since the assays took a lot of time to run (5 minutes each). The NADH may have degraded overtime. In the future assay, perhaps smaller aliquots (8 microliters) of NADH will be used. A repeat of the last assay was essentially performed at first. The results lined up with the previous assay pretty well. The only problem of concern is the steady degradation of NADH. This will have to be corrected in the next assay. Also an assay was performed on snap frozen samples of FabV as well. The data was inconclusive, because the initial absorbance reading of each were too low to determine whether or not the snap frozen samples were functional. Next week another assay will be performed comparing the snap frozen sample to the glycerol stored sample. NADH will also be stored in the -20 degree freezer unless in use or placed into individual assay aliquots until in use.

Fall: Week Four Michael - Good work on the assay! We ordered some more substrate (Crotonyl CoA) too -- Dr. B

This week an enzyme assay was run. The results looked promising. NADH was cleaved when Crotonyl CoA was added to the mixture. This suggests that the coupled assay is working. However, the NADH was being cleaved way too quickly. What needs to be determined is the proper amount of NADH, Cortonyl CoA, and enzyme that needs to be in the solution in order for the NADH to be used up completely over a span of five minutes. Also, this week the virtual refresher protocol was completed.



Michael - have you decided on a day to try this out? -- Dr. B 091812

Fall: Week Three

Made Tris base of pH 7.5 in order to prepare for enzyme assay to be run in week four. 100 mM Tris base was made using 200 mL of autoclaved nanopure, 2.42 g of Tris, and HCl (to reach desired pH). Protocol for the assay was overlooked a few times for understanding. Timing is very important as the reagents are very expensive. Pre-cooling materials may be more productive in terms of running the assay. Protein stored in -20 degree Centigrade is to be used, and if that fails testing will be done on -80 degree samples frozen using liquid nitrogen.

Fall: Week Two

http://www.sciencedirect.com/science/article/pii/S0968089606008443 (link to site that contains assay information for fabg) Made LB, and plates of cloning vectors and expression vectors with the gene for FabV. Found protocol for a FabI enzyme which is structurally similar to FabV in terms of functionality and substrates needed for a functional/inhibition assay.

Fall: Week One

























Summer Research

070212 - Nice concentrations --Dr. B 6/28/12

6/26/12



6/25/12



As a precautionary measure, I made two secondary PCRs in order to ensure myself a higher chance of success. As seen in the figure, my first secondary PCR failed for some reason. Perhaps there was an error on my part. The second secondary PCR worked just fine. Both secondary PCRs were made using the same primary PCR. The smear looks clean and smooth, as is desired. The next step after this would perhaps be to run the PCR squared.

6/21/12



This is Daniel and I's PCR gel electrophoresis of pNIC-BSa4. The results were similar for both of us. As the concentration goes up, there is an increase in band intensity. Also in there control group where there is no DNA, there is no DNA which means there isn't contamination.

6/19/12



This is Daniel, Max and I's first attempt at a PCR using our respective targets. Daniel's worked the best, I think mine may have worked and Max's did not show up on the gel. This was done using the first and last set of primers from the oligo mix. Another secondary PCR would be made using the diluted primers that were ordered online.

062112- Michael - looks good. Great captions. Do you have your image of your first PCR gel (of pGBR22).. -- Thanks, Dr. B

This is Max and I's first PCR gel of pGBR22. The results came out well. The band grows more intense as more and more plasmid is added to the mixter. For some reason though, the control sample had a band in the same region. Perhaps there could be contamination?

6/14/12



The samples that were supposed to go into wells 5 and 8 were switched which resulted in a gel that seems a bit off in terms of the bands. Also lane 9 was skipped because some of the sample from well 8 appeared to be leaking over. The use of different primers caused a shift in the band length. The M13 control resulted in a band of smaller mass. When compared with another student, it appeared to do the same thing as well. Perhaps the primers were not diluted properly which could have resulted in the difference in band size. A peculiar result is the intensity of the band for the no DNA control in well 10. Perhaps there was some contamination and some of the plasmid got into the sample. The no DNA control in well 8 had a faint band, so perhaps there was a bit of contamination in that sample as well.

6/7/12



The gels for this lab ran two samples. There appears to be a bit of contamination in the sample. With the first cut the length is around 3 kb similar to no cuts. With two and three the fragments appear add up to around 3 kb. In land 10 there is a band where the 2nd of the three cuts should be but it is very faint. This may have resulted due to experimental error or perhaps the enzyme was not able to cleave properly, The process of enzyme digestion may be used to develop primer sites to sequence DNA strands greater than 1000 bp in length.

6/12/12



As more plasmid of DNA was added to the sample, there was an increase in the number of colonies that grew in the overnight plate. The efficiency was undetermined due to a flaw in the protocol. There was too much growth to count in the plates. As a result, there needs to be a change in the protocol. However, the transformation was successful. The ampicillin resistant DH5alpha cultures were able to grow in the LB and amp plate. The next time this lab is used, perhaps less of the samples need to be added to each plate.

061212 - Nice captions and images, Michael. Do you have RE digest results? -- DR. B

6/5/12