Samantha+D.+(Springer)

Weeks 14 & 15 Thank you Samantha for submitting - Dr. B121714 Virtual Screening: Figure 7: GOLD binding poses of the top 3 scoring ligands from the NIH clinical collection library (SAM001246783, SAM001246577, and SAM001246653). Poses are shown in PyMOL with 3N28 shown as a surface, and the ligands shown as sticks and colored by element with cyan carbons. Hydrogen bonds between the ligands and active site are represented by black dashes lines.







Analysis: The vcPSP protein was screened against the NIH clinical collection, the Chembridge 306 library, and the library of in house compounds. Although these are small libraries, a few ligands had high binding scores. However, the two highest scoring ligands did not satisfy Lipinski's rule. The binding poses for the top ligands were analyzed in PyMol. It appears that GOLD did dock ligands in the active site of vcPSP properly. The next step will be to continue virtual screening by screening larger libraries.

Control Ligand Docking:





Figure 1: Active site of 3N28- Protein is shown as a surface and colored by element with green carbons. The active site is colored by element with cyan carbons. The ligand SEP is shown as sticks.

__Analysis:__ In preparation for virtual screening the active site of the vcPSP protein was defined by aligning it with a homologous protein (PDB ID: 1L7P), extracting the ligand, SEP, from that structure, and then defining the active site around this ligand. The protein was then prepared for virtual screening with MolProbity, and later with Hermes. First, a library of positive and negative control ligands was screened. Based on the GOLD docking scores for these ligands, it appears that the ligands selected were not very good controls. Many of the negative controls scored higher than the positive controls. This could be because the controls chosen were not very good, or it could be due to errors in the way that GOLD docked the ligands.

1242014- Outstanding work

Weeks 11, 12 & 13

Cloning Trial #3:







Analysis: There was some growth of colonies on the plates, although there also appears to be some contamination. The next step is to make a master plate and grow cultures overnight.

RE Digest:

Analysis: The pNIC was successfully cut with BsaI restriction enzyme. Then it was cleaned using PCR clean-up to remove contamination.

Cloning, Trials 1 & 2:



Analysis: Both of these cloning trials were unsuccessful as shown by the lack of colonies growing on the plates. There could have been errors making the pNIC accepting vector or with the transformation.

Secondary PCR and PCR Squared:

Analysis: Secondary PCR was conducted so that more PCR squared could be made in preparation for cloning. The first to attempts at PCR squared were unsuccessful, likely due to errors with PCR technique.

RE Digest:









Analysis: pNIC was sucessfully cut with BsaI as shown by the gel. The sacB gene was removed. The pNIC sample is know ready to use in cloning.

PCR Squared:



Analysis: PCR squared was conducted to amplify the vcPSP gene. Now, enough of the gene has been made to do the cloning procedure.

1162014- Nice work Weeks 8,9 & 10


 * PCR Squared: **





Analysis: The purpose of PCR squared was to amplify the gene produced by secondary PCR. I used two different secondary samples for PCR squared, and only one of them was sucessful. The next step will be to redo PCR squared and PCR clean up or possibly gel extraction. After completing PCR my gene will be ready for cloning.


 * Secondary PCR: **



















Analysis: Secondary PCR was attempted using many different thermocycling conditions. However, none of the conditions used worked for secondary PCR. This could have been because the ideal conditions for my protein were not found, or it may have been caused by problems with PCR technique or experimental error. Avery's secondary sample was then used to move on to PCR squared.

9232014- Keep up the good work

Weeks 5,6 & 7

__Figure 5:__ Gel electrophoresis results for trial 2 of primary PCR for vcPSP oligo mix. Lane 2 contains the 1Kb DNA ladder, lane 3 contains my primary PCR sample with no visible results, and lane 4 contains Cidia's secondary PCR sample.
 * Primary PCR: **
 * Trial 3:**
 * Trial 2:**


 * Trial 1:**

__Analysis:__ The first 2 trials for my primary PCR were unsuccessful and nothing was seen on the gels for those trials. The third trial of primary PCR was successful, and a smear can clearly be seen on the gel. For trial one the NEB recommended guidelines were used for PCR, and for trial 2 the other guidelines were used. For the successful PCR the annealing time was changed from 10 to 20 seconds. This likely allowed more time for the oligos to successfully attach to one another.


 * Midiprep: **

__Figure 3__: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.7 ng/uL yield for pNIC plasmid.



__Figure 2__: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.6 ng/uL yield for pNIC plasmid.

__Analysis:__ The purpose of Midi Prep was to isolate the pNIC plasmid that we grew from the cell pellet so that only the plasmid DNA remained in solution. Then the sample was nanodropped to determine the concentration of the plasmid. The average yield for my midiprep sample was 40.65 ng/uL, which is approximately what would be expected for pNIC because it is a low copy plasmid.

__Analysis:__ The PCR of the purple protein coding sequence of pGBR22 was successful The bands in lanes 3-5 appear to get darker with each sample because they have higher concentrations of DNA. The band in lane 5 does not appear darker than the band for the previous sample. This could be because the sample was not properly diluted or it may have been caused by a problem with the gel.
 * PCR- pGBR22: **

09232014- Good job, but remember next time to include more pictures

Weeks 3 & 4 __Figure 1:__ Gel showing results of restriction enzyme digest. Lane 1 contains the 1 kb DNA ladder. Lane 2 contains the uncut plasmid pGBR22. Lane 3 contains the plasmid digested with EcoRI, Lane 4 contains the plasmid digested with PvuII, and Lane 5 is the plasmid digested with both EcoRI and PvuII.

__Analysis:__ The restriction enzyme digest of pGBR22 was not successful. There are no visible bands in lanes 3-5 which should contain the plasmid cut by the restriction enzymes EcoRI and PvuII. It is possible that the restriction enzyme digest failed because there was not enough plasmid added to the samples, or possibly because the restriction enzymes were not properly deactivated and digested too much of the plasmid.

Weeks 1 & 2



Analysis: The transformation was unsuccessful because neither of the plates had any growth of bacteria. This could be because the DH5 alpha competent cells were left on ice for 1 hour after the pNIC-Bsa4 plasmid was added instead of thirty minutes. The DH5 alpha cells were also not used within 5 minutes of removing them from the -80 degree freezer, which also could have caused the transformation to fail.