Stephanie+B.

=Week 15=

12/8/12
 * completed the inhibition assay using the same YopH enzyme sample as used on the 7th. Results showed no activity (all readings at 410nm were at an absorbence of zero). This could be due to poor enzyme activity. No samples were yellow.

12/7/12
 * completed the enzyme assay test of the the phosphatase enzyme YopH (sample B in the pink container in the -20 degrees Celsius fridge).

12/1/12-12/5/12
 * Ran virtual screen of cb306 library and cb-Kin_UT library with my homology target.




 * Top ten ligands from cb306 library shown as pymol images above. Ligand shown as sticks, target (homology) shown as surface, active site shown as lines. polar contacts shown as black dashed lines.




 * Top ten ligands from cb-Kin library shown as pymol images above. Ligand shown as sticks, target (homology) shown as surface, active site shown as lines. Polar contacts shown as black dashed lines.
 * The cb-Kin library yielded better fit ligands than the ch306 library according to the fitness score assigned by GOLD.

=Week 14=

11/27/12 Stephanie Bsaibes- Samples1-6 Protein Characterization-Pagegel Lane 1: 1kb PageRuler prestained Protein ladder Lane 2: sample 0a pre-induction Lane 3: sample 0b pre-induction Lane 4: sample 1a post-induction Lane 5: sample 1b post-induction Lane 6: sample 2a soluble fraction Lane 7: sample 2b soluble fraction Lane 8: sample 3 flow through Ni-NTA column Lane 9: sample 4 Wash Lane 10: sample 5 Elution 1 Lane 11: sample 6 Elution 2 Lane 12: 1kb PageRuler prestained Protein ladder
 * the gel was messed up.

=Week 13= 112612 - Good. Dr. B 11/21/12 Elution #1 Sample 5 Elution #2 Sample 6
 * purified the protein. below is the OD of elutions 1 and 2 at A280 in 5ml

Elution #1 Sample #5 after concentrating


 * FPLC was unsuccessful

11/20/12
 * spun down the sonicated samples after adding beta mercaptoethanol to a final concentration of 0.002M. the samples were kept on ice for 3.5 hours before syringe filtering and adding 1.5mL Ni-NTA resin and storing in 4 degrees Celsius fridge overnight.

11/19/12
 * continued protein expression with sonication to lyse the cells. Then was kept in -80 degrees Celsius overnight.

=Week 12= Good - Dr. B 11/19/12 11/17/12
 * the protein was harvested by centrifugation at 6000g for 20 minutes. 10 mL of lysis buffer was added to each pellet, transferred to a 10 mL conical tube and placed in the -80 degrees Celsius freezer until I sonicate on Monday.

11/16/12
 * transferred 25mL of sample A into 500mL of LB for a final of 0.188 absorbance reading at OD600. Transferred 10mL of sample B into 480mL of LB for a final absorbance reading of 0.125 at OD600. After leaving sample A in the shaking incubator for 2 and a half hours the final absorbance reading was 0.522. Sample B was left in the shaking incubator for 3 hours, the final absorbance reading was 0.505. IPTG was added and the samples were left to further grow in a 25 degree Celsius shaking water bath with no water.

11/15/12
 * created a homology model using swissmodel.expasy.org. The model template used was 1ywf. The sequence identity is 25.57% and the QMEAN Z-score is -3.8.
 * below is the molprobity results for my template.
 * Began protein expression. let two single colonies grow for 16 hours overnight.

=Week 10=

11/2/12
 * We were running low on secondary PCR, so I made secondary using Ivys primary and my primers. I used the same settings she did when making her secondary (those in the protocol).

Stephanie Bsaibes 11/02/12 Secondary PCR Lane 1: 100 bp ladder Lane 2: my secondary PCR using Ivy's primary PCR as a template Lanes 3-5: Paul's samples Lanes 6-7: skip Lane 8: 100 bp DNA ladder that had been sitting out (not in the freezer) to check it
 * as seen above, there was no yield with the secondary. Because I did all the steps the way Ivy did (she was successful) I can only conclude that it is my primer dilutions that are affecting the results of my PCR runs. Ivy will make more secondary PCR with her primers and I will use those next week.

10/31/12 Stephanie Bsaibes- PCR squared gel check- 10/31/12 Lane 1: skip Lane 2: 100 bp dna ladder Lane 3: Ivy's PCR squared Lane 4: skip Lane 5: My PCR squared
 * ran a gel check on the PCR squared sample from October 29th
 * The gel electrophoresis equipment was not working properly, so the gel was stopped before completion. The gel check shows some contamination in the product. It is not apparent if the product is at the correct length.
 * Also did a second PCR squared reaction to have more product and did 8 tubes instead of 4. After PCR clean up I did a nanodrop to determine the concentration of the product.


 * as evidenced by the low concentration yield, this PCR was not successful. I am uncertain of the reason this could have happened. It is possible that the PCR clean up step was where the product was eliminated. It is also possible that the actual PCR reaction was not successful either because of the reagents included (maybe they were kept out too long-- not the hot start polymerase-- even though they were on ice).
 * Karthik helped me realize that I used the HCl solution for the wrong step (the wash instead of the elution). for my first PCR squared I did not use the HCl solution- I used the elution solution in the kit.

10/29/12 Stephanie Bsaibes 10/29/12 Nanodrop concentration check on PCR squared clean up
 * Ran the PCR squared off of Ivy's secondary PCR. I made the same mistake as I did on Saturday by seemingly not having the correct amount of master mix to measure out for four PCR tubes. I found out that I was using the pipettors incorrectly. I am not supposed to push all the way down on the top when measuring out solution, only when dispelling it. This may have lead to incorrect proportions of the reaction buffer, forward and reverse primers, secondary template, polymerase, dNTPs, and MgSO4. This would also explain why there wasn't enough of the master mix to spread among four PCR tubes; when using the 200 ul pipettor to take up 50ul of the master mix, it probably took up more than 50ul for the first three times.
 * Also completed the PCR clean up using the kit provided. Mistake made here was that when I realized that the PCR squared product plus the solution added to it would not fit into the tube, I split the product+solution into two tubes and continued the procedure. The only change to the procedure I made upon realizing my mistake was to add 25ul to each tube instead of 50ul of elution buffer for the last step.
 * the Nanodrop showed a concentration of 88.8 ng/ul. The slope on the graph shows little to no contamination. This concentration is ok, but not ideal. My next step will be to go ahead and gel check the product and, if it's good enough, do a gel extraction to continue to cloning.

=Week 9=

10/27/12
 * attempted PCR squared using Ivy's secondary PCR. Made a new dilution of the forward and reverse primers due to the fact that I accidentally added 4ul of the reverse primer into the forward primer. Did not run the PCR because I was rushed for time and did not know what to do after I did not have enough of the master mix to measure out 50ul into four PCR tubes-- I only filled three PCR tubes.

=Week 8= 102112 - you may re-make yoru primary using a lower annealing temp (57 vs 58 ). This may then lead to a better secondary. -- Dr. B 10/20/12
 * redid my secondary PCR (using the same primary PCR) to try to get a higher concentration
 * My second try did not even show up on the gel. The band from the first try did show up a little clearer this time. I am unsure if I should continue to PCR squared or remake my primary. I may not have mixed my reverse and forward primers enough before using them to make the 20uM dilution.

10/19/12
 * Completed secondary PCR and ran gel
 * The secondary PCR band was very faint, so I'm redoing the secondary PCR to see if I can get a better concentration. The secondary PCR band was around 900 base pairs long.

10/15/12
 * Completed the primary PCR for my target oligomix. Ran the gel for the primary to see if the temperature set up in the PCR machine would work (the PCR for Jennifer and Ivy did not work with our target).
 * The primary PCR was successful, so I moved on to the secondary PCR.
 * Ran the second part of the virtual screening refresher.



=Week 7= 101612 - Stephanie - show some type of data or results. -- Dr. B 10/10/12
 * Began the virtual screening refresher to run overnight. No complications arose, although some of the commands had to be looked up.
 * Created the oligomix for my target. Again, no complications arose. Made sure to adhere to clean techniques, to mix each well before putting into the final tube, and to switch pipette tips for every well.

=**Week 6**= 100912 - Yeah. Ok now move on to your real cloning of your target and try to catch up to some of the other guys. -Dr. B 10/4/12
 * I ran the gel on the samples from the PCR the day before.


 * Sample A was messed up again because I added 2 ul of DNA ladder to it instead of the blue juice. The gel shows that the PCR worked and the purple protein coding sequence was amplified in the pGBR22 plasmid. Sample D was the no DNA control so there shouldn't be a band in the lane (as shown).

10/3/12
 * I redid my Practice PCR #1 protocol to amplify the pGBR22 protein. This time, I used ReadyMade T7 as my reverse promoter and ReadyMade SP6 was my forward promoter. The concentration of pGBR22 was 187.9 ng/ul. After PCR, the samples were kept in the PCR machine at 4 degrees Celsius storage overnight, until they were placed in the -20 degrees Celsius refrigerator at 11:34am on 10/4/12.

=**Week 5**= 9/29/12- 9/30/12 093012 - Stephanie, ok good. You will need to get one of your PCRs to work before moving on. -- Dr. B For more detailed information please see the google doc
 * I worked on the Pymol refresher protocol. I had to look up my old Pymol labs from Spring 2012 semester to recall some of the commands. Below are the images that I made:
 * I am not sure I used the correct command for 3HBB (figure 8). I don't think there are supposed to be 3 active sites. However, I couldn't find MTX, so I thought that the inhibitor TMQ would be part of the active site.

9/28/12
 * Today I worked on the PCR primer Design for pNIC-Bsa4 cloning on Ivy's target. Her target (and now mine as well), protein tyrosine-phsophatase, functions in Yersinia //enterocolitica.//I created a virtual plasmid in which I added the target DNA sequence with primers within the accepting vector where Bsa1 cuts.
 * Forward Primer: TACTTCCAATCC__ ATG ACCACCTCTGCACTC__
 * 30 bp
 * GC content: 50%
 * Melting temperatures (degrees Celsius): 0mM Mg2+: 63.2; 1.5mM Mg2+: 70; 2mM mg2+: 70.5; 4mM Mg2+: 71.4; 6mM Mg2+: 71.9
 * Reverse Primer: TATCCACCTTTACTG __TTA TTCGAGGAAATAAGA __
 * 33 bp
 * GC content: 33.3%
 * Melting temperatures (degrees Celsius): 0mM Mg2+: 57.6; 1.5mM Mg2+: 65.6; 2mM mg2+: 66.2; 4mM Mg2+: 67.3; 6mM Mg2+: 67.8

=Week 4= 9/21/12 - Stephanie -- ok better luck on the next round! Good gel labeling and marker. -- Dr. B
 * This lab session was full of mistakes that will inevitably cause me to have to redo PCR 1.
 * When I went to pull out the PCR-ed plasmids from the freezer, I found I was missing sample B.
 * I waited too long for my agarose solution to cool and when I poured it into the gel plate, it came out a little chunky.
 * As I was preparing my samples to be pipetted into the gel wells, I put in 2uL of the 100 bp DNA ladder into sample A instead of 2uL of DNA loading dye.
 * These mistakes cause the experiment to be null and require a re-do of the experiment. The gel electrophoresis was ran anyway just to observe the effects of the multiple mistakes.




 * As evidenced by the non-existent bands on the gel, the PCR was not successful. This could be due to the mistakes made in protocol today or mistakes made on September 19th. The next step would be to redo the protocol from the beginning without any mistakes.

9/19/12
 * The goal of this protocol was to amplify the purple protein coding sequence in the pGBR22 plasmid using forward and reverse primers. This was also used as "Practice PCR Protocol #1." On this day, I used ReadyMade M13 Forward, M13 Reverse, and the pGBR22 plasmid (concentration: 187.9 ng/ul) to run a PCR. After the PCR was completed, the samples were placed in a -20 degree Celsius freezer until 9/21/12

=Week 3= Stephanie -- great job. And you got points back for last weeks. -- Dr. B 091812 9/14/12
 * The purpose of this protocol was to extract DNA from the bacterial cells transformed on 9/11/12. The protocol used came from the "Plasmid HiSpeed Midi Kit." Multiple buffers were used to extract DNA from the bacterial cells. The final product was then run through Nanodrop to determine the concentration of the DNA.
 * A concentration of 82.1 ng/uL was determined. This is a good concentration of DNA. There is little to no contamination because the 260/280 concentration of proteins is within the acceptable range, around 1.9 to 2.1.
 * **Figure 5:** Nanodrop graph of concentration of DNA. Wavelength at 260 nm. Concentration: 82.1 ng/uL ||

9/12/12 - 9/13/11
 * The goal of this protocol was to digest pGBR22 with restriction enzymes EcoRI and PvuII and then run a gel to analyze the enzymatic activity. The dilution was done on September 12th. Buffer 2 was used because it was the optimum for PvuII, while EcoRI worked 100% with all buffers. An extra control group was created and incubated. The temperature for the heat block fluctuated between 70 and 88 degrees Celsius because I had trouble understanding the use of the temperature knob. The samples were frozen until the 13th, when they were used to run the electrophoresis gel for 58 minutes at 100V.
 * The gel came out as expected with no difficulties in the procedure. Even though the plasmid cut with EcoRI has the same mass as the uncut plasmids, it remained higher up on the gel because the uncut plasmid becomes a circular shape that is twisted like a screw and moves faster in the gel than that of the straight line of the cut plasmid.
 * As expected, the plasmid cut with EcoRI has 1 band because it was only cut once and is therefore one long line of DNA. The plasmid cut with PvuII has two distinct bands because the two cuts made create two shorter lengths of DNA (which causes them to move faster through the gel and is the reason both bands are further down than the EcoRI and uncut plasmids). The plasmid cut with both enzymes, has three distinct bands because of the three cuts made. The third band is furthest down the gel and is the lightest distinct band indicating that it is the shortest and least massive length of DNA.
 * **Figure 4a:** A black and white UV created image of the plasmids cut with the restriction enzymes. ||
 * **Figure 4b:** 1 kb DNA ladder showing the length (in kilobases) and the mass (in nanograms) of corresponding bands on the gel. ||

=**Week 2**=

9/7/12
 * An analyze DNA sequence was done to virtually understand what is happening with the cutting of the plasmid. BLASTs were also run.
 * EcoRI works best in the NEBuffer Universal as well as in NEBuffers 1, 2, 3, and 4.
 * PvuII works best in the NEBuffer 2 as well as in 1, 3, and 4.
 * Both enzymes can be run in the same buffer because EcoRI can be used with any buffer. So both could be used in buffer 2.

A)B)

C)


 * **Figure 3.** Images created by NEB of cuts of the pGBR22 plasmid cut with different enzymes (A) is cut with EcoRI (B) is cut with PvuII and (C) is cut with both EcoRI and PvuII ||

9/6/12 - 9/11/12 [no image to display]
 * The transformation of competent cells for the plasmid prep pf pNIC-Bsa4 spanned the course of three days, September 6th, 10th, and 11th. On the first day, DNA was transformed into //E. coli// DH5a, plated, and allowed to grow overnight. The next day, the plate was transferred to a 4 degree Celsius refrigerator for storage over the weekend. Then, a colony was picked to grow in an LB shaker at 4 decrees Celsius and allowed to grow for 16 hours. The last step was to fill conical tubes with the LB solution and then have them spun down and frozen. The next step was done on 9/14/12.

9/5/12
 * For the primer (pNIC-Bsa4) dilution, only the forward reaction was done, Divya G. did the reverse. This [|ordering]website was used to order the sequencing required, and then the plasmid was dropped off at the proper location on campus.
 * This forward DNA sequence (shown below) has not yet been compared with Divya's reverse sequence.


 * Figure 2**: The forward sequence of pNIC-Bsa4.

=**Week 1**=

8/31/12 >> Wikispaces Page ==
 * The first protocol completed for the semester was Target Discovery. The purpose of doing this first was to narrow down target options for virtual drug screening research for the rest of the semester. To look up possible targets the NIAID, EUPATH, PATRIC, and CDC websites were used primarily to select possible organisms (bacterial or protozoan). The BRENDA, SIGMA, and PDB websites were also used to determine the relative ease of study and current projects on possible targets before
 * I chose to research YopH from the organism //Staphylococcus aureus//. I found the PDB structure, if it's "druggable," its EC number, whether or not it's assayable, and any current inhibitors. I also ran an NCBI BLAST search and found itsamino acid sequence and its


 * Figure 1:** PDB image of YopH


 * My target was not chosen for study for the rest of the semester because the information provided for YopH for the organism //Staphylococcus aureous// got confused with information for YopH for the organism //Yersinia entercolitica//