Urvashi 

= **SUMMER 2013** = 9/20 Lane 1: 100bp ladder Lane 2: WbmDXR g-block 2a @ 63 C Lane 3: WbmDXR g-block 2b @ 64 C Lane 4: WbmDXR g-block 2c @ 65 C  Made the gel with 60 mL 1xTAE and .8g agarose and .5uL Etbr. Only the third lane exhibits a bright band, indicating that the amplification reaction was successful at 65 C only.

Lane 1: 100bp ladder Lane 2: WbmDXR g-block 1a @ 56 C Lane 3: WbmDXR g-block 1b @ 57 C Lane 4: WbmDXR g-block 1c @ 58 C  Made the gel with 60 mL 1xTAE and .8g agarose and .5uL Etbr. All lanes exhibit a very bright band, indicating that the amplification was successful at all three trial temperatures.

7/3 Lane 1: 1kb ladder Lane 2: 100bp ladder Lane 3: wbm dxr block 3 @ 54 Lane 4: wbm dxr block 3 @ 53 Lane 5: wbm dxr block 3 @ 55

Ran two ladders just in case. This time the results look good! Made the gel with 60 mL 1xTAE and .8g agarose and .5uL etbr, which formed a good solid gel, that wasnt dense with agarose particles, and wells formed well. The bright bands in the last two lanes indicate that the reaction is working well at these temps. It appears as though the 54 degree sample isnt producing any amplification. The bright bands are at the right size, since they are about 400 bp. Will measure the concentration of the two bright samples, then run the rxn again at the temp with higher concentration to make more of the gblock 3. Yay!

7/2 -ran gel on dxr 3 with Tm @53, 54, 55 Lane 1: 100bp ladder Lane 2: wbm dxr block 3 @ 54 Lane 3: wbm dxr block 3 @ 53 Lane 4: wbm dxr block 3 @ 55

This gel is super sketchy bc it looks grainy and everything looks blurry, including the ladder. Some faint bands seen at bottom, but the bright bands to the far right, with the temp varied, look promising. The gel just looks smeared, which may be a result of the buffer, gel quality, or voltage. Will run another gel.

7/1 Repeated pcr rxn but did three different samples, adjusting Tm one degree above and below from before. Samples were run at 53, 54, and 55 degrees. Also, changed all durations and number of cylces to max values. -did a second dilution on gblock and added only .5 ng of gblock to each sample this time, instead of 1 ng like last time.

6/26 -ran gel on dxr3 with Tm @ 54 Lane 1: 1kb ladder Lane 2: wbmdxr block 3, amplified sample, Tm @54

The 1kb ladder is a poor standard to compare against since the fragment is much smaller than the ladder indications, will use 100bp next time. the faint band seen towards the bottom of the gel could be 'primer dimers' or it could be the amplified product, the faintness of the band indicates it was not amplified very well. Next thing to try is to vary the duration of cycles/ number of cylces, and most importantly, vary the Tm(annealing temp), bc that will have the biggest effect.

6/25 -gel fell apart 6/24 -readjusted tris pH -resupended gblocks in tris -resuspended primers in tris -pcr rxn with q5 and wbdxr block 3 w/ f and r primers to amplify block -**gel check and sequencing**
 * -**picked up primers from mbb

6/19 -made primers for Wbm2F and Wbm1R longer to increase their Tm to be within two degrees of the other primer, ordered. -looked at q5 polymerase to adapt pLIC sequencing protocol -**need to do run 2 of maybridge--**scores for ligands from run 1 are ranging from 20-50, which are low... 6/17 -analyzed primers, melting temps too far apart for wbm dxr segments 1 and 2. primers will be diluted in TE.

6/13 -took out cultures and spun down, stored in -20 as pellets. **need to midiprep**

6/12 -grew overnight culture of pNIC-Bsa4 from Young's pnic dh5alpha w/ kan plate. Used lb from fridge. Preparing vector for gibson assembly. -ordered primers for pcr amplification of wbmdxr gblocks, since there are only 200ng. Gibson assembly will require higher concentrations.

6/11 -Submitted Maybridge Virtual Screen -Submitted validation dock with dxp, obtained from pubchem and did ligprep on it. Validation dock gave a score of 20, which was very low--might need to reanalyze how the screening files were set up. Also, a validation dock with dxp and the actual molecule from which it was extracted/crystallized (dxr from similar species) should be done for comparison.

6/10 -Set up GOLD virtual screen -analyzed active site of DXR across 4 organisms, see "protocol virtual screen" on ddfe for images

6/6 -created homology model off of 1r0kb in ICM(automatic regularization) using ProModel protocol -MolProbity analysis on icm model and template (hydrogens added for both) -The icm model is better than the previous homology model made on SWISS-Model -See ICM folder on ddfe for results

6/5 -ordered gibson assembly master mix -ran template identification tool on SWISS MODEL: 1r0kB identified as best template for WbmDXR. This is the same template that was used for the first homology model. See ProMol Project for ICM hom. model protocol

6/4 -Met with Dr. B to discuss summer research plan -ordered gblocks from IDT. see gblocks word document for details

= FALL 2012 Results =

2/15

--virtual screening--make hom. model with icm --positive control ligands- get from pubchem and do ligprep

-validation dock --sci. lit. drug delivery for anti-filarial/helmynth, difference btw. microfilariae and adult worms


 * Week 14:**

-Updated Lab Nb -Submitted Final Research Report
 * 12/8**
 * -**Lab Cleanup

-Last day:-( but will be mentoring:-) -Happy Holidays!!!!


 * 12/7**
 * -**Inhibition Assay with FtHap


 * Table 1.** The sample column indicates the concentration(mM) of the inhibitor, compound 5250098. The first sample was the negative control and contained .1mM inhibitor but no enzyme. The last sample was the positive control and contained enzyme and a known inhibitor, orthovanadate. Orthovanadate is a commonly used general competitive inhibitor for protein phosphatases. The rest of the samples contained increasing amounts of compound 5250098. Two readings were taken for each sample and then averaged. The readings were pretty precise, as indicated by the small standard deviation for the two readings. The average absorbance for the samples steadily decreased as the concentration of the inhibitor increased, which indicates the inhibitor was effective, and that the enzyme was functional. However, the decrease in absorbance was not dramatic or marked, so this inhibitor is not super effective at these concentrations. The highest concentration, .6mM, of compound 5250098 yielded an absorbance higher than that obtained by just .001mM of orthovanadate.


 * Figure 1.** Results from the inhibition assay with FtHap and compound 5250098. The absorbance of the samples steadily decreased with an increase in inhibitor concentration, as seen by the negative slope of the bar graph. However, the absorbance only decreased marginally with every increase in concentration. At .6mM, the inhibitor was less effective than the positive control was at just .001mM. The data shows that the enzyme is functional and that the inhibitor works, but the inhibitor is not very effective and is likely not a good potential drug.

Even though the enzyme assay from before yielded bad data, the inhibition assay data 'makes up' for it because it is clear that the enzyme is functional. The samples for the inhibition assay were yelllow, indicating that the substrate pNPP was phosphorylated in the reaction with the Histidine Acid Phosphatase. The darkest shade of yellow occurred with no inhibitor and the lightest shade occurred with the positive control. The negative control was clear. A red tide spec (Luke) was used and a different computer from last time was used.


 * NOTE:** I performed a second replicate of the inhibition assay, however I made a very big and careless error during the second run:-( This is unfortunate because it means I will have only one replicate of the inhibition data which was actually pretty good for the first run and extra sad because it took a while to do the assay and was essentially a waste. Due to time constraints, I will not repeat the second run. The error was that I forgot to add the substrate pNPP. I measured the absorbance on LoggerPro anyway, the table is shown below. Obviously, it is impossible to tell if the enzyme was functional or if the compounds worked to inhibit the enzyme if there is no substrate for the enzyme to phosphorylate. The data from the second run was not analyzed and will be discarded. Only one reading was taken.


 * Table 2.** As expected, the absorbance is near or at zero for all samples since there is no substrate present in the samples. The reading is probably just showing signal noise that the spectrophotometer picked up.

-analyzed final results from virtual screen run 2
 * 12/6**
 * -**analyzed virtual screen run 1 results and submitted cb306run2 and cbkinrun2


 * Table 2.** The Chembridge 306 Library, which contains 306 small compounds, was virtually screened against WbmDXR using GOLD. The library was screened twice, the first time the library was reduced to 30 potential inhibitors and the second time the selectivity was increased and the number of potential inhibitors was reduced to 10. The top 10 highest ranking ligands are shown in the table above. The highest fitness score was 70.59 This score is mediocre. The highest score from this library is lower than the highest score for the CBKinLibrary. The number of polar contacts for the top 3 ligands are shown. They ranged from 1-2, indicating that the ligands are not bound very strongly within the active site. The top three ligands were docked within the active site of WbmDXR in PyMol.

There is one polar contact between the enzyme and substrate, shown as black dashed lines. This indicates the ligand is not bound very tightly. The fitness score for this ligand, as determined by GOLD, was 70.59.
 * Figure 6.** PyMol representation of the highest ranking ligand from the CB306 Library docked within the active site of WbmDXR. The ligand, compound 6322987, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket.

There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 70.35.
 * Figure 7.** PyMol representation of the second highest ranking ligand from the CB306 Library docked within the active site of WbmDXR. The ligand, compound 6013108, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket.

There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 68.36.
 * Figure 8.** PyMol representation of the third highest ranking ligand from the CB306 Library docked within the active site of WbmDXR. The ligand, compound 6318163, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket.




 * Table 1.** The Chembridge KinLibrary, which contains 4000 small compounds, was virtually screened against WbmDXR using GOLD. The library was screened twice, the first time the library was reduced to 400 potential inhibitors and the second time the selectivity was increased and the number of potential inhibitors was reduced to 40. The top 10 highest ranking ligands are shown in the table above. The highest fitness score was 74.96. This score is not extremely high but it is a decent score. The number of polar contacts for the top 5 ligands are shown. They ranged from 1-3, indicating that the ligands are not bound very strongly within the active site. The top five ligands were docked within the active site of WbmDXR in PyMol.


 * Figure 1.** PyMol representation of the highest ranking ligand from the CBKin Library docked within the active site of WbmDXR. The ligand, compound 6322987, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket. There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 74.96.


 * Figure 2.** PyMol representation of the second highest ranking ligand from the CBKin Library docked within the active site of WbmDXR. The ligand, compound 6428065, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket. There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 70.61.


 * Figure 3.** PyMol representation of the third highest ranking ligand from the CBKin Library docked within the active site of WbmDXR. The ligand, compound 6352604, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket. There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 70.28.


 * Figure 4.** PyMol representation of the fourth highest ranking ligand from the CBKin Library docked within the active site of WbmDXR. The ligand, compound 6013108, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket. There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 69.67.


 * Figure 5.** PyMol representation of the fifth highest ranking ligand from the CBKin Library docked within the active site of WbmDXR. The ligand, compound 6596240, is shown as sticks. The protein WbmDXR is shown as surface. The surface display of the protein emphasizes the hydrophobic pocket of the active site. The ligand is seen to fit well within this pocket. There are two polar contacts between the enzyme and substrate, shown as black dashed lines. The fitness score for this ligand, as determined by GOLD, was 69.12.

-fixed active site of WbmDXR -resubmitted virtual screen cb306run1 and cbkinrun1
 * 12/5**
 * 12/3**
 * -**worked on fixing active site of WbmDXR


 * Week 13: 11/26-12/2**

-examination of the top inhibitors for WbmDXR from cb306 as designated by GOLD led to discovery of a badly defined active site. Since there was no ligand present or active site defined in the homology model, the active site was defined manually and verified on the visualizer in HERMES. While the active site appeared correct in HERMES while setting up the virtual screen, the results indicate that the active site was misplaced. (Need to fix this!!! Was unable to access gold/hermes from my laptop on sunday because was having trouble using xming/xlaunch, going to come in for help monday)
 * 12/2**


 * Figure 1**. Pymol representation of //Wbm//DXR, shown in blue as ribbon, and top ligand result, shown in green as sticks, from screening cb306. As can be seen, the active site was not defined correctly because the ligand is placed on the outside of the protein! It should be in the pocket in the center of the protein. In this position, there are no polar contacts between the ligand and protein and the maximum fitness score was 52.64.

-cb306 Run 1 finally worked! -submitted cb306 Run 2 virtual screen
 * 11/30**

-performed enzyme assay with FtHap to test if the enzyme was functional


 * Table 1.** The raw data obtained from the spectrophotometer during the assay is shown in columns 'Reading 1' and 'Reading 2'. The readings represent the absorbance of the sample at 410nm. The sample contained the enzyme FtHap and the substrate pNPP, and was incubated at 37 degrees celcius for ten minutes to allow for the reaction to occur. The reaction of histidine acid phosphatase with pNPP is supposed to produce a water soluble yellow product with absorbance at around 405-410 nm. So, an increase in enzyme concentration should yield more product and thus greater absorbance. Samples A through H correspond to enzyme concentrations of 0, .125, .25, .5, .75, 1, 1.25, and 1.5 uM respectively. The average of the two readings taken at each concentration is shown. The absorbance measured when there was no enzyme present in the sample was used as a control and was subtracted from the average readings. The standard deviation of the two readings at each concentration was also calculated.


 * Table 2.** The table shows the average absorbance reading measured at each enzyme concentration. The standard deviation calculated for the two readings at each concentration is also shown. This data is depicted in the graph below. See Table 1 for more data.




 * Graph 1.** The graph displays the absorbance of the samples measured at 410nm. As enzyme concentration increased, there should have also been an increase in absorption since a higher amount of enzyme would dephosphorylate more pNPP substrate and accordingly yield a higher absorbance. The standard deviations, as calculated in Tables 1 and 2, are plotted as vertical bars on the blue columns. The standard deviation bars indicate a large amount of error, which is concerning--the absorbance readings were not precise. Next time, more readings could be taken.

The graph shows inconsistent data but indicated that the enzyme is functional, since there was an absorbance measured for some of the samples. It is likely that the Spectro Vis spectrophotometer(William) was unreliable and giving bad readings, or that it wasn't calibrated correctly. I will proceed to the inhibition assay and use a larger concentration of enzyme in order to ensure that it will be functional. I will use a different spec and computer next time.

-submitted cb306 Run 1 virtual screen with new protein file
 * 11/29**

-diluted compounds with DMSO, entered data into dilutions spreadsheet on Google Docs -recreated WbmDXR protein file with Hermes setup
 * 11/28**

112612 - Good. Dr B -Stained the protein gel from 11/19, did FPLC, collected and concentrated the protein-containing sample. Note: the gel was not destained and dried, it was simply stained to verify presence of the protein FtHap.
 * Week 12:**
 * 11/20**

Graph obtained from Fast Protein Liquid Chromatography of FtHap. The buffer used was 100 mM Tris, 150 NaCl, pH 8.0. Approximately 5ml of concentrated FtHap sample was injected into the column. The first portion of the graph, from -5 to 10 mL, shows initial 'noise' and equilibration of the column. The pressure, shown in green, remains constant at less than .2MPa. The UV absorbance, measured at 280nm, is shown in solid blue, and exhibits a prominent hump from tube numbers 32-38(tube numbers shown in red). The large hump is presumably the protein of interest, and is at about the right location on the graph as compared to previous FPLC results with FtHap. Tubes 32-38 were saved, combined, and concentrated. The rest of the tubes were discarded.

NanoDrop reading for the absorbance of the FtHp sample following FPLC, before concentration. The sample was obtained from tubes 32-38, as indicated by the FPLC graph. The absorbance of the protein at 280nm was .47mg/ml. Though this is low, the absorbance should increase after concentrating the sample to 1 ml via centrifugation.

NanoDrop reading for the absorbance of the FtHp sample following FPLC, after concentration. The absorbance of the protein at 280nm was 2.26 mg/ml. The absorbance increased almost five-fold after concentrating the sample.

-purified the soluble fraction, concentrated the purified sample, ran a protein gel using premade gel and samples from protein expression
 * 11/19**

NanoDrop reading of Elution 1(upper) and Elution 2(lower) of FtHap. The absorbance of Elution 1 at 280nm was 1.78mg/ml. This absorbance is 'okay'. The absorbance of Elution 2 at 280nm was .25mg/ml. The absorbance is much lower for Elution 2 because most of the protein was eluted out with Elution 1. The next step is to run Elution 1 and 2 in a protein gel to verify the size of the protein bands and to perform FPLC to purify the protein sample.

Ok, try to include some type of 'data' - e.g. virtual results from CB306. - Dr. B 11/19/12
 * Week 11:**

-sonicated the cell lysate, spun down samples in JA17 rotor, and obtained soluble fraction (pH 7.96 and 8.00), then stored at 4 degrees C.
 * 11/18**

-monitored cell density until OD600 reached .5, at which point expression was induced with IPTG, and colonies were grown overnight at 25 degrees C. Janice spun down the samples the next day, on 11/17, and stored at -20 degrees C.
 * 11/16**

-Picked two colonies from FtHap transformation plate and began overnight growth in shaking incubator at 37 degrees C.
 * 11/15**

-Planned out protein expression
 * 11/14**

-Discussed using a new approach to clone Wbm DXR gene: Make fresh FOR and REV primers, perform PCR with first and last oligos, use pUC19 as cloning vector, and work with Janice and Daniel. Also, I will express, purify, and characterize someone else's protein target (Joey's target protein, FtHap) and perform an enzyme assay on the protein in order to gain experience with FPLC and assays.
 * 11/12**


 * Week 10: 11/5-11/11**

-Looked at run submitted on 11/9, for some reason the job was aborted before it finished, at around 98 ligands (306 ligands total). Resubmitted run. -Ran a test docking job with DHFR and 20dhfr-ligsRT using ICM
 * 11/10**


 * 11/9**

-Resubmitted virtual screen //Wbm//DXR vs. cb_306 -Looked at Daniel and Janice's results on my target. After creating a new oligo mix, primary and secondary PCR Rxns were carried out. Though there was a successful Primary PCR, the Secondary PCR failed. These results are consistent with what I have been seeing. Since Primary seems to work most of the time and Secondary usually does not work, the primers may be the source of the problem. They may need to be rediluted or reordered, or they may have been constructed incorrectly. However, these same primers were used when Secondary //did// work, so it is not likely that their sequences are incorrect. Overall, the PCR steps of //Wbm// DXR have yielded bad results during the fall semester, while in the summer, I was actually able to obtain a PCR insert without contamination and proceed to cloning. If possible, I would want to start over by ordering a new well plate containing new oligo primers. I think that the primers themselves are contaminated, which is why it has been impossible to amplify the gene successfully.

-Janice and Daniel are now working on //Wbm// DXR, debriefed them on my research so far.
 * 11/7**

-Performed a Gel Check on Secondary PCR Rxn from 11/2 Lane 1: Skip Lane 2: 100bp ladder Lane 3: Second Secondary PCR Sample

Nothing can be seen on the gel for the Second Secondary PCR. This is the second negative result. The Secondary PCR sample from 10/26, off of which the Second Secondary had been done, simply had too low of a concentration, with only 8.3ng/uL.

-Looked at Virtual Screen results from 11/2. Run had a fatal error, found the source of the error.


 * Week 9: 10/29-11/2**


 * 11/2**

-Redid Second Secondary PCR Reaction on Sample from 10/28. This is the second try, done to see if the Gel Check results will come out the same as from 10/31

-Submitted a virtual screen on GOLD, screened cb_306 against WbmDXR, ran job on one processor. As mentioned before, this is a 'test' virtual screen to validate the homology model. The active site on the homology model was defined by first aligning Wbm DXR with the PDB Structure 1QOL, a related DXR structure(//E.coli)// crystallized with the inhibitor Fosmidomycin.
 * Note: GOLD was accessed using Linux!

PyMol representation of WbmDXR(homology model based off of 1R0K), shown in magenta as ribbon, aligned with //E. coli// DXR(1Q0L), shown in blue as ribbon. The two are aligned in order to determine the active site of the homology model since it contains no ligands or inhibitors. The RMs value was 1.729. 1Q0L was used because it was a closely related structure that was crystallized with the inhibitor fosmidomycin. Fosmidomycin is shown in green, as sticks. The active site of the aligned proteins is shown as sticks, with magenta residues belonging to WbmDXR and blue residues belonging to EcDXR. The xyz coordinates of the magenta residues were used to locate the active site of the model in HERMES for the purpose of virtual screening.


 * 10/31**

-Gel Check on Second Secondary PCR

Lane 1: Skip Lane 2: 100bp ladder Lane 3: Second Secondary PCR Sample

The method of performing a second Secondary PCR on the gel extract appears to have not worked. It is possible that it was simply a bad sample or a bad thermal cycler machine, so I will attempt the second Secondary PCR reaction on the sample from 10/28 once more.

-Began preparing for virtual screen by setting up directory, running homology through MolProbity, etc., but was unable to access GOLD to submit a run. GOLD was running extremely slow, could not access interface on Windows PC.

Image 1. MolProbity Kinemage of Wbm DXR Homology model, shown as ribbon. The pink indicates error in the model. All of the pink error regions were due to 'bad overlap'.

A large concern for this homology model is its high percentage of error. The homology model was created based on a template that most resembled Wbm DXR. However, this template PDB structure was crystallized without a ligand, so it will be a little more difficult to define the active site. Because there is a chance this model may not dock ligands at all, I will first perform a 'test' virtual screen with the cb_306 library rather than screen the entire Chembridge Diversity Library. Then I will analyze these results.


 * 10/29**
 * -'**Second' Secondary PCR reaction using 1 uL from gel extraction sample from 10/28


 * Week 8: 10/22-10/28**

-Used PCR Cleanup Kit on gel extraction sample from 10/26.
 * 10/28**

Figure 1. NanoDrop results for Secondary PCR Sample following gel extraction and PCR cleanup. As expected, the concentration was very low due to loss of massive amounts of DNA in both gel extraction and pcr cleanup steps. However, this clean sample will be used to redo Secondary PCR, so a high concentration is not necessary for this step! Redoing Secondary PCR with this clean sample should increase the concentration but have (ideally) no contamination, and from there, PCR SQ will be carried out.

The current plan is to perform gel extraction on PCR 2, redo PCR 2, then continue to PCR SQ, because this method will remove contamination at an earlier step and will hopefully result in a higher PCR SQ yield.
 * 10/26**

-Performed PCR 2 with PCR 1 Sample A from 10/3. 4 identical tubes, Tm 58 C. -Ran 8 lanes on the gel. *new agarose worked well *used a different brand of 100bp ladder -Cut out desired bands from gel -Used Gel Clean Up Kit to extract DNA from gel

Lane 1: Skip Lane 2: Fermentas 100bp Ladder Lane 3-11 Secondary PCR Sample, Tm 58 degrees C

Bands were not as bright as desired, some lanes are extremely faint. The first two lanes came from the first sample tube, the second pair of lanes came from the second sample tube, and so on. It appears as though sample tubes 1 and 3 were the most successful. The appearance of the ladder is different because a different brand than usual was used. The Fermentas ladder looks 'scrunched up' compared to the NEB ladder. The next step is to excise the desired band from the gel. This will be important as it will isolate the desired DNA from the contaminants.

-Target Meetings
 * 10/24**
 * -**Open House
 * in the near future, could possibly work with Janice and Aldo on FrtDXR, or work with Daniel on different target within Wolbachia, or have them join my target

102112- Urvashi, ok homology model stuff looks good. Glad they both gave the same template. Not sure on the wetlab stuff. Did you also try lowering the annealing temp in the PCR squared ? The confusing thing is that why you wouldn't see that in secondary? One option is doing gel purification on Secondary PCR before doing PCR Squared. -- Dr. B -cut pNIC-Bsa4 accepting vector. Accepting Vector had concentration of 106ng/uL and had some deletions in the sequence(mostly in the middle SacB region), with a 83% query coverage and 98% max identity with the pNIC-Bsa4 FASTA sequence, according to BLASTn. Incubated at 37 C for 2 hrs. -analyzed Homology model results -PCR cleanup on pNIC-Bsa4, eluted with 30uL pre-warmed elution solution. -updated notebook
 * Week 7: 10/15-10/21**
 * 10/21**

NanoDrop results for cut pNIC Bsa4. The concentration was 9.4 ng/uL.

This is probably too low to proceed to cloning with. Unsure of why the yield is so low. Possible reasons are bad PCR cleanup reagents and bad technique.

The next step will be to continue to work on getting a PCR insert with a high concentration. This time, I will make more Secondary PCR and perform gel extraction on it before performing PCR Squared.

-Created Homology model using SWISS-MODEL.
 * 10/20**
 * -**Ran gel check on Secondary PCR from 10/19

Secondary PCR Gel Check Lane 1: 100bp ladder Lane 2: Secondary PCR Sample, Tm 59° C Lane 3: Secondary PCR Sample, Tm 60° C As the gel shows, raising the annealing temperature to 59° C did not decrease contamination, but rather, seemed to increase it—there are a lot more extra bands showing up than before. Raising the annealing temperature to 60° C did not work either—there is zero amplification seen in Lane 3. This indicates that this temperature is too high. One reason for the large amounts of contamination seen might be the fact that the lid cover of the deep well plate had been placed on incorrectly, noticed on 9/19/12, when the most recent oligo mix was made. Since the lid cover was on backwards, cross contamination is a definite possibility, and it is possible that primers from another gene(not Wbm DXR)from the same deep well plate are in the Wbm DXR oligo mix and are the cause of the extra bands that are being seen consistently.

The template identification results showed 1r0kB, which is DXR from Zymonas Mobilis, as the top hit for the Gapped Blast. The image above shows the alignment of the Wbm DXR sequence with the Zymonas Mobilis DXR. The score was 293, with a 42% identity match and 63% positives.
 * SWISS-MODEL Template Identification Results**


 * SWISS-MODEL Automated Modelling Results**

The results show a modelled residue range of 2-383. The model template was 1r0kB, which is DXR from Zymonas Mobilis. There was a 42.34% sequence identity, a QMEAN Z-Score of -.421, and a QMEANscore4 of .741.

MolProbity Analysis of homology model for Wbm DXR created with SWISS-MODEL Auto Modelling, with 1r0kB used as the template. The MolProbity analysis is not very good, the percentages of Ramachandran outliers, residues with bad bonds, and residues with bad angles are all significantly higher than the listed 'Goal' percentage.

Regularization with ICM will be done to further optimize the model.


 * BLASTp Results**



The BLASTp results are consistent with SWISS-MODEL. Both results give DXR from Zymonas mobilis as the best match for Wbm DXR.

-Ran Secondary PCR Rxn with Sample A from 10/5: Tube A with annealing temperature at 59 degrees C and Tube B with annealing temperature at 60 degrees C -Got new Agarose from BioSci Storeroom
 * 10/19**

101612 - ok this PCR quantity is rather low. But you may be able to make it work. I would recommend doing gel extraction next time if you can't get the contaminating bands out with a change in temp on PCR squared. Also, PCR cleanup won't get rid of those contaminating bands unless they are really small or really big. -- Dr. B PCR Cleanup with new GenElute Kit(9/10/12) on PCR SQ Samples from 10/8 -all 8 samples combined into binding column -eluted with 50 uL prewarmed(37 degrees celcius) Elution Solution
 * Week 6: 10/8-10/12**
 * 10/9**

NanoDrop reading for Wbm DXR following PCR cleanup. The concentration was 13.6 ng/uL.

This concentration is too low to proceed to cloning. It is possible that a significant amount of DNA was lost in PCR Cleanup, or that there wasn’t much DNA in the PCR Squared sample to begin with. PCR SQ will be redone, and this time, I will vary different factors in an attempt to minimize contamination and maximize amplification of Wbm DXR, in order to obtain a high concentration.

Performed gel check on PCR SQ
 * 10/8**Performed PCR Squared Rxn with the Secondary PCR Sample A from 10/5/12. Tm=58 degrees C.

PCR Squared Lane 1: Skip Lane 2: 100bp ladder Lane 3-10: PCR Squared Sample, 50uL aliquots

The reaction was successful, and Wbm DXR was amplified in 6 of the 8 aliquoted samples, as seen above. The bright bands seen at about 1200 bp(second band from top on ladder) are the gene of interest. Unfortunately, there is some contamination, including primer dimers, above and below Wbm DXR, but hopefully PCR cleanup will be able to remove most of this contamination. . The reason for only 6 of the 8 aliquoted samples is unclear, since the samples were part of the same master mix and same PCR reaction. Perhaps the samples that show no bands on the gel check did not contain the necessary reagents as a result of improper mixing and aliquoting. Another reason is that the samples were not heated and cooled properly during the PCR reaction—the samples were distributed across the thermal cycler block and the ones that didn’t work could have been placed in a bad spot. Finally, it is possible that EtBr did not diffuse into that one area of the gel and so no bands are showing.

The next step is to proceed to PCR Cleanup. All 8 samples will be combined into the binding column.

100912 - So, maybe use a higher melt temp for your PCR^2 ?? - perhaps you could get rid of some of the spurious bands. - Dr. B Also We have new T4 DNA Poly that may work for your cloning (new dGTP and new dCTP also)

100912 - ok nice PCR!. Performed gel check on Primary and Secondary PCR
 * Week 5: 10/1-10/5**
 * 10/5** Performed Secondary PCR on Peltier Thermal cycler with Primary template from 10/3 and FOR and REV primers from 9/20, with annealing temp. at 58 degrees C.

Primary and Secondary Lane 1: Skip Lane 2: 100bp ladder Lane 3: Primary PCR Sample A Lane 4: Secondary PCR Sample A Lane 5: Primary PCR Sample B Lane 6: Secondary PCR Sample B

Note: Sample A and B are simply duplicates, carried out the same way (to serve as back up in case of error)

Sample A Primary and Secondary PCR was successful. Sample B Primary was successful but Secondary was not. Lane 4 shows a bright band at about 1200 bp, which is Wbm DXR. There is some contamination above the amplified band. I will proceed to PCR SQ with Sample A, but I will not perform gel extraction since the band appears to be clean enough. Skipping gel extraction will allow me to lose a lot less DNA. Changing the Tm to 58 degrees celcius seems to have improved the brightness of the band as well as decreased the smear above the band that was seen in the last attempt.


 * 10/3** Performed Primary PCR on Peltier Thermal cycler with oligo mix from 9/19, with annealing temp. at 58 degrees C.


 * 10/2** Used PCR Clean Up Kit (Gen Elute) to clean up DNA extracted from gel. Eluted with 50uL 10mM pH 8 Tris HCl.

Nanodrop reading for Wbm DXR after PCR clean up. A lot of DNA was lost, as can be seen by the very low concentration of 16.6 ng/uL. The concentration is too low to proceed to cloning.

The PCR reactions will have to be redone again. Stocks of Primary and Secondary PCR reactions from the last attempt were depleted, so the PCR reactions will be redone beginning with Primary. The stocks of the oligo mix and FOR and REV primers were not depleted, so those will be reused. Also, the annealing temperature for the PCR reaction will be changed in order to prevent some of the contamination that was seen in the last attempt. **Tm will be changed from 57 degrees C to 58 degrees C.**


 * Week 4: 9/23-9/28**
 * 1001112 - Urvashi, ok the band instensity is not great. But let's see what you get on Nanodrop after gel extractions. -- Dr. B**

Performed Gel Check for PCR SQ--put all of the sample into the wells Performed gel extraction to cut out desired band and used GenElute Kit to clean up the gel
 * 9/29** Performed PCR SQ Rxn with Secondary PCR Sample A from 9/28, used same thermocycler as on 9/28

PCR Squared Lane 1: 100bp ladder Lane 2-8: PCR SQ Sample, 50 uL aliquots

The PCR Squared Reaction, performed with Secondary PCR Sample A from 9/28, was successful. Wbm DXR was amplified, as can be seen by the bright bands in all 7 lanes at the second marker (from the top) of the 100bp ladder. However, there is a heavy amount of contamination both above and below the gene of interest. The reason for this is unclear, but in order to get rid of this contamination and increase the chances of just the gene of interest being inserted into the pNIC-Bsa4 accepting vector later on, gel extraction and gel cleanup was performed.

The next step is to perform PCR Cleanup and go on to cloning the gene using pNIC-Bsa4.

Performed Gel check for Secondary PCR
 * 9/28** Redid Secondary PCR Rxn, using a different thermocycler machine. Used Primary PCR samples A and B from 9/21.

Secondary PCR Lane 1: Skip Lane 2-4: AB Samples Lane 5-6: Skip Lane 7: 100bp Ladder Lane 8: Secondary PCR Sample A Lane 9: Secondary PCR Sample B

Since the last attempt at secondary PCR failed, the reaction was redone using the successful primary PCR samples A and B from 9/21, and a different thermocycler was used. The gel shows that Sample A worked, while Sample B did not. The Wbm DXR gene was amplified and can be seen at about 1200bp, at the second marker on the 100bp ladder. There is a smear above the gene of interest, this may be due to leftover fragments from Primary PCR. I will proceed to PCR Squared with Sample A.


 * Week 3: 9/17-9/21**

Urvashi - Janice had luck using a different PCR machine. You may try that out -- Dr. B

um474_UrvashiMitbander_92112_VirtualScreenRefresherPt2, because the file was too large to upload.
 * 9/22** Analyzed Virtual Screen results. Results on Google Drive. **Note:** The results are posted in two parts, um474_UrvashiMitbander_92112_VirtualScreenRefresherPt1 AND

Performed Gel Check for Primary and Secondary PCR Submitted Second Run for Virtual Screen Refresher Cleaned out personal freezer box--got rid of most old samples from the summer
 * 9/21** Performed Secondary PCR Reaction, second attempt

Primary and Secondary PCR Gel Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Primary PCR Sample A Lane 4: Secondary PCR Sample A  Lane 5: Primary PCR Sample B  Lane 6: Secondary PCR Sample B

The second attempt for Primary PCR was successful but once again, the Secondary PCR was unsuccessful. Since fresh FOR and REV primers were used, the reason for the gene of interest not amplifying is unclear. The protocol was carried out in the same way as over the summer, when the secondary PCR had been successful. The Secondary PCR reaction will have to be redone. It will be helpful to perform the secondary reaction with with the first and last oligos from the oligo mix, to determine whether the primers are causing the problem or if the oligo mix is causing the problem.

Diluted Wbm FOR and REV primers with autoclaved nanopure. Performed Primary PCR Reaction, second attempt Submitted First Run for Virtual Screen Refresher
 * 9/20**


 * 9/19** Primer Assembly step performed. (Oligo mix for Wbm DXR remade.) Lid of deep well plate was on backwards--possibility of cross contamination.


 * Week 2: 9/10-9/14**

Urvashi - ok - try again with fresh everything. - Dr. B 091812

PCR Primer Overlap Protocol

PCR Squared Gel Lane 1: Skipped Lane 2: 1kb ladder Lane 3: PCR SQ Sample 1 (A) Lane 4: PCR SQ Sample 2 (A) Lane 5: PCR SQ Sample 3 (A) Lane 6: PCR SQ Sample 4 (A) Lane 7: PCR SQ Sample 5 (B) Lane 8: PCR SQ Sample 6 (B) Lane 9: PCR SQ Sample 7 (B) Lane 10: PCR SQ Sample 8 (B)
 * 9/15**:

PCR Squared was done using the Secondary PCR from Sample A (lanes 3-6) and Secondary PCR from Sample B (lanes 7-10) as templates. As predicted, the reaction failed. No band can be seen at around 1170 bp, the size of the Wbm DXR gene that should have been amplified. Some contamination can be seen at the bottom the gel. These may be primer dimers. This step will be redone with new Secondary PCR templates.

The PCR Squared Reaction was carried out for Samples A and B (before the gel below had finished running). However, since the secondary PCR for both reactions failed the gel check, it is highly unlikely that PCR squared was successful for either Sample. The gel check for PCR Squared will be performed tomorrow. Secondary PCR will be redone with 'fresh' forward and reverse Wbm DXR primers.
 * 9/14:**

Primary and Secondary PCR Gel Lane 1: Skipped Lane 2: 1 kb ladder Lane 3: Primary PCR Sample A Lane 4: Secondary PCR Sample A  Lane 5: Primary PCR Sample B  Lane 6: Secondary PCR Sample B

This was attempt 2. This is the second gel check for the same PCR reactions (Sample A and B) that were carried out on 9/12/12. This time, the image was successful since you can see the ladder and samples. The Primary PCR reaction can be seen as a streak in Lanes 3 and 5. The Sample B Primary PCR Reaction was more successful than Sample A because the streak in Lane 5 is brighter and longer, indicating that the oligos were assembled correctly. However, the Secondary PCR reactions for both Sample A and B were unsuccessful. Nothing is seen in Lanes 4 and 6. This indicates that the gene Wbm DXR was not amplified. It is possible that the forward and reverse primers responsible for this amplification had degraded, because the primers used were 3 weeks old.

Primary and Secondary PCR Gel
 * 9/12:**

Lane 1: 100bp ladder Lane 2:Primary PCR Sample A Lane 3: Secondary PCR Sample A Lane 4:Primary PCR Sample B Lane 5: Secondary PCR Sample B Lanes 6-9: Max M.'s Samples

This was attempt 1. Since nothing can be seen on this gel, including the ladder, there was probably something wrong with the gel itself, rather than the samples. EtBr that is contaminated or not concentrated enough is the most likely cause for this failed gel image.


 * Week 1: 8/29-9/7**

PyMol Refresher Lab

Results are posted in Google Drive: [|um474_UrvashiMitbander_090712_PyMolRefresher]

= SUMMER 2012 Results = __**BLAST Results**__

​


 * Above: Codon Optimized gene sequence for Wbm DXR aligned with the forward read of Wbm DXR. There was an 18% query coverage with 100% identity match. This shows that only a part of the gene(tail end) was inserted into the vector. The cloning was unsuccessful.**




 * Above: Codon Optimized gene sequence for Wbm DXR aligned with the reverse read of Wbm DXR. There was an 18% query coverage with 100% identity match. This shows that only a part of the gene(tail end) was inserted into the vector. The cloning was unsuccessful.**

__**DNA Sequencing Results**__

NNNNNNNNNNGNNNNNTTTAGNNGAGNNNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCG AGAACCTGTACTTCCAATCCATGAAGAAAATTTCCGTTCTGAACGCTGCGAACGAAGTTGCAGTGGACAAATTTCTGAAA TCTCGCATCGATTTCCTGGAGATTATCAAAGTTGTTAAACTCACCGTTGAAAACTTCGACTCTTATACCGACATCAACTC CCTCTCTGACATC ATTAACATTGACCTCGAAAGCCGTGCGATTGCAAAAGAGATCATCAAAAACAAAGTTCTGGCGTACT CTTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACC ACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAA CCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCG CCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCC CGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG CCCTGATAGACGGNTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCNAACTGGAACAAC ACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGANTTTGCCNATTTCGGCCTATTGGTTAAAAAATGAGCTGA NTTAACAAAAATTTAACGCGAATTTTAACAAATATTAACGCTTACNNTTTNNNNGCACTTTTCCGGGGAAATGTGCGNNN NCCNNNNTTGTTNNTTTTNNAANACNNNCNANTATGNANCNCTCATNNNNNNNNTANAAAAANTCNTCNANNNTNNANNN ANCNNCANTNNNTCNNNNNCNNGNNTNTNNNNCNNNNTTTNAAAANNCNNNNTNNNGNNNNNNNNNNAANNNNNNNNNNN NNNNNNNNNANNNNNANNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNCNNNNNNN
 * //Above: DNA Sequencing Result for the combined samples 1 and 2. Wbm DXR was read with the primer pLIC-for. This is the forward sequence of the gene.//**

NNNNNNNNNNNNNNNCNNNNGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGAT CCGTATCCACCTTTACTGTTAAGAGTAC GCCAGAACTTTGTTTTTGATGATCTCTTTTGCAATCGCACGGCTTTCGAGGT CAATGTTAATGATGTCAGAGAGGGAGTTGATGTCGGTATAAGAGTCGAAGTTTTCAACGGTGAGTTTAACAACTTTGATA ATCTCCAGGAAATCGATGCGAGATTTCAGAAATTTGTCCACTGCAACTTCGTTCGCAGCGTTCAGAACGGAAATTTTCTT CATGGATTGGAAGTACAGGTTCTCGGTACCCAGATCTACACCAGAAGAATGATGATGATGATGGTGCATATGTATATCTC CTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATT TCGCGGGATCGAGATCTC GATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCG CCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATG GTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGG CCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGG CGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCNNGGTGGTGAATGTGAAACCAGTAACGTT TACGATGTCGCANANTATGCCNGTGTCTCTTATCANANCGTTTCCCGCGTGGNGAACCNNNCCNGCCNCGTTTTCTGCG AAACGCGNNAAAAANTNGGAANNNGGCNATGGCGGANCTGAATNNNNTTNCCCANNNNNGTNNNNNNNCNACTGGNNNNN NNNNNTCNNNNCNGATTGNNNTTGCCNNCNNCCNGNNNNGGNCCNGCACGNGNCNNNNNCNAANNNNNNNNNNNNNNNTN NAANNNNNNNNCNANNNNNGGNNNNCNNNNNNNNGNNNNNNNNGNNNAANNNNNNNNNNNNNNNNNANNNNNNNNNNNNN NNCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNN
 * //Above: DNA Sequencing Result for the combined samples 1 and 2. Wbm DXR was read with the primer pLIC-rev. This is the reverse sequence of the gene.//**

These reads are significantly better than last time. The next step is to BLAST these sequences.

__**Gene Insert**__

The nanodrop reading above shows the concentration of the pNIC Bsa4 plasmid containing the Wbm DXR gene. The image above is for samples 1 and 2 combined. There were only two //E. coli// colonies on the growth plate. The two samples were combined at the PCR cleanup step.

__**Cutting pNIC Bsa4**__

Lane 1: Skipped Lane 2: 1kb ladder Lane 3: pNIC Bsa4 plasmid treated with BsaI

The gel image indicates the plasmid was cut correctly by the restriction enzyme. The two fragments of the plasmid can be seen as bright bands in Lane 3.




 * Fig. 1** The above nanodrop reading is for the cut pNIC bsa4 plasmid. The concentration was 43.8 ng/uL. The concentration was checked to ensure there was enough plasmid within the sample.

Since pNIC was cut properly and has an acceptable concentration, the Wbm DXR gene will now be inserted within the plasmid.

__**PCR Cleanup**__
 * Fig. 1** Above is the nanodrop reading for Wbm DXR. The PCR Squared samples were combined and cleaned using the PCR Clean Up Kit. The concentration was 51.9 ng/uL.

__**PCR Squared, Trial** **3**__



Lane 1: skipped Lane 2: 100 bp ladder Lane 3: PCR Squared Rxn A Lane 4: PCR Squared Rxn B Lane 5: PCR Squared Rxn C Lane 6: PCR Squared Rxn D

The gel indicates that the PCR Squared reaction was successful for three of four samples. Faint primer dimers are seen towards the bottom. The bright bands indicate the amplified gene of interest, at about 1200 bp. These samples will proceed to PCR Cleanup.

__**Primary and Secondary P**____**CR, Trial 3**__

Lane 1: skipped Lane 2: 100bp ladder Lane 2: Primary PCR Sample 1 Lane 3: Secondary PCR Sample 1 Lane 4: Primary PCR Sample 2 Lane 5: Secondary PCR Sample 2

Two samples were made. Primary PCR failed for Sample 2, and accordingly, Secondary PCR for Sample 2 failed as well. Sample 1, however, had a successful primary and secondary PCR. There is little to no contamination seen for Sample 1 Secondary PCR. Sample 1 will proceed to the PCR Squared reaction.


 * __Protein Purification__**
 * //7/13/12//**

The protein mTb PSTP (Sadhana's protein) was expressed and purified. The following NanoDrop results show the OD for Elutions 1 and 2. The results show that most of the protein was released with Elution 1.

Figure 2. The OD of Elution 2 was .01 mg/mL.

Figure 1. The OD of Elution 1 was .46 mg/mL.

A master plate containing //E.coli// colonies transformed with WoBm DXR was created. 8 samples from the master plate were grown in LB and spun down. The protein pellets obtained after centrifugation were then eluted using the MiniPrep kit. The concentrations of the 8 samples are shown below. Some samples had larger pellets than others and accordingly had higher concentrations of WoBm DXR.
 * __Miniprep with WoBm DXR__**
 * //7/11/12//**

Figure 1. Concentration of WoBm DXR for Sample 1 was 70.2 ng/uL. This was the highest concentration.

Figure 2. Concentration of WoBm DXR for Sample 2 was 47.8 ng/uL.

Figure 3. Concentration of WoBm DXR for Sample 3 was 44.8 ng/uL. Add Link Figure 4. Concentration of WoBm DXR for Sample 4 was 26.3 ng/uL.

Figure 5. Concentration of WoBm DXR for Sample 5 was 53.4 ng/uL.

Figure 6. Concentration of WoBm DXR for Sample 6 was 31.1 ng/uL.



Figure 7. Concentration of WoBm DXR for Sample 7 was 25.3 ng/uL. This was the lowest concentration.

Figure 8. Concentration of WoBm DXR for Sample 8 was 28.8 ng/uL


 * __Clonin__****__g wit__****__h__** **__pNIC-Bsa4__**
 * //7/11/12//**
 * Figure 1.** NanoDrop results for pNIC-Bsa4 plasmid (accepting vector). The sample containing the plasmid was eluted using the PCR cleanup kit. The concentration was 22.8 ng/uL.

Lane 1: 100bp ladder Lane 2: pNIC-Bsa4 plasmid cut with BsaI
 * Figure 2.** The gel above was run to ensure that the accepting vector, pNIc-Bsa4, was cut correctly. The two bright, clearly defined bands lane 2 show that the plasmid was cut successfully with the restriction enzyme BsaI.

The gene for WoBm DXR was then inserted into the vector and cloned using ligation independent cloning.


 * __PCR Cleanup__**
 * //7/06/12//**
 * Figure 1.** NanoDrop results for WoBm DXR after PCR cleanup. The PCR Squared reaction was eluted using the Sigma PCR cleanup kit. The concentration of the sample was 21.6 ng/uL.

The concentration was a little lower than expected. The next step is to clone the WoBm DXR gene into the accepting vector, pNIC-Bsa4.

__**PCR Squared**__
 * //7/03/12//**

Lane 1: 100bp ladder Lane 2: PCR Squared Reaction A Lane 3: PCR Squared Reaction B Lane 4: PCR Squared Reaction C Lane 5: PCR Squared Reaction D

4 PCR squared reaction samples are shown in lanes 2-5. This step was performed to create more PCR product in preparation for PCR cleanup. The gene for the target protein is seen as bright bands at about 1200 bp. There are faint bands seen below, which are either from contamination or unwanted gene fragments inserting themselves into the vector. The next step is PCR cleanup.

__**PCR Target Primer Overlap (Option B)**__
 * // 7/02/12 //**

Lane 1: 100 bp DNA ladder Lane 2: Secondary PCR sample

Secondary PCR with custom primers(sticky ends) amplified gene of interest, seen as a bright band above 1000 bp mark. The gene itself is about 1200 bp long. Two smaller fragments can be seen lower down. These will hopefully be taken care of with PCR cleanup and will not insert themselves into the plasmid vector during the cloning process.

__**MidiPrep**__


 * Figure 1. The two graphs above show the concentration of pNIC-Bsa4 plasmid DNA, as measured by Nanodrop. The concentration for the first measurement was 53.2 ng/uL and 65.4 ng/uL for the second measurement. The average concentration was 59.3 ng/uL. **

The Midiprep kit was used to make pNIC-Bsa4 stock plasmid from //E. coli// colonies that were previously transformed with the plasmid, grown overnight, and spun down. A sample of the stock plasmid was submitted to ICMB Core facilities for sequencing. The sequencing results are shown below.

NNNNNNNNTNNACTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGA ACCTGTACTTCCAATCCATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTCCGCGACGTGGCTGACACCGCGACCGCG CTGCGTCCGGGTCGTCTGTTCCGTAGCTCTGAACTGTCTCGTCTCGATGATGCCGGTCGTGCCACCCTGCGCCGTCTGGG TATCACCGACGTTGCCGATCTCCGCTCTTCCCGTGAAGTTGCTCGTCGTGGTCCAGGTCGTGTTCCAGATGGTATCGACG TTCACCTGCTGCCGTTTCCTGATCTCGCGGACGATGACGCGGATGACTCTGCGCCTCACGAAACCGCATTCAAACGTCTG CTGACCAACGACGGCTCTAATGGCGAATCTGGTGAATCTTCTCAGTCTATCAATGATGCAGCCACTCGTTACATGACCGA CGAATATCGTCAATTCCCGACCCGTAATGGTGCGCAGCGTGCCCTGCATCGTGTTGTAACCCTCCTGGCCGCTGGCCGTC CGGTTCTGACCCACTGCTTTGCGGGTAAAGACCGTACCGGCTTCGTTGTTGCACTGGTTCTGGAAGCGGTTGGTCTGGAT CGTGACGTAATTGTGGCGGACTACCTCCGTTCTAACGACAGCGTTCCGCAACTGCGTGCGCGTATCTCTGNNTGATCCAG CAGCGTTTCGACACCGAACTGGCACCGGAGGTTGTTACCTTCACCAAAGCGCGTCTGTCTGACGGCGTTCTCGGTGTTCG TGCGGAATACCTGGCAGCGGCTCGTCAAACCATCGACGAAACTTACGGTTCTCTGGGTGGTTACCTGCGTGATGCGGGCA TCTCTCNNNGACCGTTAACCGTATGCGTGGTGTTCTCCTGGGTTAACAGTAAAGGTGGATANGGATCCGAATTCGAGCTC CGTCGANAGCTTGCGNCNCACTCGAGCACNCNCNCNCNCTGANANNNNCTGCTANNAAGCCGAAAGNNCTNANTNNTGCT GCACGNTGANCATANTAGCATANCCTNGGNNCNNNNGNNNTGNNGGNTTTTNNNNNNNNNGACTNNNTCNNNGNNANGGN NNNNNNNNNNNNGNNNTAGCNNNNNNNNNGNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NTNNNNNNNNNNNNNNNNNNNN

A nucleotide BLAST analysis for highly similar sequences revealed that the plasmid was not, as previously thought, just pNIC Bsa4 but rather pNIc Bsa4 with the mycobacterium tuberculosis gene for serine threonine protein phosphatase cloned into it, mTb PSTP. The initial colonies chosen to grow into large cultures prior to isolating the plasmid with MidiPrep were picked from the incorrect plate (Sadhana's plate from last year).

__**PCR Target Primer Overlap (Option A)**__

Lane 1: skipped Lane 2: 100 bp DNA ladder Lane 3: Primary PCR sample 1 (w/ Thermopol buffer) Lane 4: Primary PCR sample 2 (w/ Thermopol buffer) Lane 6: Secondary PCR sample 1 (w/ Thermopol buffer) Lane 7: Secondary PCR sample 2 (w/ Thermopol buffer) Lane 8: Secondary PCR sample 3 (w/ KOD buffer)well broken Lane 9: Secondary PCR sample 3 (w/ KOD buffer)well broken
 * Lane 5:** Primary PCR sample 3 (w/ KOD buffer)
 * Lane 10:** Secondary PCR sample 3 (w/ KOD buffer)

Primary PCR of assembled target oligos resulted in piecing together of full length DNA for WoBm DXR, seen as a long bright smear in Lane 5. The products of the primary PCR contained DNA fragments in addition to the full length gene. Secondary PCR with forward and reverse primers(blunt ends) resulted in amplification of full length gene of WoBm DXR, seen as a bright bar in Lane 8.

__**PCR pNIC-Bsa4**__



Lane 1: 100bp DNA ladder

PCR for amplification of cloning DNA sequence of pNIC-Bsa4 with pLIC-for and pLIC-rev primers. //PCR failed//

__**PCR pmCherry**__



Lane 1: 100bp DNA ladder

PCR cloning of pmCherry into pGBR22 multiple cloning site. //PCR failed.//

__**PCR pGBR22**__



Lane 1: 100bp DNA ladder (not seen) Lane 2: Sample A containing .3 ng of pGBR22 Lane 3: Sample B containing 3 ng of pGBR22 Lane 4: Sample C containing 30 ng of pGBR22 Lane 5: Sample D containing 0 ng of pGBR22

M13 for and M13 rev primers were used to amplify the purple protein coding sequence of pGBR22. The amplification is seen as bars of varying brightness in lanes 2-5. Samples A-C should be increasing in brightness since the plasmid concentration of the samples increased. Sample D should not be seen because there was no plasmid in the sample.


 * __DNA Sequencing__**

//pNIC-Bsa4// //forward sequence found using the primer pLIC-for, results from ICMB DNA Sequencing Facility// code NNNNNNNNNNNTTACTTTAGANGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA

GAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTAT

GATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGA

AACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATA

GACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGG

CAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCG

TAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACG

ATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGC

GTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAA

TCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAA

GGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTT

TGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTG

ACAGCTGGNAAAACGCTGGNNGCGTCTTTAANGACAGCGACAANTCGATGCANTGATNNATCCTAAANACCAANCNNANA

NNGNCNNNNAGCCNCNTTTNCNTCTGANNNAAANCNTTNNTNCTANNCTGATTTNNNNCNGNNNNNNNNGNNNNAANCNC

NNNNNNTNNNNANNNNNNNNNNCANCATCNNNNNNNNNTNNNNNNNNNNNNNNTNNNNNNNTNNNNNNNNNNNNNNNNNN

ANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNN code

//pNIC-Bsa4// //reverse sequence found using the primer pLIC-rev, results from ICMB DNA Sequencing Facility//

code NNNNNNNNNNNNNGNNCTCNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGG

ATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGT

TAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAA

ACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGT

ACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGG

CTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTT

TTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACT

GTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTT

TGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCAT

AGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTG

TGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTG

CTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGT

CTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTNGTTTGCCGTANNNTTANCGNAGAAATCANTGNANAATAAAC

GGATTTTTCNTCNGANGTAAATGNNNNGNNNNNNNTNNNNGNGNTTNNNNTTTNGNNNNANCNTTNGCATCNANNNTCNC

NNNNTTTNNNNNNNNNNGNNNTNCNNCNNNNANNNNANNNNCNANTTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNTT

NNNNANNNCNNNCNANNNNNNNNNNNNNNNNGNNGNNNNNNNN code


 * __Primer Design__**

FORWARD PRIMER: TACTTCCAATCC __ ATG __ __ AAGAAAATTTCCGTTCTG __ // GC Content 36.4%  // REVERSE PRIMER: TATCCACCTTTACTG __ TTA ____ AGAGTACGCCAGAAC __ //GC Content 42.4%//

The primers were designed for the WoBm DXR gene and will be used for PCR amplification and ligation independent cloning. The coding DNA sequence of the gene is underlined. The leading or lagging portions of the primers are not underlined. The start and stop codons are highlighted in green and red. The melting temperatures of the two primers are within a few degrees of each other.

__**Transformation Efficiency**__

Figure 1. 15 uL of DH5alpha //E.coli// bacterial cells transformed with 25 ng of the plasmid pmCherry plated on an LB agar +amp plate. The plasmid contained a gene for ampicillin resistance. The plate was incubated overnight at 37 degrees celcius. Approximately 230 colonies were counted.

Figure 2. 15 uL of DH5alpha //E.coli// bacterial cells transformed with 5 ng of the plasmid pmCherry plated on an LB agar +amp plate. The plasmid contained a gene for ampicillin resistance. The plate was incubated overnight at 37 degrees celcius. Approximately 124 colonies were counted.

Figure 3. 15 uL of DH5alpha //E.coli// bacterial cells transformed with 1 ng of the plasmid pmCherry plated on an LB agar +amp plate. The plasmid contained a gene for ampicillin resistance. The plate was incubated overnight at 37 degrees celcius. No growth was seen, this could be due to experimental error.

The plates exhibited varying amounts of bacterial growth because they contained different amounts of plasmid. Plate A in Fig.1 exhibited the most growth because it had the greatest amount of plasmid. The bacterial growth confirms that the transformation was successful; if the bacteria had not taken up the plasmid containing amp resistance they would not have grown on the LB agar + amp plate. These colonies were then grown overnight in flasks and spun down to form a pellet.

__**RE Digest**__

Figure 1. Visualization of restriction enzyme digest of plasmid pGBR22. The agarose gel was run at 110V for 40 minutes in TEA buffer. Lane 1 was skipped, Lane 2 contained 1kb DNA ladder, Lane 3 contained uncut plasmid, Lane 4 contained the plasmid and restriction enzyme EcoRI, Lane 5 contained the plasmid and restriction enzyme PvuII, Lane 6 was unintentionally skipped, and Lane 7 contained the plasmid and both enzymes.

Figure 2. Virtual gel for pGEMT plasmid containing pGBR22 gene cut with restriction enzymes is shown above. The 1kb ladder is labeled and is shown in the first lane. The three following lanes from left to right show the plasmid cut with EcoRI, the plasmid cut with PvuII, and the plasmid cut with EcoRI and PvuII. This is what the gel should look like.

Figure 3. Diagram of pGBR22 plasmid cut once by ECORI and twice by PvuII.

Unfortunately Lanes 3, 4, and 5 did not run through the gel because the sample remained in the well, this was probably due to experimental error. However, we should have seen one band for the uncut plasmid, one band for sample 1, and two bands for sample 2. The uncut plasmid will remain as one piece. Sample 1 would be one elongated piece since EcoRI cuts the plasmid only once. Sample 2 would contain two pieces since PvuII cuts the plasmid in two places. We can see three bands for sample 3. The third, lowest band is faint. The three bands are a result of the plasmid being cut into three pieces; it was cut twice by PvuII and once by EcoRI in the double digest.

__**DNA Sequencing**__

//pgbr22 forward sequence found using the primer M13F-20, results from ICMB DNA Sequencing Facility//

NNNNNNNNNNNGGGCGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTG ATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACAT CCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAA TAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGT GTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGT AGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCC CATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTA TTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCC CCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAG GTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGAC CATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATG GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGANCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGNAAANCTGTCG NGCCAGCTGCATNNNGANCGGNNNNNNNNGGGGNNAGGCGGNTNGCGTNNTGGGNGCTCNTCNCTTCCTCNNNNNNTGAN TNNNNNNNCNTNNNNNNNNNNGCNNNNNANNNNNNNNCAGNNNNNNNNNANGGNNGNNNTNCNGTNNNCNNNNNNCNGGG NNANNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNANNNNNNNNNNNNNNNNGNNNNNNNNN

//pgbr22 reverse sequence found using the primer M13R-24////, results from ICMB DNA Sequencing Facility// ` NNNNNNNNNGANNATAGANTACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCC GCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTC AGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGC TCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTC ACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTT TGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTG GTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCA CGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATC TACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACA AGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAA AATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGG CCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTNCCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCC AGCTGGCGTAATAGCGAANAGGCCCGCANCGATCGCCCTTCCCANAGTNGCGCANCNGAATGGNGATGGNNGNNNCCNGN AGCGNGCATNNNNNGCGGNNGNNGNNGNNCNCGCAGCGNGACNCTACNNTNNNGNNNCNANNNCCNNTNNNTNNNTTNNN NNNNTTNNNCNCCNNNNNNCGNTTNCNNNANNNNNAANNNGGGNTNNNNNNNNNCNANTNNNGNTTNNNNNNNNNNNNNT NNNNNGNNNNGGNNNNNNTNNNNNNNNNTCNCNNNNNN

__**NanoDrop**__






 * Figure 1. The two graphs above show the concentration of pGFP plasmid DNA, as measured by Nanodrop. The concentration for the first measurement was 1583.9 ng/uL and 1166.1 ng/uL for the second measurement. The average concentration was 1375 ng/uL. The actual concentration of the stock plasmid pGFP was 1580 ng/uL. **

__**Target Page**__

http://vdsstream.wikispaces.com/Target+-+1-deoxy-D-xylulose+5-phosphate+reductoisomerase+%28Wolbachia%29
 * Wolbachia [Brugia Malayi] DXR**