Justin+S.+(Springer)

=Week 14 & 15=

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== = = Control library of positive and negative ligands for validating the accuracy of GOLD docking was created. This library was run through GOLD and the rankings are shown in Figure 1. GOLD was validated by this control library because the positive ligands dominated the top rankings as expected. The ligands that were already tested experimentally through inhibition assays and found to be decent inhibitors had a higher fitness score. The random negative control ligands and aspirin demonstrated lower scores. Figures 2&3 show the GOLD docking of the negative and positive control docking to mtPtpA respectively in PyMOL. The docking done by GOLD appears to be legitimate as PyMOL shows that the ligands docked fairly well into the active site defined. The range of scores, however, was only from about 38 to 68. This relatively small range may be either a result of choosing not so great positive ligands, or the preparation of the protein was not done well.
 * Analysis**

The control library demonstrated that GOLD is an accurate tool to use for virtual screening of mtPtpA. After validation by the control library, the next step would be to complete the virtual screening of several libraries of ligands via GOLD to find novel ligands to test in an inhibition assay.
 * Conclusion**

=12042014- Don't forget to include an analysis and conclusion, nice work= = = =Weeks 11, 12, & 13=

= =



Third round of cloning was started. Growth can be seen on the plates, **Figure 9 & 10**, but there were smears all over the plates as well. The colonies also had white spots, so the plates may contain contamination, not any positive clones.











Second round of cloning failed as well. Growth occurred on the plates, seen by **Figures 4** and **6**, but a gel was run on the Minipreped pellets of these colonies. The gel, **Figure 8**, showed a band above 10,000 base pairs. This is much larger than the expected size of the gene sequence desired, which is only about 5800 base pairs.






 * Figure 2** and **3** show the concentrations of the cleaned up digested pNIC-Bsa4 (accepting vector) and the mtPtpA gene sequence (insert). These materials were used for the second and third rounds of cloning performed later.




 * Figure 1** shows that the first round of cloning mtPtpA gene into the DH5alpha competent cells failed. The purified DNA from tubes #1 and #3 was not the expected band size. The bands shown are larger than 10,000 base pairs, while the expected band size of the desired gene sequence is only around 5800 base pairs.

1162014- Great Job







After Miniprep of #1 and #3 pellets from below, the concentrations were determined using Nanodrop, with #1 having a very low concentration. These purified DNA samples were then run on a gel alongside the controls, cut and uncut pNIC-Bsa4, as seen in **Figure 13.** No bands can be seen for the purified DNA sample of #1, indicating either that no plasmid was obtained from the pellet or that the concentration used for the gel was too small to be noticed. #3 DNA sample showed a slight smear, but no definite band at 5848bp, the size of the expected complete sequence for pNIC-Bsa4 with the gene sequence for mtPtpA.

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Transformation of BL21 (DE3) competent cells appears to be successful. The plates use negative selection via sucrose on the colonies, so only the colonies which have successfully taken in a plasmid pNIC-Bsa4 removed of its SacB can grow on the plate. One large colony has grown on plate A (**Figure 7**) while two colonies have grown on plate B (**Figure 8**). These colonies were transferred to the Master plate and also incubated in LB/Kanamycin, allowing us to see that #2 from plate B was not a good colony or not a colony at all since no growth was observed. **Figure 10** shows the pellets from #1 and #3 that will be used in Miniprep to purify the DNA for analysis using DNA sequencing.





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[[image:jws nanodrop 102014.jpg width="478" height="298" caption="Figure 3- Nanodrop measurement of RE digested pNIC-Bsa4 on 10/20/2014; concentration was 27.8ng/ul"]]
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[[image:jws 1kb ladder.jpg caption="Figure 1- NEB 1kb DNA ladder"]]
The RE digest of pNIC-Bsa4 finally succeeded after several repeated tries. **Figures 2** and **4** show the failed cutting of pNIC-Bsa4. This uncut plasmid has about 7284 base pairs, but when cut by BsaI restriction enzyme, the SacB is removed. If the digest is successful, two bands should then show up, one at around 5000 base pairs and another near 2000 base pairs. This is seen in **Figure 5**, in alternating lanes of 4, 6, 8, and 10, which used the plasmid prepared by Ahn and Alberto instead of my own, which is most likely the cause for the previous RE digest failures.

10232014- Good job, try to include more pictures

Week 5, 6, & 7


After PCR squared and PCR clean-up of that reaction, Nanodrop was performed on the purified solution of the mtPtpA gene sequence. The resulting concentration is 316.5ng/uL. Once verification of the purity and concentration of DNA sample are found and determined to be good, the gene can be inserted into a cut plasmid pNIC-Bsa4 through Ligation Independent Cloning.



Secondary PCR was performed on the sample from primary PCR. Seen in **Figure 3,** only one distinct band is seen both lanes 4 and 7. Lane 4 contained the primary PCR sample mixed with the end oligo primers while Lane 7 contained primary PCR sample mixed with the custom tail primers. Both should be observed as a single band at about 500bp size, which is what is shown in **Figure 3.** To be noted is that Lane 7 has a fainter band, which is due to a far less volume of sample inserted into the well, about 2ul compared to the 10ul of the sample in well 4. After the complete sequence is obtained, amplification of the amount of mtPtpA gene is required for clean-up and later cloning.



Primary PCR was performed on the oligo primer mix (consisted of 12 primers). As shown on **Figure 2**, a faint smear can be seen on Lane 3, where the oligo primer mix sample after primary PCR was inserted. The smear shows that the oligo primers have overlapped, resulting in a continuous chain of different sized gene sequences. As overlap occurred, this sample can now undergo secondary PCR to obtain the complete gene sequence for mtPtpA.



Midiprep was performed on the plasmid pNIC-Bsa4 obtained from the transformation of E. coli. From the Nanodrop, the concentration of the solution containing pNIC-Bsa4 was around 40-50ng/ul. The peak occurred around the 260nm wavelength, indicating that DNA is present within the solution. Now, this pNIC-Bsa4 can be cut during RE digest to allow insertion of gene of interest, mtPtpA, for cloning of that gene.

09232014- Phenomenal work!

Week 3 & 4


The gel for the PCR of pGBR22 shows that the PCR was successful. The gel is expected to have one band at the same location for Tubes A, B, and C. Tube D was the control, where no DNA template was inserted. The gel shows the expected bands, but they are not in the correct lanes. This indicates a possible error in that the tubes were mistakenly deposited in the wrong wells. Lane 3 (Tube A) and Lane 7 (Tube D) were most likely switched because the Lane 7 should have no bands while Lane 3 should have one. The band's color should get darker as the DNA template gets more concentrated in the tube. It isn't seen in this gel, probably because the volume of mixture was not constant for each well.



The gel for the RE digest of pGBR22 using EcoRI and PvuII failed. The gel should have shown one band on Lane 4, two bands on Lane 5, and three bands on Lane 6. The EcoRI should have cut the plasmid once while the PvuII should have cut it twice. This would result in the number of bands stated above. This gel, however, shows no bands for Lanes 4-6. This might be due to an error in making the DNA cocktails, where the plasmid itself was not sufficiently inserted in.







The over-expression process of the transformation of DH5alpha E. coli is seen in **Figures 1-3.** The E. coli can be visibly seen in all three images. In **Figure 1,** the growth of the colonies resulted in the turbid liquid. This liquid was later spun down to separate the solid (the pellet seen in **Figure 2 and 3**) from the liquid (LB + Kanamycin). The pellets also indicate that E. coli has successfully multiplied from the initial first colony taken from the 50ul plate. Though the growth of the competent cells is confirmed, the integration of pNIC-Bsa4 into the plasmid is still not determined.

982014 - Good Job, don't forget both of the pictures of the Nanodrop.





The transformation of DH5alpha E. coli can be seen in both **Figure 1 and 2.** The colonies grown on the plate covered in 10 microliters of bacteria mixture was about 20 colonies. For the plate covered in 50 microliters of mixture, the number of colonies is about 150. The growth of the colonies in the LB + Kan agar was evident, but whether the plasmid pNIC-Bsa4 was actually inserted into the bacteria's genome cannot be determined as of this step. The next step would be growing a large amount of the bacteria in order to over-express the desired protein coded by the inserted plasmid.



The Nanodrop was used to verify purity of DNA sample. This is done by examining the ratios of absorbance at different wavelengths (260/230 and 280/230). The ratio for a pure pGBR22 sample should be 260/230 = 1.8 and 280/230 = 2.1. From **Figure 1**, the ratios almost match these values, indicating a fairly pure sample. Since the pGBR22 sample was determined to be pure, it can be sent off to DNA sequencing for further analysis of the exact composition of the plasmid. Any contaminants present would not significantly interfere with the results of the sequencing.