Keenan+W.+(springer)

=**__Drug Discovery & Virtual Screening of Phosphoserine Phosphatase of //Vibrio cholerae//__**= Great work Keenan - your written work has always been great and now your lab work is catching up to it. - Dr. B 121214
 * __Week 14 & 15__**


 * CLONING INTO DH5alpha**
 * Figure 1.** Master plates with kanamycin and sucrose present in agar. Successful growth of transformed DH5alpha //E.coli// cells with cut pNIC annealed with gene of interest //V. cholerae// (VcPSP). Two plates with different ratios of plasmid to cell respectively. In the center, there is a 2:1 ratio of plasmid to cell and on the right there is a ratio of 8:1. The small colonies appear milky white in color and there are more colonies located in 2:1 ratio of plasmid to cell.

In cloning, we prepared our vector pNIC by cutting out the toxic SacB gene using BsaI RE leaving an open space for our gene. Then our vector is modified to create longer sticky ends to enhance annealing of the vector with our gene of interest (VcPSP) in a process known as cohesive end generation. Once our gene and pNIC are annealed, the plasmid and DH5alpha cells are transformed by heat shock/hot water bath. I used a ratio of 2:1 and 8:1 of plasmid to cells respectively. They are then plated on agar with kanamycin and sucrose. When cutting pNIC, there was some doubt as to whether or not to proceed with cloning when there is some uncut plasmid. However, the kanamycin and sucrose present in the agar plates will kill off cells not transformed with the modified plasmid. After overnight incubation, colonies grew on both 2:1 and 8:1 agar plates suggesting that I was successful in generating clones. Whether or not if they are positive clones or other extraneous bacteria, a positive clone is determined when sent to DNA sequencing facility to generate its gene sequence. The results are compared though BLAST to see if it matches the known gene sequence with 100% identification and query cover.
 * Analysis of Results:**


 * VIRTUAL SCREENING**
 * Figure 1a, b & c** (left to right). **a)** PDB crystal structure of Phosphoserine Phosphatase of V. cholerae (3N28) colored in chainbows. **b)** Aligned/superimposed image of target 3N28 and homologous structure 1L7P (archaea). 3N28 and 1L7P are displayed as cartoons and colored green and blue respectively.**c)** Defined active site (cyan) of 3N28, using SEP ligand of 1L7P displayed as surface and colored by element; carbon green, nitrogen blue and oxygen red.


 * Table 1.** Table of positive and negative controls found for phosphoserine phosphatase of //V. cholerae// in virtual screening. Included in the table is general identification of the compounds and information regarding Lipinski's Rule. 2D images of compound's structure are not present in the table.

The goal of virtual screening in lab is to generate and find possible compounds that can inhibit phosphoserine phosphatase of //V. cholerae.// In figure 1, we were able to locate the active site of phosphoserine phosphatase of //V. cholerae// by using a dummy ligand from a similar structure 1L7P (archaea) because the active site was not defined in the crystal structure. Both positive and negative controls were discovered (through research and ZINC respectively) to test our prep protein structure though GOLD. The results were scattered as expected because the negative controls were found on ZINC based on variations of the averages of the positive control compound's properties. Trifluorperazine, a positive control, was the best scoring compound predicted by GOLD and compound 19166762 (neg3) was the best scoring negative control. Running a library of In-house compounds, the top 4 of 40 compounds (top 10%) are 7911340, 7637680, 7833474 & 7785278. These could be possible inhibitors of phosphoserine phosphatase but however their scores ranged in the 60. They are not real working inhibitors because GOLD can only make predictions based on algorithms. We need to then take these compounds and test them through enzyme and inhibition assay.
 * Analysis of Results:**


 * 12042014- Great job **

__**Week 11, 12 & 13 **__


 * CUTTING PNIC VECTOR**
 * Figure 1**. Gel electrophoresis run of pNIC sample with restriction enzyme BSAI and buffers to remove SacB gene in pNIC. Well 2 contains 1 kb ladder, well 4 contains pNIC sample.

The purpose of this lab is to prepare our vector (pNIC) in order to create transgenic cells by removing the toxic SacB gene of pNIC and inserting our gene of interest. The SacB gene is toxic to the cell and will kill the cell if not removed. Restriction enzyme BSAI was used to cut into the plasmid and remove the gene. The gel shows three separate bands in well 4. The band closest to the bottom represents the SacB gene. It is much smaller in size than the plasmid itself and therefore it moves though the gel the fastest. The cut plasmid is just above the SacB gene. We were only expecting two bands to appear but a faint third band is shown faintly above the cut pNIC. Other than contamination particles, this could only mean that some of our plasmid was not cut by RE. Nonetheless, cloning will be attempted assuming that the agar plates containing sucrose will kill off any cell that hasn't had its SacB gene removed. The smearing on the gel is created by human error: waiting too long before pouring the solution into the mold (already solidifying), creating an uneven terrain when DNA flows though the gel.
 * Analysis of Results:**


 * Finding Template Homology Model **

>gi|14719642|pdb|1F5S|A Chain A, Crystal Structure Of Phosphoserine Phosphatase From Methanococcus Jannaschii MEKKKKLILFDFDSTLVNNETIDEIAREAGVEEEVKKITKEAMEGKLNFEQSLRKRVSLLKDLPIEKVEK AIKRITPTEGAEETIKELKNRGYVVAVVSGGFDIAVNKIKEKLGLDYAFANRLIVKDGKLTGDVEGEVLK ENAKGEILEKIAKIEGINLEDTVAVGDGANDISMFKKAGLKIAFCAKPILKEKADICIEKRDLREILKYI K
 * BLASTP Template **

>sp|A0QJI1|SERB_MYCA1 Phosphoserine phosphatase OS=Mycobacterium avium (strain 104) GN=serB PE=1 SV=1 MNSPPKVSVLITVTGVDQPGVTATLFEVLSGHGVELLNVEQVVIRHRLTLGVLVCCPADV ADGPALRHDVEAAIRKVGLDVSIERSDDVPIIREPSTHTIFVLGRPITAAAFGAVAREVA ALGVNIDLIRGVSDYPVIGLELRVSVPPGADGALRTALNRVSSEEHVDVAVEDYTLERRA KRLIVFDVDSTLVQGEVIEMLAAKAGAEGQVAAITDAAMRGELDFAQSLQQRVATLAGLP ATVIDEVAGQLELMPGARTTLRTLRRLGYACGVVSGGFRRIIEPLAEELMLDYVAANELE IVDGTLTGRVVGPIIDRAGKATALREFAQRAGVPMAQTVAVGDGANDIDMLAAAGLGIAF NAKPALREVADASLSHPYLDTVLFLLGVTRGEIEAADAIDGEVRRVEIPPE
 * SWISS-MODEL Template **

While there is a crystal structure of 3N28 (phosphoserine phosphatase of //Vibrio cholerae)//, the active site was not identified. The homology model 1F5S (template from BLASTp) will be used to help identify the location of the active site in order to identify possible inhibitors that may work well for the similar structure of 3N28. The compounds, found though virtual screening, that bind well to the similar active site of 1F5S, may also bind well to the active site of phosphoserine phosphatase (3N28) in //Vibrio cholerae// and hinder its function. The above information are just two similar templates generated from BLASTp and SWISS-MODEL. The BLASTp model 1F5S will be used to locate the active site of 3N28.
 * Analysis of Results:**


 * PCR CLEANUP**
 * Figure 1.** Nanodrop results of four trail runs of PCR sample after PCR Cleanup. Concentration of DNA plasmid from trials one to four are 45.6, 48.6, 50.0 and 48.1 ng/ul respectively. The average concentration of the sample after PCR cleanup yields to be 48 ng/ul.

The concentration for our plasmid is very low after PCR Cleanup as shown in figure 1. The average concentration only yields approximately 48 ng/ul. Even after all the PCR replication/amplifying stages, we still end up with a low amount of our full gene sequence of interest due to PCR clean up. The process is a way to remove unwanted fragments/contamination within our sample but we also lose mush of our gene sequence as well. This average concentration is expected because during cleanup, the sample was separated into two different filters and spun down, instead of having one sample. Having only one sample introduces more of our plasmid present yielding a higher concentration of DNA when comparing data with peers. Also, pNIC is a low copy plasmid which was used as the vector.
 * Analysis of Results:**

__**Week 8, 9 & 10 **__


 * PRIMARY AND SECONDARY PCR & PCR SQUARED**

**Figure 1.** Primary PCR Gel Run. Well 2 contains the 1 kb ladder and the 4th well contains the oligo mix sample.
 * Primary PCR**

In primary PCR, we prepared our sample of plasmid in conditions for PCR: dilutions and buffers as well as our oligos from DNA Works. Through electrophoresis, different components of our gene sequence are separated and a smear appeared on our gel. This is what is expected because the oligos we created are just fragments of our gene at various sizes and length. The smear represents these small chucks that are not yet fully connected together though our full length sequence is in here somewhere in our sample.
 * Analysis of Results: **


 * Secondary PCR**
 * Figure 1.** Gel electrophoesis of secondary PCR (Trail 1).


 * Figure 2.** Secondary PCR electrophoresis gel. Samples contain primary PCR sample along with forward and reverse primers to amplify full length gene sequence. 1kb ladder is in well 2 and original sample is located in well 4. Wells 6 and 8 are used by peers.

In figure 1, the experiment obviously failed. Nothing showed up for my sample. In figure 2, there is a faint band belonging to my sample located in well 4. Wells 6 and 8 are occupied by peers doing the same experiment. There should be only one band this time, instead of a smear, located on the gel because the forward and reverse primers only amplify our full length sequence. Therefore, there should only be a single noticeable band that should appear. A single band did appear but however, it isn't as bright as Avery's in well 6. This could mean that during PCR, the gene sequence was not amplified enough or correctly. It could also mean that there wasn't much of my full gene sequence to begin with.
 * Analysis of Results:**


 * PCR Squared**
 * Figure 1.** PCR Squared electrophoresis gel. Sample contains secondary PCR sample along with forward and reverse primers. A master mix of 200ul was made and split into 4 tubes used in wells 4-7. Well 2 contains 1kb ladder.

In figure 1, we can faintly see four single bands in each well 4-7. This is expected because we are again amplifying our full length gene with one size. However, the bands are depressingly very faint which could perhaps suggest that there isn't much of my gene, especially using the faint sample from secondary PCR. However, with the approval of a mentor, cloning will be attempted using this sample.
 * Analysis of Results: **

10232014- Good work

__**Week 5&6**__ lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll PCR Primer Design Tails for pNIC-Bsa4 Cloning 5’ TACTTCCAATCCATGTCTCTGGACGCG 3’ 27 bp GC Content 51.9% 0.0 mM Mg2+ Tm 62.3 oC 1.5 mM Mg2+ Tm 68.8 oC 2.0 mM Mg2+ Tm 69.3 oC 4.0 mM Mg2+ Tm 70.4 oC 6.0 mM Mg2+ Tm 70.9 oC lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll 5’ AGCTGGAAATCTAAACCGTAACAGTAAAGGTGGATA 3’
 * Forward Primer:**
 * Reverse Primer:**

5’ TATCCACCTTTACTGTTACGGTTTAGATTTCCAGCT 3’ 36 bp GC Content 38.9% 0.0 mM Mg2+ Tm 61.8 oC 1.5 mM Mg2+ Tm 69.5 oC 2.0 mM Mg2+ Tm 70.0 oC 4.0 mM Mg2+ Tm 71.0 oC 6.0 mM Mg2+ Tm 71.5 oC lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
 * Reverse complement it:**
 * Figure 1.** Results of tail primer design of forward and reverse primer.

The goal of this lab is to design a forward and a reverse primer for PCR amplifying the gene sequence of interest and synthesizing compatible ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the accepting vector for eventual expression. After virtually designing our tail primers, they are then requested to be ordered.
 * Analysis of Results:**


 * Figure 1.** Three trial runs from MidiPrep (from left to right) of nanodrop results of our purified plasmid from all four spun down //E. coli// DH5alpha tubes transformed with pNIC-Bsa4.

It seems that, compared to other classmates, that the more bacteria that you use = the higher the concentration of DNA plasmid you have. It makes sense because more bacteria contains more plasmid. Also, this is a low copy plasmid, which means that it doesn't produce much DNA.which might be difficult when doing primary PCR which is why its better to use more plasmid. The average concentration that was collected though nanodrop was 148.6 ng/ul which is relatively close to what needs to be expected for our low copy plasmid. Therefore it is safe to assume that we are successful in purifying our DNA (rather extracting our plasmid from the cell) and is ready to go through primary PCR.
 * Analysis of Results:**


 * Figure 1 & 2.** One colony was selected from the bacterial //E. coli// DH5alpha and grown up in LB and KAN overnight. On the left is our cultured bacteria of pNIC-Bsa4 after incubation. On the right is the pellets of E. coli transformed with pNIC-Bsa4. Contents were spun down leaving the pellets on the bottom of the tubes.

**Analysis of Results:** The solution was cloudy following overnight incubation shown in figure 1. The cells were likely transformed by the pNIC-Bsa4. The antibiotics should have killed off any unsuccessfully transformed bacteria. After the tubes were spun down, pellets remained at the bottom of each tube which contains all parts of the cell and our desired plasmid. The plasmid will be taken and purified in another lab experiment.


 * Figure 1.** Used wells 6-10 for RE digest. 1-5 wells are used for another lab experiment. Aragose gel for pgbr22 plasmid with enzymes EcoI, PvuII and both enzymes together.

The results on the RE digest gel are inconclusive and unreadable and no observations or assumptions can be made with the following results. In lane 10, there should be one mark for the uncut pGBR-22 plasmid, but there is nothing visible in that lane along with lanes 7-9. In lane 7 there should be one band corresponding to the one piece of linear DNA obtained when the pGBR-22 plasmid is cut by EcoRI at one restriction site. Lane 8 should have two bands since PvuII cuts the plasmid in two places, resulting in two smaller pieces of DNA.Lane 9 should have three bands corresponding to the three pieces of plasmid. However, none of the bands showed for any lane except the 1 kb ladder (even that was smeared). Many things could go wrong in this process especially when we are working with enzymes which may degrade or become inactive due to the wrong environment that it is in. Other than that, human error is the biggest problem.
 * Analysis of Results:**

__**09232014-**__Great Job, don't forget more pictures next time

__**Week 3&4**__
 * Figure 1.** Gel electrophoresis of purple protein pgBR22 plasmid coding sequence using M13 F & R primers. Each well (3-6) contains a different amount of concentrations of DNA template. Lane 1- empty, Lane 2- 100bp ladder, Lane 3 - Tube A (1:10000), Lane 4 - B (1:1000), Lane 5 - C (1:100), Lane 6 - D (uncut plasmid).

The gel for PCR of pgBR22 failed. After the 100bp ladder, there was suppose to be a single band that followed from wells 3-6 varying in opacity depending on how heavily concentrated the DNA was. However, well 3 (Tube A 1:10000), the most dilute sample, was the only sample that showed the band while sample B, C and D did not. This is unusual because this was the most dilute concentration of DNA we had. It would be expected that the one that is least diluted would be brighter than the rest (sample C or D). The results are inconclusive and no apparent relationship/comparisons can be made.
 * Analysis of Results:**

982014 - Great Job, don't forget the pictures next time

__**Week 1&2**__ separate petri dishes: a 10ul sample (left) and 50ul sample (right) of our //E.coli// with inserted DNA.
 * Figure 1.** Bacterial culture of //E.coli// DH5alpha with our plasmid DNA pNIC-Bsa4 growing on two

Our bacteria did grow as shown in figure 1. There is less bacterial growth in the 10ul dish and a lot of bacterial colonies in the 50ul dish. Nonetheless, despite the amount of bacteria added to each dish, //E.coli// bacteria did in fact grow. But whether or not if it contains our DNA pNIC-Bsa4 is to be determined.
 * Analysis of Results (Day 1):**

(Human error: We forgot to take a picture of our second graph)

DNA absorbs light wavelegth of 260. In figure 1, there is a peak around the mark 260nm which indicates the maximum absorbency of light wavelength by pgbr22. Therefore we know that our plasmid is consisted with DNA. We took two measurements of our sample and found out that for trial 1 and 2 for concentration is 302.9 ng/ul and 327.6 ng/ul respectively. The 260/280 value is 1.88 and 1.87 and the 260/280 value is 2.34 and 2.44 respectively. This indicates that our sample is pure with respect to proteins and other contaminants because the results are considerably close to the recommended score of 1.8 and 2.1.
 * Analysis of Results:**