Mihir+S.

= Week 15: = Worked on Protein Purification. Figure 1: Elution 1 from Purification

Figure 2 : Elution 2 from Purification

I also worked on Characterization.



= Week 14: = This week I finished protein expression and finally got my Virtual screening to work. The fitness scores were pretty low, indicating that these ligands do not bind well. Run 2 is currently in progress.

Figure 1: Some of the top scores from run 1
 * Fitness Score ||
 * 56.18 ||
 * 55.76 ||
 * 53.88 ||
 * 57.79 ||
 * 51.26 ||
 * 46.46 ||
 * 45.73 ||
 * 41.77 ||
 * 66.52 ||
 * 41.77 ||
 * 66.52 ||
 * 66.52 ||

= Week 13: =

I began working on protein expression, using the alternate protocol of incubating for 18 hours at room temperature as opposed to 4 hours at 37 C. = Week 12: = 112612 - Good. Dr B code TARGET   1        KLEGAVN VRDLGGYKTT DGLTIKPHKL IRSAELANLS DSDKKKLVNT 2oz5A    4     r--elpgawn frdvadtat- ---alrpgrl frsselsrld dagratlrr-

TARGET                     ssshhhh            s ss         hhhhhhhhh 2oz5A                      ssshhh       sss  ss sssss      hhhhhhhhh

TARGET   48    YDLSHIVDFR TSSEV-ATKP DPKLTDVDYT HDSVMKD -- -- 2oz5A    47    lgitdvadlr ssrevarrgp grvpdgidvh llpfpdladd addsapheta

TARGET            sssss                     sss ss 2oz5A           h  ssssss   hhhhhhh          sss ss          hhhhhhhhh

TARGET         -- -- -- -- -- 2oz5A    98    fkrlltnges sqsindaatr ymtdeyrqfp trngaqralh rvvtllaagr

TARGET 2oz5A          hhhhh      hhhhhhhhhh hhhhhhhhhh h   hhhhhh hhhhhhh

TARGET         -- -- -- -- -- 2oz5A    155   pvlthcfagk drtgfvvalv leavgldrdv ivadylrsnd svpqlraris

TARGET 2oz5A           sssss      hhhhhhhhh hhhh   hhh hhhhhhhhh    hhhhhhhh

TARGET         -- -- -- -- -- 2oz5A    205   emiqqrfdte lapevvtftk arlsdgvlgv raeylaaarq tidetygslg

TARGET 2oz5A          hhhhhh       hhhhhhhh hh           hhhhhhhh hhhhh   hh

TARGET         -- -- -- 2oz5A    255   gylrdagisq atvnrmrgvl lg

TARGET 2oz5A          hhhhhh   h hhhhhhhhhs ss

Figure 1: Homology alignment code



Figure 2: Molprobity
I also worked on protein expression of my surrogate: FtHap. I overincubated my bacteria because I grew it overnight for 18 hours. I re-did the protocol and grew my bacteria up for exactly 12 hours, then followed that up with an 18 hour growth at room temperature. I proceeded to centrifugation and stored my pellet after adding sonication lysis buffer.

= Week 11: = This week I worked on the cloning protocol. After many failed attempts at creating an accepting vector with high concentration, I used 12.6 ng/uL pNIC-Bsa4.

Figure 1: Bacterial plates. Did not spread the bacteria correctly, overincubated. No colonies resulted = Week 10 = This week I worked on cloning, however the week was filled with failure. I focused on making the accepting vector for cloning, however when I did PCR clean up, my concentration for pNIC was very low each time.

Figure 1: I used 146 ng/uL pNIC, but clean up only yielded a result of 19.7.

Figure 2: Using 69.7 ng/uL pNIC (newly made to compensate for faulty 146 ng/uL), the result was 14.3 ng/uL, which is very low. = Week 9: = This week I began my cloning protocol. However, my pNIC only yielded a nanodrop result of 4.6 ng/uL.

= =  Fig. 1: Failed pNIC

I will try this procedure again, if it fails, I will remake pNIC.

WEEK 8:
102112- Mihir, show some VS results - Dr. B This week was largely focused on doing the Virtual Screening Refresher. I did my initial runs incorrectly and spent some time making sure I ran the screenings correctly. = Week 7: = 101612 - Mihir - ok good job. the PCR squared curve doesn't 'look' great - but the quanitity is high enough to justify going to cloning. Good luck. Dr. B

This week I worked on PCR squared, PCR Cleanup, and I began the Virtual Refresher.

Below is my nanodrop from my PCR squared. I had to apply wash solution, elution solution, and follow the protocol of the Sigma elution kit. After following the procedures, I was left with a small amount of eluate, which I nano dropped.

Figure 1: Nano Drop Result.

My concentration is somewhat low (31.3 ng/uL), but it is enough. My 26/280 absorbance is 1.64, and my 260/230 is .83.

Below is my result from PCR squared.

Lane 1: 100 bp ladder Lane 2: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 1 Lane 3: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 2 Lane 4: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 3 Lane 5: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 4

Analysis of Gel: Lanes 3-5 seemed to turn out pretty well. However, lane 2 did not show up. This may be due to the fact that I used a faulty PCR machine, but that doesn't explain why all of the other lanes turned out fine. I treated all of the samples with the same conditions (temperature/mix). It could be that I may have inserted the sample into the well incorrectly (maybe I poked through the gel) resulting in the improper gel outcome.

Below is my result from secondary PCR.



Lane 1: 100 bp ladder Lane 2: Shane Lane 3: Akhi Lane 4: Mihir

Analysis of Gel: The sizes seem to be fairly consistent between all three of our samples. My sample is slightly dimmer than the rest, this could be due to some error in preparation, or failure of as many primers to connect at that size.

= Week 6: = 100912- Mihir, ok good. Include your Secondary PCR too. - DR. B Also, when you move on to cloning (after PCR^2) use the new T4 DNA Poly and the new dCTP and dGTP.

I worked on Primer Overlap PCR this week.


 * 1) [[image:GEL GEL.png]]

Lane 1: 100 bp ladder Lane 2: Shane's mix Lane 3: My mix Lane 4: Akhilesh's mix

The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.

Analysis of gel: My lane (lane 3) showed a spectrum of sizes, varying from small (towards the bottom) to large (top) primers. The size varies because not all of the primers join together to form the ideally long strand, and those that do are towards the top. = Week 5: = Mhir - address why you think PCR 2 didn't work. Go back and get PCR #1 to work. If you get that to work. then you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. B

This week I worked on PCR #2. Unfortunately, the gel did not show anything. Gel may not have worked due to an error in making the samples, such as insufficient, or prolonged storage.

Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG Downstream: TATCCACCTTTACTG TTAGTACAGGTATGCTTTT Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA

Below is my FASTA:

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTT TAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACC TGTACTTCCAAT CCATG AAAAACTGGGTTAAAGTTACCGGTGCGGGCGTTCTCTCTGCTACTCTGCTGCTCGGT GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCAAACGTCAAGACCGAACAGACCCTG AAGCCGGGTAGCCAGATCAAACTGGAAGGTGCCGTTAATGTTCGCGACCTGGGTGGTTAC AAGACTACCGATGGTCTGACCATCAAACCGCACAAACTGATCCGCTCTGCGGAACTCGCA AACCTCTCTGACTCTGACAAAAAGAAGCTGGTTAACACCTACGACCTGTCTCACATCGTT GACTTCCGCACGTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTGGAC TACACCCACGACTCTGTTATGAAAGACAACGGTACGTCTACCTCTACCCAGGACCTGACG GCGTCTCTGGCCAAGATGGACAACCCGGAAACCTTCCTGATTAACGCGAATAAATCCTTC ATTACCGACGAGACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCGAAC CAGGACGGTTCCGTTCTGTGGCACTGCACCGCCGGTAAAGACCGTGCCGGTTTCGGCACC GCGCTGGTTCTGTCTGCGCTGGGTGTTGATAAAAACACCGTTATCGACGACTACATGCTG TCTAACAAATACCGTGCTGACGAAAATAAGAAAGCGATCGAAGCGGTAGCGGCGAAAACC GACAACAAAAAAGTGATCGACGGTATGACGGCAGTTATGGAAGTTCGTGAATCTTACATC AACGCGGCGTTCGATGAGATCAATGCGAAATATGGTTCTATGGATAACTTCCTCAAAGAA AAGCTGGGCCTCACTGATGCGAAGAAAGAGCAGCTGAAA AAAGCATACCTGTACTAAC AGTAAAGGTGGATACGG ATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCC GGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATG GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT TAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGA ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTG CGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGT CGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTA CCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGG CCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGAC AGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCG CTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC GTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATG TCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGC GGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCA GATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCG GTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGA CCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCG CGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGG TTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTG ATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGA AAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTA CCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATC GTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGAC ATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAA TGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTC TGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACA GGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTA ATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGC GCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTAT GCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGG TGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAA CCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG AAAT

= Week 4: = Mihir - good. may end up needing ot make more pNIC for your cloning. Good job on second attempt of PCR. You bands look good and are at the ~1,000 bp range - which is right for pGBR22. Looks like you got some carry over or contamination into the 4th lane which is negative control. Try to crop your gel images some. -- DR. B

I extracted my plasmid, pNIC-Bsa4, from my bacteria (E. Coli), using the Midiprep technique of filtration. Then I tested the concentration of my plasmid by using the Nanodrop procedure.

Figure 1: Nanodrop results of my pNIC-bsa4. My yield was 34.1 ng/uL.

PCR #1 was a failed procedure.

Figure 2: Failed PCR. DNA Ladder showed up, but none of the samples did. This could be due to errors in the mix, in the PCR, or due to the fact that the samples were not kept on ice when prepping the gel.

Redid PCR #1. Below is the result. Figure 3. Successful gel

Lane 1: 100 bp ladder Lane 2: Sample A (pgbr-22 template and m13 forward and reverse) Lane 3: Sample B (same, different amount of template and water) Lane 4: Sample C (same, different amount of template and water) Lane 5: Sample D (same, different amount of template and water)

Analysis of Gel: The bands all seem to be the same size. The heavier bands tend to be higher up on the gel (towards the top), while the lighter bands tend to be towards the bottom. In this case, all the bands are at about the middle, indicating that their size is similar.

= Week 3: = Mihir - ok good. You can move your legend for the gel to below the image (see Suman and Divyas) also include a little more analysis of gel and also include a mention of the transformation you did .-- Dr. B 091812

I worked on restriction enzyme digest. The enzymes I used were EcoR1 and Pvu-II. The buffer I used was NEBBuffer-2 (NEB stands for New England Bio Lab). Below is the result from my gel. The first lane was a skip, the second lane is the ladder, the third lane is the uncut plasmid (pgbr-22), the fourth lane is Eco-R1, the fifth lane is PVU-II, and the sixth lane is the Eco-r1+Pvu-II. Figure 1: The largeness of the top bands indicates a larger molecule. The 4th lane had one cut, the 5th lane had 2 cuts, and teh 6th lane had 3 cuts.

After RE Digestion, I worked on a practice nanodrop.

I also began my PCR #1.

= Week 2: = I worked on dilution of my primer. I submitted my result to the sequencing core. Later in the week, I translated my protein onto a gel plate, using E. Coli as a control, comparing it to E. Coli with pNIC. I also worked on analyzing DNA sequence in the computer lab.

Analyzing DNA sequence:


 * ORIGINAL:** NNNNNNNNNNNTATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANCNNNNNCNTTCNNTTNTNCNNNTNNNNNNNNNNNNNNNTTNCCGNNAGNTNNAANNGGGGNNNNNNNNNATNNGNTNNNNNNNNNNCCAAANTGNNNGNNNNGNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNGANCNNNNNNNNNNNNANNNNNNNNNNNCNNNNNAANNNNNCCNNTNNN

Above is the original sequence. TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
 * CHOPPED:**

Above is the original sequence with many of the 'N's chopped out, because they are unreadable.

REVERSE COMPLEMENT of ORIGINAL CHOPPED:

GNTNGNNGCNGNAANTGTAGNGTCNCGCTGCGNGNNCNCNNNCCNGCCGNGCTAATGCGN CGNTNCAGGNNGCGTCCATNGCCATCAGGCTGCGCAACTGTGGGAAGGGCGATCGGTGCG GGCCTNTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTG GGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATA CGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGG GATTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCAC ACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACAT AGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCAT CTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAG GTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTT GGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACT TGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCA GAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTT TTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTA GTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTT GAGTATNCNATA

Reverse Complement: pGEM vector

TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC

CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT

AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG

ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA

CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA

GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC

CATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG

GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT

AAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA

ACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTAC

GCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA

CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAAC

CCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG

TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC

TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGG

ATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA

GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC

AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG

AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA

GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCG

CTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA

ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGT

TGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT

GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGT

TTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG

GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC

TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA

AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT

TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT

TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT

GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGC

AGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTG

TAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG

ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT

CGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAAC

TGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGG

ACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG

GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGAT

TTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTT

TACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTG

ATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAA

CGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGC

CTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGA

AAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG

CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTC

ACACAGGAAACAGCTATGACCATGATTACGCCAAGCTATTTAGGTGACACTATAGAATAC

TCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGC

ACTAGTGATAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCC

= Week 1: = We worked on target discovery and creating our target pages. Below is my target page. = =

Organism: Staphylococcus aureus

Target: NMN phosphatase; Class B acid phosphatase precursor

EC:3.1.3.2 :Acid phosphatase

[]

Background and Disease: S. Aureus has a role in causing food poisoning, atopic dermatitis, staphylococcal scalded skin syndrome, or Ritter's disease in neonates.

Enzyme page on Brenda: []

Reaction pathway: [|http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.2&Suchword=t.+cruzi&organism[=Staphylococcus+aureus&show_tm=0]]

enzyme assay info: []

= ** REAGENTS: ** =

Sodium hydroxide solution: 199.00

 * Gene #:12865799 **

Essentiality of protein: None

Inhibitors: Vancomycin

Image of protein:



Coding DNA Sequence link: []

Amino Acid sequence: 1 mraaplllar aaslalascf cffcwldrsv lakelkfvtl vfrhgdrspi dtfptdpike 61 sswpggfggl tglgmeqhye lgeyirkryr kflndsykhe qvyirstdvd rtlmsrmtnl 121 aalfppegvs iwnpillwqp ipvhtvplse dqllylpfrn cprfqelese tlkseefqkr 181 lhpykdfiat lgklsglhgq dlfgiwskvy dplysesvhn ftlpswated tmtklrelse 241 lsllslygih kqkeksrlqg gvlvneilnh mkratqipsy kklimysahd ttvtglqmal 361 gpvipqdwst evmttnshqg tedstd

Primer Design:

1 ATGCGTGCAGCACCTCTGCTCCTGGCCCGTGCGGCCTCTCTGGCGCTGGCATCTTGC 57 2 TCTTTCGCCAGAACAGAGCGGTCCAGCCAACAGAAGAAGCAGAAGCAAGATGCCAGCGCC 60 3 GCTCTGTTCTGGCGAAAGAACTGAAGTTCGTTACTCTGGTTTTCCGTCACGGTGACCGTT 60 4 GAAGATTCTTTAATCGGATCGGTCGGAAAGGTATCGATCGGAGAACGGTCACCGTGACGG 60 5 GACCGATCCGATTAAAGAATCTTCTTGGCCGCAGGGCTTTGGTCAACTGACGCAGCTGGG 60 6 GCGCTTACGGATGTACTCGCCCAGTTCGTAGTGCTGTTCCATACCCAGCTGCGTCAGTTG 60 7 CGAGTACATCCGTAAGCGCTACCGTAAATTCCTGAACGACTCCTACAAGCATGAACAGGT 60 8 GACATGAGGGTACGGTCAACGTCAGTAGAACGAATGTAAACCTGTTCATGCTTGTAGGAG 60 9 GTTGACCGTACCCTCATGTCTCGTATGACGAACCTGGCGGCACTCTTCCCGCCTGAGGGC 60 10 ACCGGGATCGGCTGCCACAGCAGGATTGGATTCCAGATAGAGACGCCCTCAGGCGGGAAG 60 11 GCAGCCGATCCCGGTTCACACCGTTCCACTCTCTGAAGATCAACTCCTGTACCTGCCGTT 60 12 AGGGTTTCAGATTCGAGTTCCTGGAAACGCGGGCAGTTGCGGAACGGCAGGTACAGGAGT 60 13 GGAACTCGAATCTGAAACCCTGAAATCTGAGGAGTTCCAGAAACGTCTCCACCCGTACAA 60 14 CCGTGCAGGCCAGACAGTTTGCCCAGGGTCGCGATGAAGTCTTTGTACGGGTGGAGACGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">15 TGTCTGGCCTGCACGGTCAGGATCTGTTTGGTATCTGGTCTAAAGTTTACGACCCGCTGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">16 CCCAAGATGGCAGAGTGAAGTTGTGAACAGACTCGGAGTACAGCGGGTCGTAAACTTTAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">17 CTTCACTCTGCCATCTTGGGCGACTGAAGACACCATGACTAAACTGCGTGAGCTGTCTGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">18 TCTTTTTGTTTGTGGATACCGTACAGAGACAGGAGGCTCAGTTCAGACAGCTCACGCAGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">19 GTACGGTATCCACAAACAAAAAGAAAAATCTCGTCTGCAAGGTGGCGTTCTCGTTAATGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">20 GCTCGGGATCTGGGTCGCACGTTTCATGTGGTTGAGAATTTCATTAACGAGAACGCCACC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">21 CGACCCAGATCCCGAGCTACAAAAAACTGATCATGTATTCTGCGCATGACACGACCGTTA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">22 GCAGCAGGCCGTTGTAAACGTCCAGGGCCATCTGGAGGCCGGTAACGGTCGTGTCATGCG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">23 TTACAACGGCCTGCTGCCGCCTTACGCCTCTTGTCACCTCACCGAACTGTACTTCGAAAA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">24 TCTCGTTGCGATAGTACATTTCAACGAAATATTCACCTTTTTCGAAGTACAGTTCGGTGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">25 TGAAATGTACTATCGCAACGAGACCCAGCACGAACCGTATCCGCTGATGCTCCCAGGTTG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">26 CCAACGAGCTCCGCGAAACGTTCCAGCGGGCAAGACGGAGAGCAACCTGGGAGCATCAGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">27 TCGCGGAGCTCGTTGGTCCGGTTATCCCGCAGGACTGGTCCACCGAAGTTATGACCACTA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">28 GTCGGTAGAGTCCTCGGTGCCCTGGTGAGAGTTAGTGGTCATAACTTCGGTGG 53