Akhilesh+P


 * __Week 15 - 12/3/2012__ **

Wrap up of the purification and transformation protocol. Started the enzyme assays. One of which failed, consequently tried again. Characterization results indicate weight of about 47kDa.

This is the figure from the successful enzyme assay. This is the data results from the unsucessful enzyme assay.

Worked a bit on the surrogate procedure. Predominately spent time on the virtual screening of the target and creating a swiss model. Ligand binding continues to prove unsucessful. Will recheck gold coordinates next week.
 * __Week 14 - 11/26/2012__ **

 Figure 1: Homology model achieved using the SWISS protocol.

QMEAN Z-Score: -5.05
 * Modelled residue range: || 67 to 221 ||
 * || Based on template: || [3lj8A] (2.70 Å) ||
 * || Sequence Identity [%]: || 16.67 ||
 * || Evalue: || 9.80e-8 ||

More data pending (left in the VDS class laptop).

112612 - Good - show some 'data' or image. Dr B Worked a bit on the latter portion of the surrogate protocol. No other work due to Thanksgiving Break. Data images pending.
 * __Week 13 - 11/19/2012__ **

Akhi, ok - would like to see graph or data from virtual here. -- Dr. B 11/19/12
 * __Week 12 - 11/12/2012__ **

Moved onto the surrogate protocol. Did the day one and day two steps.

mL required for 0.1 OD reading - 40mL. (More specific data impending) 1.5 hours required to achieve .346 OD reading.

Weight of Bacteria - 1.5g, 1.19g, 1.16g.

**__Week 11 - 11/5/2012__** Started the cloning and transformation protocol. Then decided it would be more efficient if Shane took care of most of it and I made more backup PCR products instead. First I though I found some leftover PCR sqared, so I tested it, however it was not PCR product at all (picture below confirms it). Then I used PCR secondary and started the to create PCR squared. However, Shane tested it and found out that the PCR did not work. 

Results indicate anonymous samples were not PCR squared. work.

__**Week 10 - 10/29/2012 **__ PCR squared was finally a success. Two concentrations were achieved. <span style="font-family: Arial,sans-serif; line-height: 0px; overflow: hidden;"> <span style="font-family: Arial,sans-serif; line-height: 0px; overflow: hidden;">PCR cleanup concentration 1: Gained through using 100uL of PCR product during cleanup.

<span style="font-family: Arial,sans-serif; line-height: 0px; overflow: hidden;"> PCR cleanup concentration 2: gained through using another 100uL of PCR product during cleanup.

<span style="font-family: Arial,sans-serif; line-height: 0px; overflow: hidden;">Both concentrations are acceptable, though the 56.4 ng/uL is preferable. My group plans to utilize this latter.

__**<span style="font-family: Arial,sans-serif;">Week 9 - 10/22/2012 **__ <span style="font-family: Arial,sans-serif;">Did two PCR cleanups. Unfortunately, both results yielded 0 ng as a concentration. This would mean that either I am missing some important component in my PCR protocols, or I am getting very unlucky. Will try another PCR cleanup next week.

__**<span style="font-family: Arial,sans-serif;">Week 8 - 10/15/2012 **__ <span style="font-family: Arial,sans-serif;">Predominately dedicated time to Virtual Drug Screening Refresher. Results are posted on my Google docs. Was going to start PCR cleanup on Saturday but fell ill.

__**<span style="font-family: Arial,sans-serif;">Week 7 - 10/8/2012 **__ <span style="font-family: Arial,sans-serif;">101612 - Akhi - ok good job. Good luck with PCR cleanup step and cloning. -- Dr. B

The final four lanes are my PCR^2. The results correlate with the results from PCR secondary and also match up with the predicted length of my gene sequence. I also worked on virtual drug screening and finished part 1. Currently waiting for results.

__**<span style="font-family: Arial,sans-serif;">Week 6 - 10/1/2012 **__ 100912 - Akhi, ok great. I am glad you guys got this to work. Crop your gels a little more so the gel is more visible. When you guys move on to cloning - use the NEW T4 DNA Poly and the new dGTP and new dCTP. - Dr. B

Ran primary PCR and got these results.

PCR Primary Results Lane 1 - 100bp ladder Lane 2 - Shane's sample Lane 3 - Mihir's sample Lane 4 - My sample (not even visible), perhaps attributed to the low temperature I used during the pcr program.

<span style="font-family: Arial,sans-serif; line-height: 0px; overflow: hidden;">

Secondary PCR (done using Mihir's stock) Lane 1 - 100bp ladder Lane 2 - My sample Lane 3 - Shane's sample Lane 4 - Mihir's sample

__**<span style="font-family: Arial,sans-serif;">Week 5 - 9/24/2012 **__ 100112 - Akhi - ok good. Does your Midiprep sequence line up with anything? Is it the right plasmid? I think your primer design looks good. We will order those. For you PCR gel - be sure to add that it is PCR of pGBR22 with M13for/M13rev primers. Also try to crop your gel images better. -- Dr. B

Sequencing Results from Midi Prep: NNNNNNNNNNNNNNNNNNNNNCTNNGTGGTGGNGGNGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGNNNNNNNNNNNGNNNCCACCTTTACTGGAGACCGTCAATGCCAATANNNTNNNGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGNCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGGTTGTCGCCTGAGCTGTAGTNGCCNTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCNNTGATGTTCAAAGAGCTGTCTGATGCTGATACGTTAANNTGNGCAGTGNCAGTGTTNGNTTNNCGNAANGTTTACCGGANAAATCAGTGNANANNAAACGGATTTTTCCGTCANANGNNNTGNGCNTNAACCNNACCNNTNCTTGNNTTNNGNNNTTTTNAGGATANNNTCATTTNCAATCGAAATTTNNCNCNNNNCTTTAAANNNNNNNNNNNNNTTTTTTNCNNNNNNNNNNANNAANTTNNNNNCCNANTTTTTGNNANNAANNNNNAANNCNNNNNNNCNNNNNNNNTTTNNNNTNNNNNNNNNNNNAANNNNAANNNNGNNNNNNNNNNNTTNNNNNNNNNNNNNCCNNNNNNNNNNTTNTNNANNNNNGNCN

It is the right plasmid.

<span style="font-family: Arial,sans-serif;">Design Primer Protocl Results: <span style="font-family: Arial,sans-serif;">Also posted on google docs.

<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Forward: <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 0 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 1.5 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 2 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 4 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 6
 * **LENGTH:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">31 ||
 * **GC CONTENT:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">35.5 % ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">57.9 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">65.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">65.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">66.3 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">66.3 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.4 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.4 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.0 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.0 ºC ||

<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Downstream: TATCCACCTTTACTG TTAGTACAGGTATGCTTTT Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 0 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 1.5 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 2 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 4 <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Mg++ 6
 * **LENGTH:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">34 ||
 * **GC CONTENT:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">35.3 % ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">59.2 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.1 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">67.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">68.7 ºC ||
 * **MELT TEMP:** || <span style="font-family: Verdana,sans-serif; font-size: 7.5pt;">69.3 ºC ||

PCR 1 1st Lane - DNA 1 kb ladder 2nd Lane - .3ng 3rd Lane - 3ng 4th Lane - 30ng 5th Lane - negative control (i might have accidentally added some plasmid)

__**Week 4 - 9/16/2012**__ Akhi - ok concentration on midiprep is good. Depending on how much you use for cloning, you may end up needing to make more? -- Dr. B Did midi prep and have currently sent the obtained plasmid to sequencing. Will post results when available. In the meantime, nano drop reveals



Concentration 53.6ng/uL

Also did the PCR protocol for pLIC sequencing vectors of pNIC-Bsa4. Currently let the samples run through the thermal program and stored in the -20 freezer, next step is to run it with the agarose gel. Problems occurred. Seems that the machine was programmed wrong, 5min heat for 30 cycles instead of 1 min for 30 cycles. Will see to it on Monday.

__**Week 3 - 9/10/2012**__ Akhi - ok good. Include analysis of your RE digest results. Do your pLIC results match up to what they should be? -- Dr. B 091812

Data pending (talk about transformatio

Did a transformation pNIC- Bsa4, left the conicals in the -20 freezer.

RE Digest Results:

9/13/2012 Akhilesh Padhye RE Digest pGBR22 Lane 1 skip Lane 2 1kb DNA ladder NEB Lane 3 1kb DNA ladder NEB Lane 4 Uncut Plasmid Lane 5 EcoRI Lane 6 PvuII Lane 7 EcoRI + PvuII

Questions for RE Digest: Why was the 3rd band a bid higher even though there was no reduction in size? The uncut plasmid curled up during the process and was able to navigate more quickly, compared to the stretched out EcoRI. Why did the last lane have a fading band in the end? Possibly due to that sequence being a bit lighter than the rest of the bands.

Did pLIC primer dilution first. Sequencing Results for The pLIC reverse sequence did indeed match with what was expected. Blast results yielded a 99.8% identity.

LICrAAP-pLIC-rev : NNNNNNNTGNNNNNTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAG AACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGA TATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAA CAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACC AGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAA GACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTA ACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGA ACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTT GCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCNNTCCCTG AACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGA CGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGC CGGAGATCCTAAAAATGCGGATGANNCATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAA ACGCTGGCNGCGTCTTTNANGACAGCGACAANTCGATGCAATGATNCTATCCTAAANACCAANCNCAANNATGNCNNNNAN CNCNTTTNCNTCTGANGAAAANCCNTNNTCTANNCTGATNNNCNNANNNTNNGNANNNANNNCTGANANTGNNNANNANNN ATCANCNTNNNNNNNNNNNNANNNNNNNGNNNANNGNNNNNNAANNNNNNTNNNNGGNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNGNNNTNNNNNNNGGNNNNNNNNNNCNNNNNNNNNNNNNNNNNNN

LICfAAP-pLIC-for NNNNNNNNNNNNGGNNCTCNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCG GATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATT GTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATT GTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGA GTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAA AGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTCAGTGAA CAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCA CTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCA GAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCAT CTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGC CTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAANNATTGATTTNNNATCCTCT ACACCGTTGATGTTCAAAGAGCTGTCTGANGCTGATACGTTAACTTNTGCAGTNTCAGTGNTTGNTTGNCGTANGNTTACCGNA NNAATCANTGNNNAATNAACGNATTTTNCGTCNNATGTAAATGNGNTGANNTGANNNTTNNNNNNTNNNNNTTNNNNNNNNCAT TNGCATCNNNNGTCNNNNNNNTTNNNNNNNGNNNNNNTNNNNCNNNNNNNNANNNNNCCNANTTTNNNNNNNNNNNNNNNNA NNNNNNNCNNNNNNNNNNNNNNNGNANNNNNNNNNCNNNNNNNNNNNNNNNNNCNNNNN

__**Week 2 - 9/3/2012**__

Not much lab work - only diluted primers and got them ready for primer DNA sequencing. Analyzed a DNA sequence in the computer lab.

__**Week 1 - 8/27/2012**__

Basic Info:

Target (protein/gene name): <span style="font-family: Arial,Helvetica,sans-serif;">Protein Tyrosine Phosphatase - lmo1800
*NCBI Gene # or RefSeq#: Project:[|61583], <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">NC_003210.1, Gene ID: 985934

*Protein ID (NP or XP #) or Wolbachia#: <span style="background-color: #ffffff; font-family: Calibri,sans-serif; font-size: 11pt;">NP_465325.1

*Organism: <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Listeria monocytogenes

*EC#: <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">3.1.3.48

*Amino Acid Sequence:

>gi|16803840|ref|NP_465325.1| hypothetical protein lmo1800 [Listeria monocytogenes EGD-e]

MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRTSSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGVDKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY

*CDS Gene Sequence:

Partial

>gi|16802048:c1873620-1872724 Listeria monocytogenes EGD-e, complete genome <span class="ff_line" style="background-color: #ffffff;">ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA

<span class="ff_line" style="background-color: #ffffff;">Full Info: http://vdsstream.wikispaces.com/Target+-+Protein+Tyrosine+Phosphatase+%28Listeria+monocytogenes%29