Nicolet+F.

=__ **Fall 2013** __= __ **Week 13 & 14** __

__**Week 11 & 12**__

Missing analysis. more data? -UM

Figure 27: Table of positive and negative control ligands used for virtual screening of MTcanA protein.
 * __Positive Control Ligands__ ||
 * MZM ||
 * EZL ||
 * GRE ||
 * a09 ||
 * HQE ||
 * 1QV ||
 * TOR ||
 * BEW ||
 * AZM ||
 * IE2 ||
 * __Negative Control Ligands__ ||
 * Aspirin ||
 * < 34375942 ||
 * < 6425551 ||
 * < 84844675 ||
 * < 8698501 ||
 * < 11535796 ||
 * < 8698501 ||
 * < 11535796 ||

code NNNNNNNNNNNNNNNNNNNNNNNNNNNTNNGNNNNTTGNCNGNNNCNTNNTCNNCNTCTTTNTTCTGGCGNNNATCNGGN NNNNNNNNNNNGNNNNTNNAATCNANGNCCGTTANNNNNACNTNNNNNNNNNAAAGGNNATCTCTCTTTNTNGCNCNTNN CNCGTGAAATCNTNCNGNNNCNCNANNNCNAAGNGGNNNNNNNNNNNNNNNNNNNNGACTTNNNTNNNNATTCNNGANNA ANCCCGTATNNNTCCNANCNGNTNNCCNGNNTCNNCNCCGTNNNTTCCACNTNNNNCGTTTTAGCTNNNNNNNNN code || Figure 28: MTcanA_rev sequence for cloning attempt #1. Sequence results were abnormally shorter than gene length. Sequence also contains a large amount of unknown nucleotide bases. Query was

code NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGNANNANNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNC NNNNGNNNNNNNNANGATTTTNCNNNATTCNAANNNANCCTTTTTCTNNCAANAGNGGGCCNANNNNTTTTTTTNAANNN CNNAATTNNCNCTTTCNTTNNNNNGCNNNNNTNAANCCTTGTAANANNANCGGTTTTNNNNNCACANTGCCCTTTTTTTT NNNNNNNNNCCCNGCATTTTTGGGCNNAAAAGGGNCCCCTTTTTTTTTNNNNNCCCCCCCCCGAAAAGAAAAANTNGACC CCCCCNCAANCANAAAAANNTTTGTGGTANTGGAGGGANCCCANNNNTTTTTTTTAACNGNGGCCTTNNAAANNNNGNTT TNNNNNNNNNNNNNNNNGNCNNNNAAAAGCNNNN code || Figure 29: MTcanA_for sequence for cloning attempt #1. Sequence results were abnormally shorter than gene length. Sequence also contains a large amount of unknown nucleotide bases.

When submitting a nucleotide BLAST on MTcanA for cloning attempt #1, a message of "No significant similarity" resulted.

code NNNNNNNNNNNNNNGNNCTCNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCG GATCCGTATCCACCTTTACTGTTACGGGGTAACCTCGTTCAGTTTACCGGTCGCAACGTCAAAGACGAAACCACGCAGAG AGGTGTGTTTAGTAACGAACGGGTTCACTTCAATGCGGCGCAGGCTCTGACGTACATCTTCAACCGCGTCCGGGTAAGAT TCCGGAGACCAGGTCGGACGGATACCGGTTTCATCCTGAATCGCGCGTTTGAAGTCGTCGTCGGTGAAGTGAGCATACCA CAGTCGGTGTGGTGCAGCAGAATGATTTCACGGGTACCGAGCAGACGCTGAGAGATCGCCAGAGAACGGATAACGTCATC GGTAACGGTCATGGATTGGAAGTACAGGTTCTCGGTACCCAGATCTACNCCAGAAGAATGATGATGATGATGGTGCATNT NNNTTTCTCCTTCTTAAAGTTNAANNNAATTNNTTCTAGAGGGGAATTGNNNTACGATNNNNGNNNNCNTGTANANNGNG ANAANNNANNNNNATAATCANNANGANNGAGNNNNNNAANTN code || Figure 30: MTcanA_rev sequence for cloning attempt #2. Sequence results looked better than the first attempt, however, the identity was only 93% and the query was 61%.

code NNNNNNNNNNNNNACTTTAGNNGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA GAACCTGTACTTCCAATCCATGACCGTTACCGATGACGTTATCCGTTCTCTGGCGATCTCTCAGCGTCTGCTCGGTACCC GTGAAATCATTCTGCTGCACCACACCGACTGTGGTATGCTCACTTCACCGACGACGACTTCAAACGCGCGATTCAGGATG AAACCGGTATCCGTCCGACCTGGTCTCCGGAATCTTACCCGGACGCGGTTGAAGATGTACGTCAGAGCCTGCGCCGCATT GAAGTGAACCCGTTCGTTACTAAACACACCTCTCTGCGTGGTTTCGTCTTTGACGTTGCGACCGGTAAACTGAACGAGGT TACCCCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCA CCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAG CATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGG ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCANCGTGACCGCTACACTTGNCAGCGCCCTA GCGCCCGCTCNTTTCGCTTTCTNCCCNTCCTTTTCACGCCACGTNCGCCGGCTTTCCCCGTTAAGNTCTAAATCNGGGGN NTCCCNTTAGGGNTCNNANNNNNGCTANNNNNGNACCNCNACNNNAAAANNNNTNANNGNNATNNGNTNTTNNNANTNNN NCNTN code || Figure 31: MTcanA_for sequence for cloning attempt #2. Sequence results looked better than the first attempt, however, the identity was only 93% and the query was 61%.



__ **Week 9 & 10** __

Oligo mix was remade from original primers. Tail primers, MTcanA_for and MTcanA_rev were also remade to rule out any possibilities for primer dimers. This time, secondary PCR showed much less primer dimers, though they still appeared. PCR squared will be performed to see if gel extraction will be possible.







Virtual work has been started on the 1YLK protein with positive and negative control ligands. So far, only the control library of compounds has been concatenated.

__ **Week 7 & 8** __

Secondary PCR was performed on a temperature gradient from 58 °C to 68°C. Primer dimers still formed within all samples.



Secondary PCR was then done using 0.5uL of forward and reverse primers instead of 1.0uL. A lower annealing temperature of 56 °C and the original annealing temperature of 58.8°C were used.




 * __Week 5 & 6__**


 * Gel extraction and PCR cleanup were performed using the PCR squared gels. Insufficient amount of MTcanA was retrieved due to contamination of the sample.**




 * Secondary PCR was repeated using extended annealing times. There was no contamination, however, the gel indicates that primer dimers have been created the secondary PCR sample. To solve this issue, a temperature gradient will be used for the annealing temperatures.**

__Week 3 & 4__


 * pNICbsa4 from week 2 was nanodropped. The concentration was found to be 69.8 ng/uL. This sample of pNICbsa4 was submitted for DNA sequencing with the forward and reverse primers pLIC_for and pLIC_rev. **




 * Primary and secondary PCR on MTcanA were completed and run on a gel. Secondary PCR shows two bands. Band at 516bp is the needed MTcanA. Lower band is a contaminant. PCR squared will be done to amplify MTcanA, however, gel extraction may need to be done if contaminant still remains after PCR squared.**

__Week 1 & 2**__

New tail primers were ordered (MTcanA_for and MTcanA_rev) for target. These primers were each diluted from the original amount of 33.2 nMoles (MTcanA_for) and 33.4 nMoles (MTcanA_rev) to a stock dilution of 100uM. From this stock dilution, working dilutions of 20uM were made of each. An oligo mix for MTcanA was also made.

pNICbsa4 was grown up overnight and midiprepped.

=__**Summer 2013**__=

__**Week 8**__





New tail primers were ordered and these will be used to re-run secondary PCR.

__**Week 7**__

Updated target page: β-carbonic anhydrases

__**Week 6**__



__**Week 5**__ PCR on pGBR22 was successful.



Target Assigned: Beta-carbonic anhydrase (canA) (Mycobacterium tuberculosis) Primers ordered: mtcanARev1284_FOR, mtcanARev1284_REV

__**Week 4**__

__**Week 3**__ PCR was done using pGBR22 from Week 2 and run on a gel for better results. This time it showed contamination.



BL21(DE3) cells were used to transform an overnight culture of pNIC-Bsa4 in 100 ml LB. The next morning, the culture was transferred to a 500mL LB flask. 20 ml of the culture was transferred to a larger culture, stored in the shaking incubator, and checked every 30 minutes until an OD 600 of 0.625 was reached. This culture was then stored in the shaking incubator for 4 hours. The pNIC-Bsa4 culture was spun down and re-suspended in buffer to be stored at -80 degrees Celsius.

__**Week 2**__



__**Week 1**__

Transformation Efficiency: Plate A, Fig. 1 (1 ng pGBR22): 3 Colonies Plate B, Fig. 2 (5 ng pGBR22): 252 Colonies Plate C, Fig. 3 (25 ng pGBR22): 1,920 Colonies

ATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACAT CCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAA TAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGT GTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGT AGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCC CATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTA TTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCC CCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAG GTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGAC CATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATG GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANNTGTCNN GNCAGCTGCATTAATGATCGGCNANNNNCGGGGNNANGNNNNTNGCGTNTGGGNNCTCTTCNNNTCCTCNNTCANNGACT CNNNNNNNTCNNNCNNNNNNNNNNNNNNNNNGNNTCANNNNNNNNNANGNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNN ANNNNNNNGNAANANNNNNNNNNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNN NNN || Figure 4: DNA sequencing-pGBR22.
 * NNNNNNNNNNNNNNCGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTG