Serena+Z.


 * FALL 2013 **


 * Weeks 9 & 10: October 21 - November 3 **

// October 21-25, 2013: Inhibition Assay I // // October 26, 2013: Rice Undergraduate Research Symposium // // November 2, 2013: V //// irtual screening against novel compounds: ChemBridge-diversity3D library //



'In house' top-ranking compounds were acquired. Compound 5107893 was tested for inhibition in an enzyme assay. Inhibitory activity was not indicated, as normal nitrocefin hydrolase activity was detected at 486 nm. Additional concentration of compound 5107893 will be tested, along with other top-ranking 'in house' compounds. Top ranking cb306 compounds will be ordered. Virtual screening round against ChemBridgediversity3D library is currently being completed.

Have you collected any more data for weeks 7 & 8? Thank you. -Max 10/21/13
 * Weeks 7 & 8: October 7 - October 20 **

//October 10, 2013: Synergy HT Biotek Reader to determine optimal [NDM-1]// // October 11, 2013: Determining control set of ligands // //October 14-18, 2013:// //LigPrep protocol, GOLD & ICM control ligands docking, virtual screening against novel compounds: cb306 library & 'in house' library//

Table 4. Virtual screening against novel compounds: top 7 'in house' library ligands. Best GOLD fitness scores 80-89.55. Compounds ranked 1, 3, 4, and 7 pursued further with favorable LogP values (<2.7).

Table 3. Virtual screening against novel compounds: top 14 cb306 library ligands. Four compounds with GOLD fitness scores >90. All top 14 ligands follow Lipinski's rule of 5. Compounds ranked 1, 2, 5, 9, and 11 pursued further with favorable LogP values (<2.4).

Table 2. ICM validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. Positive and negative control ligands are interspersed, with a few unexpected results. GOLD evaluated control ligands more accurately.

Table 1. GOLD validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. All positive control ligands scored higher than negative control ligands as expected, thus validating GOLD virtual screening software.

Positive control ligands were acquired from PubChem after referring to literature. Negative control ligands were established by the Zinc database after inputting physico-chemical properties of positive control ligands. These ligands were ligprepped and control ligand docking on GOLD & ICM was conducted. NDM-1 PDB ID: 4EY2 was utilized and prepared in GOLD Hermes (2254 Hs added, 227 H2Os deleted). GOLD scored control ligands more accurately than ICM. Virtual screening against novel compounds was conducted using cb306 and 'in house' libraries. Potential inhibitors from both libraries will be acquired and tested for inhibition concurrently.

Great Captions and Great analysis. Keep up the nice work. Thank you. -Max 10/07/2013 Good Results Serena, keep up the good work! - Dr. B
 * Weeks 5 & 6: September 23 - October 6 **


 * //September 26-27, 2013: Optimal substrate (nitrocefin) dilution to establish absorbance signal, NDM-1 enzyme assay II//**
 * //September 30, 2013: NDM-1 enzyme assay III :)//**
 * //October 1, 2013: NDM-1 1.5 hour enzyme assay IV//**
 * //October 3-4, 2013: Virtual screening: ICM control docking//**


 * Optimal substrate (nitrocefin) dilution was established to register absorbance signal during enzyme assays. 1:5 nitrocefin dilution determined using results from Fig. 1 above (optimal absorbance of 0.5-1.0 at 485 nm). 1:5 substrate dilution was utilized during the second attempt of the enzyme assay. Results in Fig. 2 illustrate that the absorbance change was insignificant during the 10 minute time-based assay, as NDM-1 dilutions created assay conditions with too little enzyme. Enzyme assay III was successful after using 2ug un-diluted NDM-1, which resulted in an expected increase in absorbance through NDM-1 hydrolase activity. NDM-1 enzyme assay results (9/30-10/1/2013) validate reproducibility of assay data for same aliquot of NDM-1. Future research will involve determining optimal [NDM-1] to create a plot with varied substrate concentrations to ultimately determine the Km value. In addition, other aliquots of NDM-1 will be tested to ensure functionality. Lastly, these results indicate that the NDM-1 enzyme purified on 7/31/2013 is indeed functional and can be utilized for enzyme inhibition assays after virtual screening is completed. **

Week 3 & 4: September 9 - September 22
 * Serena - ok good. Show a pretty picture (screenshot is ok) of an enzyme assay graph though. Dr. B 100113**


 * //September 12, 2013: Planning NDM-1 enzyme assay//**
 * //September 13, 2013:// // Prepared buffers: NDM-1 enzyme assay //**
 * // September 19, 2013: NDM-1 enzyme assay I with substrate nitrocefin //**

Preparation of assay buffers


 * Source: Thomas, P.; Zheng, M.; Wu, S.; Guo, H.; Liu, D.; Xu, D.; Fast, W., Characterization of Purified New Delhi Metallo-β-lactamase-1. //Biochemistry//** 2011**, 50, (46), 10102-10113.**


 * Dilute NDM-1 in Buffer A: 50 mM Tris-HCl, pH 7 and 150 mM NaCl**
 * Buffer B: 50 mL 0.1 M Hepes, pH 7 and 20 µM ZnSO4 solution**
 * 0.1 mM colorimetric nitrocefin substrate in water (VWR, Radnor, Pennsylvania) from 100 mM stock solution of 5 mg nitrocefin in DMSO**
 * Prepare 0.16-0.33 µg of NDM-1 in total volume of 300 µL and monitor product absorbance at ~485nm **

Results of NDM-1 enzyme assay I with nitrocefin:


 * Spectrophotometric time-based assay (spec: Chipper) resulted in 0 absorbance signal at 486.00 nm for samples:**
 * Buffer B only,**
 * Buffer B + nitrocefin, and**
 * 4 µL NDM-1 enzyme + buffer B + nitrocefin. **


 * Full spectrum assay conducted and illustrated 0 absorbance for buffer B only. 0.02 absorbance at ~400 nm and 0 absorbance for all other wavelengths observed for buffer B + nitrocefin sample. In addition, the following samples were tested at full spectrum and had an insignificant absorbance output (maximum 0.01 absorbance at 400 nm): **
 * 4 µL NDM-1 + buffer B + nitrocefin, **
 * 5 µL NDM-1 + buffer B + nitrocefin, **
 * 20 µL NDM-1 + buffer B + nitrocefin, **
 * 80 µL NDM-1 + buffer B + nitrocefin,**
 * 90 µL NDM-1 + buffer B + nitrocefin, and**
 * Cuvette with Vf = 600 µL of NDM-1 + buffer B + nitrocefin. **




 * These results are worrisome and do not correlate with the cell-based overnight NDM-1 growth assay results. Spectrophotometric signal should result with nitrocefin. Substrate amount may be too minimal to register an absorbance signal. In addition, these results indicate that the NDM-1 enzyme may be inactive (purified 7/31/13). The ExPASy ProtParam tool establishes the estimated half-life of NDM-1: 30 hours (mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo), and >10 hours (Escherichia coli, in vivo). A second enzyme assay should be conducted with increased substrate concentration along with a second spec to elucidate accuracy of Chipper. An additional cell-based overnight NDM-1 growth assay should be completed to test whether the NDM-1 protein is still active. If results are negative, a new batch of purified NDM-1 will need to be utilized. **


 * Serena, ok good. Dr. B 090913 **

Week 1 & 2: August 26 - September 8
 * //August 29, 2013: Cell-based overnight NDM-1 growth assay//**
 * //August 30, 2013: Results of overnight NDM-1 growth assay//**

Results:


 * 2 transformation tubes (+Kan, -Amp): bacterial growth**
 * 2 transformation tubes (+ Kan, +Amp): no bacterial growth**
 * 2 transformation tubes (+Kan, +Amp, 250 uL NDM-1 protein): medium bacterial growth**
 * 2 transformation tubes (+Kan, +Amp, 500 uL NDM-1 protein): large bacterial growth**
 * 2 transformation tubes (+Kan, +Amp, 1.5 mL NDM-1 protein): no bacterial growth**


 * Transformation tubes post 16 hours in shaking incubator displayed, for the most part, expected results. However, 1.5 mL NDM-1 protein tubes should have theoretically contained greater bacterial growth than tubes containing 250 uL and 500 uL NDM-1. These results provide confidence that the NDM-1 protein is functional.**

WEEK 9


 * //July 29, 2013:// -**
 * //July 30, 2013: NDM-1 protein expression (day V) - sonication & spin down//**
 * //July 31, 2013: NDM-1 Ni-NTA affinity purification//**
 * //August 1, 2013: -//**
 * //August 2, 2013: NDM-1 protein characterization; gel dried on 8/5/2013 - K.R.//**

NDM-1 Protein Nanodrops Post-Purification
 * //Elution 1, Trial 1.// Concentration = 0.30 mg/ml, absorbance = 0.299.**
 * //Elution 1, Trial 2.// Concentration = 0.26 mg/ml, absorbance = 0.264.**
 * //Elution 2, Trial 1.// Concentration = -0.02 mg/ml, absorbance = -0.019.**
 * //Elution 2, Trial 2.// Concentration = -0.04 mg/ml, absorbance = -0.038.**

WEEK 8


 * //July 22, 2013:// // Results available - cloning #2; s //// tarter culture of transformed BL21(DE3) E. coli with //// pET27b(+) NDM-1(d35): protein expression (day II) //**
 * //July 23, 2013: NDM-1 protein expression (day III) - K.R.//**
 * //July 24, 2013: -//**
 * //July 25, 2013: Preparation of purification lysis, wash, & elution buffers//**
 * //July 26, 2013: -//**


 * // Table 2. // pNIC-Bsa4 cloning round 2 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). + clone from sample #8! **

+ Clone Pairwise Alignments & Chromatograms pNIC-Bsa4 Cloning #2 with NDM-1 Insert Nanodrops


 * //Colony #1.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.70, Concentration = 87.1 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility. **


 * //Colony #2.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.94, 260/230 = 1.65, Concentration = 107.8 ng/uL. 260/230 ratio could be improved. Excellent concentration - sample submitted to DNA sequencing facility. **


 * //Colony #3.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.79, Concentration = 79.3 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility. **


 * //Colony #4.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.99, Concentration = 76.0 ng/uL. Purity ratios are within 0.25 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility. **


 * //Colony #5.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.98, 260/230 = 1.72, Concentration = 82.7 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility. **


 * //Colony #6.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.89, Concentration = 69.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility. **


 * //Colony #7.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.84, Concentration = 64.4 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility. **


 * //Colony #8.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.53, Concentration = 72.5 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility. **


 * //Colony #9.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.92, Concentration = 65.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility. **

//NOTE**: Samples 1-9 eluted correctly in 50 uL Elution Buffer. All Nanodrops illustrate lower concentration value of 2 trials completed for each colony.**//

WEEK 7


 * //July 14, 2013: Spin down 8 cloning samples//**
 * //July 15, 2013: Mini-Prep 8 cloning samples//**
 * //July 16, 2013: Submit 10 cloning samples - DNA sequencing facility//**
 * //July 17, 2013: Results available - cloning #1//**
 * //July 18, 2013:// // Prepared overnight culture of 8 colonies from Masterplate A - cloning #2 //**
 * //July 19, 2013: Submit Midi-Prepped// //pET27b(+) NDM-1(d35) samples - DNA sequencing facility; spin down, Mini-Prep, submit 9 cloning #2 samples//**


 * //Table 1.// pNIC-Bsa4 cloning round 1 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). No + clones - perform round 2 of cloning.**


 * //NOTE: Changes for round 2 of cloning: elute in accurate amount of Elution Buffer (50 uL); use correct transformation tubes; when eluting - insert buffer directly into the center of column to maximize amount of eluted DNA.//**

pNIC-Bsa4 Cloning #1 with NDM-1 Insert Nanodrops


 * //Colony #1.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.70, 260/230 = 0.93, Concentration = 70.6 ng/uL. 260/230 purity could be greatly improved. Good concentration - sample submitted to DNA sequencing facility. **


 * //Colony #2.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.92, 260/230 = 1.29, Concentration = 41.3 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility. **


 * //Colony #3.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.07, 260/230 = 1.66, Concentration = 35.6 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility. **


 * //Colony #4.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.70, Concentration = 33.2 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility. **


 * //Colony #5.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.93, 260/230 = 1.52, Concentration = 40.2 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility. **


 * //Colony #6.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.96, 260/230 = 1.81, Concentration = 18.3 ng/uL. 260/230 purity could be improved. Very low concentration - sample submitted to DNA sequencing facility. **


 * //Colony #7.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.79, 260/230 = 1.11, Concentration = 45.5 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility. **


 * //Colony #8.// 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.75, 260/230 = 0.95, Concentration = 56.3 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility. **

//NOTE**: Samples 1-8 eluted mistakenly in 100 uL Elution Buffer - incorrectly referred to Mini-Prep kit manual. Should elute in** 50 uL **Elution Buffer. This most likely caused low, non-ideal concentration values in above Nanodrops, particularly for Colony #6.**//

WEEK 6


 * //July 8, 2013: Secondary PCR, 1//° // and 2 //° // PCR agarose gel electrophoresis verification, pNIC-Bsa4 overnight culture preparation, //**
 * // Naturally Obsessed: The Making of a Scientist documentary viewing //**
 * //July 9, 2013: PCR squared reaction, PCR cleanup, pNIC-Bsa4 Midi-Prep - submitted to DNA sequencing facility //**
 * //July 10, 2013: Cloning: preparation of pNIC-Bsa4 as accepting vector + PCR cleanup, YopH spin down, transformation of DH5//α // & BL21(DE3) E.coli with pET27b(+) NDM-1(d35) (Day I) //**
 * //July 11, 2013: pNIC-Bsa4 preparation I gel check, p//// reparation of pNIC-Bsa4 II & III, PCR combination cleanup, gel check II, FPLC - ECDHFR, overnight culture of //// DH5 //α // E.coli with pET27b(+) NDM-1(d35) (Day II) //**
 * //July 12, 2013:// //Spin down & Midi-Prep of// // DH5 //α // E.coli with pET27b(+) NDM-1(d35) (Day III), cloning: cohesive end generation on PCR insert & accepting vector, annealing & transformation step //**
 * //July 13, 2013: Master plate & overnight culture of 8 colonies//**

DNA Sequencing Results: pET27b(+) NDM-1(d35) forward sequence post-Midi-Prep code NNNNNNNNNNTTCCCNNCTAGNNAATTTTGNTTAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCT GCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGGCCAGCAAATGGAAACTGGCGACCAACGGTTTGG CGATCTGGTTTTCCGCCAGCTCGCACCGAATGTCTGGCAGCACACTTCCTATCTCGACATGCCGGGTTTCGGGGCAGTCG CTTCCAACGGTTTGATCGTCAGGGATGGCGGCCGCGTGCTGGTGGTCGATACCGCCTGGACCGATGACCAGACCGCCCAG ATCCTCAACTGGATCAAGCAGGAGATCAACCTGCCGGTCGCGCTGGCGGTGGTGACTCACGCGCATCAGGACAAGATGGG CGGTATGGACGCGCTGCATGCGGCGGGGATTGCGACTTATGCCAATGCGTTGTCGAACCAGCTTGCCCCGCAAGAGGGGA TGGTTGCGGCGCAACACAGCCTGACTTTCGCCGCCAATGGCTGGGTCGAACCAGCAACCGCGCCCAACTTTGGCCCGCTC AAGGTATTTTACCCCGGCCCCGGCCACACCAGTGACAATATCACCGTTGGGATCGACGGCACCGACATCGCTTTTGGTGG CTGCCTGATCAAGGACAGCAAGGCCAAGTCGCTCGGCAATCTCGGTGATGCCGACACTGAGCACTACGCCGCGTCAGCGC GCGCGTTTGGTGCGGCGTTCCCCAAGGCCAGCATGATCGTGATGAGCCATTCCGCCCCCGATAGCCGCGCCGCAATCACT CATACGGCCCGCATGGCCGACAAGCTGCGCGCTAGCCAGCCAGAACTCGCCCCGGAAGANCCCGAGGANGTCGAGCACCA CCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAANCTGANTTGGCTGCTGCCACCGCTGANCAATAAC TAGCATAACCCCNNGGGGCNNNTAAACGGGTCTTGNNNGGGNTTTTNGCTNANNNANGANTATATCCNNATNNNANGGGN NNNNNCCNNNANNNNNNCATANNNNNNNNNNGNNNNNNNNNANNNNNNNNCTANNNNNNCNNNNCNANNNNNNNNNNNTN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNGNGNNNNNNNNNANNNNNTNNNNCNNNANCNNANNNN NNNNNNNNNNNNN code pET27b(+) NDM-1(d35) Transformed in DH5α //E. coli// Midi-Prep Nanodrops


 * //Trial 2.// 260/280 = 1.94, 260/230 = 3.37, Concentration = 41.5 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility. **


 * //Trial 1.// 260/280 = 1.98, 260/230 = 3.15, Concentration = 42.4 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility. **

Preparation #2, 3 of pNIC-Bsa4 as Accepting Vector post-PCR combination cleanup Nanodrops
 * //Trial 2.// 260/280 = 1.90, 260/230 = 2.31, Concentration = 65.6 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector. **


 * //Trial 1//. 260/280 = 1.87, 260/230 = 2.14, Concentration = 63.3 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector. **

Note: ** 260/280 assesses purity of DNA/RNA, optimal value ~ 1.8 (DNA) and ~2.0 (RNA). 260/230 is a secondary measure of nucleic acid purity, optimal value ~2.0-2.2. **
 * // Source: [] //**

Preparation of pNIC-Bsa4 as Accepting Vector post-PCR cleanup Nanodrops


 * //Trial 2.// 260/280 = 1.66, 260/230 = 1.40, Concentration = 18.3 ng/uL. Low concentration & purity requires 2 more rounds of pNIC-Bsa4 preparation.**


 * //Trial 1.// 260/280 = 1.71, 260/230 = 1.09, Concentration = 19.0 ng/uL. Low concentration & purity requires 2 more rounds of pNIC-Bsa4 preparation.**

DNA Sequencing Results: pNIC-Bsa4 Midi-Prep code NNNNNNNNNNNNNNNNCTCNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGG ATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGT TAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAA ACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGT ACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGG CTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTT TTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACT GTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTT TGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCAT AGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTG TGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTG CTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGT CTGANGCTGANNCGTTAACTTGTGCAGTNGTCAGNGNTTGNTTGNCGTAATGNTNANCGGANAAATCANTGNAGAATAAA NNNATTTTNNNTCNNANGNAANNNNGNNNNNNNACNTNCNNNNGNTNNNNNTTTNNNNNNANNNTTNGCATCNNNNNNNN CNNNCNTNNNNANNNNNNNNNNTTNNNNCNNNNANNNNNNNNNNNCNANTTTTNNNNNNNNNNNNNNNNNNNNCNNNNNT TTNNNANNCNNNNNNNNNNNNNAANNNNNNNNNNNNNN code pNIC-Bsa4 PCR cleanup Nanodrops


 * //Elution 1 Trial 2//**


 * //Elution 1 Trial 1//**

PCR^2 cleanup Nanodrops


 * //Elution 1 Trial 2//**


 * //Elution 1 Trial 1//**



WEEK 5 // //


 * //July 1, 2013: Protocol primer design - pNIC-BSA4 - New Delhi metallo-beta-lactamase-1 (computer lab)//**
 * //July 2, 2013: Protein characterization - drying the SDS-PAGE gel, Restriction enzyme digest - preparing samples//**
 * //July 3, 2013: Restriction enzyme digest - agarose gel electrophoresis verification, PCR protocol for pLIC sequencing vectors of pNIC-Bsa4 //**
 * // July 4, 2013: Holiday //**
 * //July 5, 2013: pLIC PCR - agarose gel electrophoresis verification, Primer overlap PCR with Q5 Polymerase - p rimer dilutions for assembly step //**
 * // July 6, 2013: Optional session - Primary PCR //**



WEEK 4 // //


 * //June 24, 2013: Analyze DNA sequence exercise (computer lab)//**
 * //June 25, 2013: Protein expression scaled up - day 5: sonication & spin down steps//**
 * //June 26, 2013: Protein purification, SDS-PAGE gel production//**
 * //June 27, 2013: TACC tour - Pickle Research Campus//**
 * //June 28, 2013: Protein characterization - results available next week//**




 * Elution 1 average absorbance: 0.847 **
 * Elution 2 average absorbance: 0.0385**
 * Molar extinction coefficient: 18910 M-1 cm-1 **
 * Molecular weight: 33512.8**


 * Beer's law (A= e bc) calculations to determine yield of YopH: **


 * Elution 1 concentration at 280 nm: **
 * Average Abs: (0.838 + 0.856) / 2 = 0.847 **
 * Concentration = 0.847/[(18910 M-1 cm-1)(1 cm)]= 4.48 E-5 mol/L (33512.8 g/mol) = 1.50 mg/mL **
 * Yield = (1.50 mg/mL)(5 mL) = 7.51 mg**


 * Elution 2 concentration at 280 nm: **
 * Average Abs: (0.016 + 0.061) / 2 = 0.0385 **
 * Concentration = 0.0385/[(18910 M-1 cm-1)(1 cm)]= 2.04 E-6 mol/L (33512.8 g/mol) = 0.0682 mg/mL **
 * Yield = (0.0682 mg/mL)(5 mL) = 0.341 mg**

WEEK 3 // //


 * //June 17, 2013: Agarose gel electrophoresis - PCR I for RE cloning pGFP, D ay I transformation of YopH and FtHAP into BL21(DE3) cells, preparation of 500 mL LB for larger scale protein expression //**
 * // June 18, 2013: PCR II for RE cloning pGFP, Day II overnight culture for larger scale protein expression //**
 * // June 19, 2013: Larger scale protein expression YopH in pNIC-Bsa4 //**
 * // June 20, 2013: FF205 training, PCR III for RE cloning pGFP (samples only, no gel) //**
 * // June 21, 2013: Lab closed //**

Larger scale protein expression YopH in pNIC-Bsa4:


 * 6/18: 2 small 100 mL LB + Kan+ BL21(DE3) with YopH plasmid overnight cultures prepared ( ≈ 16 hours in shaking incubator) **
 * 6/19: -10 mL of overnight culture added to 500 mL of LB in a 2L flask**


 * -Sample #0 cell lysate before induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge **
 * -250 uL IPTG added to 2L flask to induce expression of T7 polymerase**
 * -Sample grown for 4 hrs at 37 °C in shaking incubator **
 * -Sample #1 cell lysate after induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge **
 * -JA-10 rotor in centrifuge reserved and pre-cooled; sample from 2L flask transferred to 500 mL cylindrical bottle **
 * -Centrifuged sample for 20 min at 6000 g **
 * -Disposed supernatant, pellet weight: 1.88 g stored in -80 °C fridge after resuspending pellet in 10 mL buffer **

pGFP PCR II:



pGFP PCR I:



WEEK 2 // - // // Serena - like the pink! - Good work and good documentation. Do you have your DNA Sequence result to show? - Dr. B //
 * //June 10, 2013: Primer design overlap assembly//**
 * // June 11, 2013: Agarose gel electrophoresis (check PCR I) //**
 * //June 12, 2013: Midi-Prep, DNA sequencing submission II, ordering target primers //**
 * //June 13, 2013: PCR for RE cloning pGFP/pmCherry//**
 * //June 14, 2013: Optional session//**

DNA sequencing result II post-MidiPrep: code NNNNNNNNNNNNNNNNNNNNNNNNNNCNNGCCATGGNNNNGGNNGNNNNCCCAATGCCCAAAAAACGAGGTNNNANTAAC GGGNNNNNAGGNGACATTNNNNNGNCCTCNGNCNCCCNNNNNNNCNNNNNNACTGGNNTCGGCNNGACNGGGGCNTGGNN NNNGCTGATCTACNNNTNNATCNNNNNGCNNNGANANANNNNANNNNNNGNNNNGANCNNNGNCTNGGNCNANGNNANNA GANNTNNANCNTCCCTTGGAGATCNANANCNCGANTTANACNTNAGTGCANCTNNTGNACCNGAACCCCGGTCCCAACAA ANGTNNGCTTCTTCNGNNNNAAAGAANCCNAATTNCANGTNGNANGTGGAGGGNTGTAAGGGGNNNNTGNNCNNNNNGNN NNANNNCNNNNANNNNNNNNNGAANAAGGTGGNNANCNNCNTTNCCNNTCCNGANNTTTNNGNNNNTNNNNGCNNNNNNN NNNNNCNNANNCAACANCTTNNNNNCNNANTNNNTNAGGGANGACANANNNNNNNNNNTNNNTNNNAGGNTTATNTNCTA NNCTCATGCAANNNNNNCNNCNNANNNANNANACGTCNNGNTTNNNGATAANAAAAANAGNNAGTGCTTCTGCTCNTTTN CNNATCNCACANNNNCAGGAAGANNNACTCGATNNTNTGNTGTTCTGCCCCCATCNNNTTNNTNANACNTGCTNNNANNT NNANATNAANANTNNGNNNNGTNT code pGBR22 PCR:



Midi-Prep Nanodrop:





WEEK 1 // //
 * //June 3, 2013: Primer dilution, DNA sequencing submission I//**
 * //June 4, 2013: LB + LB agar plates, Nanodrop plasmid DNA//**
 * //June 5, 2013: Transformation efficiency//**
 * //June 6, 2013: Target discovery (computer lab)//**
 * //June 7, 2013: pGBR22 PCR I//**

DNA sequencing result I: code NNNNNNNNNNNNNNNNNNNNNNNNNTNNNCTCTGANTCNTGCATTCNCGNCTGGCGTGGCAGTCCATTGGGAGTCTGTAC ACCTGGGCACTGGTGCCCGCCNCGTACCGCTGCCNACCTATCCGTTTCGCNCGAACGCGTGTGGCTGCGTCCGAAACTAG TGGCCTCCGCGCCNANGGACGCCNAGTTCTGGGCAGGGANCNNGCGGGCGGACGTNNGNNTNNNCANNGANGGNCTCNNC CCCCNCNANGANNGNATCCNNTCNAANNTGCNCGGNGGCNNNNCNTGNCNAATANNNNGNGGNNNNGCNCNATNCTANG code






 * Transformation Efficiency = # of colonies / ng of plasmid on each plate **

__ Calculations: __

Plate A (1 ng pmCherry plasmid DNA): 1,008 colonies / 1ng = 1,008 colonies/ng Plate B (5 ng pmCherry plasmid DNA): 3,861 colonies / 5ng = 772.2 colonies/ng Plate C (25 ng pmCherry plasmid DNA): 1,215 colonies / 25ng = 48.6 colonies/ng