Melissa+H.

FALL 2013  Week 13 & 14: 11/18/2013 - 12/1/2013 Summary of Week 13 & 14: Another sample of PfFabG was expressed, purified, and characterized. However, when the SDS-Page gel was analyzed, there was little to no band at the correct location (~33 kDa) on the gel which means that expression was likely unsuccessful. More virtual screening was conducted and the data was combined together in an excel document. When the samples (Elution 1, Elution 2, Concentrated Sample) were analyzed using the Nanodrop, the conentrations determined were very low. This provides more evidence that the protein did not expressed or was in the -80 degree celsius freezer for too long to be viable. From left to right: ColorPlus Protein Ladder 0: Before IPTG Induction 2: Soluble Fraction 3: Flowthrough 4: Wash 5: Elution 1 6: Elution 2

PfFabG is supposed to elute around the 33 kDa mark. From the gel you can see that the protein appears up until the wash sample and is missing in Elution 1 and 2. Based on the previous gel, I do not believe that there is a problem with the His-tag adhering to the Ni ions because there was a band at 33kDa in Elution 1 and 2 in the previous gel. There may have been a problem with purification in general. I used the same purification column as before so there could be an issue with the the age of the column or the incubation step.

Week 11 & 12: 11/4/13 - 11/17/2013 Good--nice job on the protein -UM **Summary of Week 11 & 12**: GOLD docking of the CB306 library was conducted and analyzed. An enzyme assay was conducted on the enzyme collected two weeks ago to test for enzyme functionality. Due to the results collected from the time based trial, it appears that the protein is indeed functional. **Table 1**: Gold docking analysis of CB306 compound library during the initial screening. The first 33 compounds will be further analyzed for more thorough analysis. **Table 2:** Gold docking scores of the second run of the top 33 compounds in the CB306 library. The top four scores/10% of the second run are shown.
 * = Ligand name ||= Score ||= S(PLP) ||= S(hbond) ||= S(cho) ||= S(metal) ||= DE(clash) ||= DE(tors) ||= intcor ||
 * = '5761107' ||= 66.83 ||= -59.11 ||= 2.68 ||= 0 ||= 0 ||= 0 ||= 0.18 ||= 0.02 ||
 * = '7722615' ||= 67.72 ||= -62.47 ||= 1.99 ||= 0 ||= 0 ||= 0 ||= 0.52 ||= 0.13 ||
 * = '7757183' ||= 65.44 ||= -55.07 ||= 3.74 ||= 0 ||= 0 ||= 0 ||= 1.55 ||= 2.23 ||
 * = '9027311' ||= 65.13 ||= -57.29 ||= 1 ||= 2 ||= 0 ||= 0 ||= 0.62 ||= 0.08 ||
 * = '7575772' ||= 64.69 ||= -49.8 ||= 5.55 ||= 0 ||= 0 ||= 0.5 ||= 0.74 ||= 0.21 ||
 * = '7676009' ||= 64.5 ||= -56.59 ||= 4 ||= 0 ||= 0 ||= 0 ||= 3.11 ||= 2.09 ||
 * = '7938694' ||= 63.67 ||= -61.61 ||= 1 ||= 1 ||= 0 ||= 0 ||= 2.04 ||= 0.1 ||
 * = '7842136' ||= 62.75 ||= -38.7 ||= 8.17 ||= 0 ||= 0 ||= 0 ||= 0.28 ||= 0.1 ||
 * = '5531766' ||= 62.69 ||= -55.43 ||= 3.11 ||= 0 ||= 0 ||= 0 ||= 2.21 ||= 2.36 ||
 * = '7558664' ||= 62.53 ||= -55.08 ||= 3.26 ||= 0 ||= 0 ||= 0 ||= 1.55 ||= 0.77 ||
 * = Rank ||= Ligand name ||= Score ||= S(PLP) ||= S(hbond) ||= S(cho) ||= S(metal) ||= DE(clash) ||= DE(tors) ||= intcor ||
 * = 1 ||= 7558664 ||= 70.73 ||= -65.45 ||= 3.13 ||= 0 ||= 0 ||= 0 ||= 2.36 ||= 0.61 ||
 * = 2 ||= 6629394 ||= 70.59 ||= -49.11 ||= 6.41 ||= 1 ||= 0 ||= 0.01 ||= 1.57 ||= 2.41 ||
 * = 3 ||= 7938694 ||= 70.01 ||= -64.31 ||= 2 ||= 1 ||= 0 ||= 0 ||= 1.81 ||= 0.27 ||
 * = 4 ||= 7575772 ||= 69.45 ||= -51.76 ||= 6.75 ||= 0 ||= 0 ||= 0 ||= 1.31 ||= 0.07 ||

During the second run of the CB306 docking, the top ligands that were determined in the previous Run 2 were not ranked as highly in the second run. There is however a low standard deviation between each score. Although the scores may have ranked differently, the GOLD score between each ligand does not change by a large percentage. Because there is a subtle decrease in the absorbance of NADPH at 340 nm, this may indicate that the protein is functional. For the light blue line, the peaks that appear in the line are times when more enyme was added to the measured sample.

Week 9 & 10: 10/23/13 - 11/3/2013 **Good work Melissa! Do you have your FPLC results that you could post on here? Also, it would be good to include analysis on your virtual screening results. - Suman 11/4/13** Summary of Week 9 & 10: Protein was expressed, purified, and characterized during week 9. The protein was loaded into the FPLC for further concentration. Then the protein was stored in 20% glycerol. Protein expression was re-conducted due to a low concentration in protein. For virtual screening, 2C07 was analyzed through MolProbity and control and CB306 docking was conducted. Then the DH5alpha cells from cloning during the summer were regrown on a master plate and midiprepped for a larger stock of plasmid concentration. Fig: Concentration analysis of PfFabG after protein purification. The average concentration after Elution 1 was 0.645 mg/mL. Fig 2: Concentration analysis of the concentrated elution 1 with an averge concentration of 1.59 mg/mL and an average 260/280 of 1.825. Fig 3: The average concentration of Elution 2 was 0.28 mg/ml with an average 260/280 value of 0.94. Fig: Concentration analysis of PfFabG after FPLC but before concentrating the sample. The average concentration is very low at 0.155 mg/mL. Overall, protein purification will have to be scaled up in order to collect a larger yield of protein during round 3 of expression. **Molprobity Analysis** Resolution: 1.5 angstroms Clashscore: 11.95 Poor rotamers: 0 Ramachandran outliers: 0 Ramachandran favored: 236 Cbeta deviations >0.25 A: 0 MolProbity score ^: 1.69 Residues with bad bonds: 0/982 Residues with bad angles: 1/1224 Error in Active Site: Contacts, McSc contacts, Hets contacts, 1 angle Overall there are few errors in active site and the protein structure was modified with flips and without hydrogens. **Table 1**: Gold score table of control docking of PfFabG
 * Ligand name || Score || S(PLP) || S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor ||
 * NADPH || '15983949_NADPHpos8' || 103.54 || -71.38 || 10.18 || 1.86 || 0 || 0 || 2.93 || 1.89 ||
 * Catechin Gallate || '6419835_catechingallate' || 81.46 || -60.12 || 7.22 || 0 || 0 || 0 || 0.16 || 0.02 ||
 * Epigallocatechin gallate || '65064_epigallocatechingallate' || 77.64 || -59.07 || 6.92 || 0 || 0 || 0 || 1.11 || 0.04 ||
 * Myricetin || '5281672_myricetin' || 64.71 || -42.32 || 7.47 || 0 || 0 || 0 || 0.91 || 1.81 ||
 * Chloroquine || '2719_chloroquine' || 62.98 || -61.23 || 0.84 || 0.4 || 0 || 0 || 1.07 || 0.15 ||
 * Heptadecynoic Acid || '22600366_heptadecynoicacid' || 61.67 || -56.09 || 2.19 || 0 || 0 || 0 || 0.57 || 0.1 ||
 * AG-690/10423014 || '17399864_AG690' || 60.74 || -56.91 || 1 || 0.79 || 0 || 0 || 0.77 || 0 ||
 * Morin || '5281670_morin' || 59.68 || -44.93 || 5.03 || 0 || 0 || 0 || 1.12 || 1.91 ||
 * Luteolin || '5280445_luteolin' || 58.41 || -42.15 || 5.71 || 0 || 0 || 0 || 0.43 || 0 ||
 * AC1NU03A || 5442438_AC1NU03A' || 55.91 || -58.66 || 0 || 0 || 0 || 0.44 || 1.43 || 0.25 ||
 * ZINC19166762 || 28082271_ZINC19166762' || 53.49 || -49.45 || 1.99 || 0 || 0 || 0 || 1.08 || 0.12 ||
 * Hexachlorophene || 3598_hexachlorophene' || 51.17 || -48.47 || 1 || 0 || 0 || 0 || 0.15 || 0 ||
 * AI-204/31720014 || '16654126_AI204' || 50.23 || -47.25 || 0.61 || 0.47 || 0 || 0 || 0.12 || 0 ||
 * Chrysin || '5281607_chrysin' || 49.85 || -44.16 || 2 || 0 || 0 || 0 || 0.16 || 0 ||
 * Aspirin || '2244_aspirin' || 41.94 || -36.39 || 1.95 || 0 || 0 || 0 || 0.54 || 0.78 ||

Week 7 & 8: 10/07/2013 - 10/20/13  **Good work Melissa. Try to include a brief analysis of the Nanodrop data. Do you have any virtual data? Thank you. -Max 10/21/13** Lane 0: Elution 2 Lane 1: Elution 1 Lane 2: Wash Lane 3: Flow through Lane 4: Soluble Fraction Lane 5: Cell lysate after IPTG induction Lane 6: Cell lysate before IPTG induction Lane 7: ColorPlus Prestained Protein Ladder In the Elution 1 and 2 lane, there is a band at approximately 80 kDa. PfFabG has a molar mass of 76626.6 g/mol. This shows promise of a successful expression of PfFabG. However, there is large amounts of contamination and not strong band at the correct size. The intensity of the band contradicts the concentration determined by Nanodrop. FPLC will either be pursued or re-expression may be necessary.
 * Summary of Week 7 & 8**: SDS-PAGE gel was analysed. PfFabG was spun down in the concentrator, yielding an average concentration of 2.52 mg/mL. Protein expression was attempted but due to time constraints, it could not be achieved. More preparation towards virtual screening was conducted. The positive and negative control ligands were collected. 3-oxoacyl reductase was modified in PyMol by aligning the protein structure 4I08 and adding NADPH to 2C07. Large scale protein expression was conducted on 10/19/2013. The cell lysate collected after spin down (JA10 rotor, 6000 rpm, 4degrees Celsius, 20 min) was transfered into a 15 ml conical tube and placed in the -80 freezer. 3.33 g of cell lysate was collected.

Week 5 & 6: 09/23/2013 - 10/06/2013 Nice Job on your page. Try to elaborate a bit more on your analysis such as if you have collected sufficient data to move onto next steps. Thank you. -Max 10/07/2013

The average concentration of Elution 1 is 2.01mg/mL with an average 260/280 value of 1.895. It was determined that the concentration of eluted protein demonstrated a large yield.
 * Summary of Week 5 &** 6: The BL21 (DE3) cells expressing PfFabG were sonicated to lyse the cells and release the protein into solution. The cell lysate was then spun down at 20,000 g at 4 degrees Celsius for 30 minutes total. The supernatant was collected and prepared for protein purification. Elution 1 and 2 of protein purification was collected in two 15 mL conical tubes and a SDS-PAGE gel was conducted for protein characterization. The protein gel is not shown here though because Grace was nice enough to dry the gel for me since I'll be out of town.

Week 3 & 4: 09/09/2013 - 09/22/2013 Melissa - good work and well layed out. Dr B 100113 Because the area that shows 100% confidence in both sequences overlap, Sample B2 can be concluded that it is indeed a positive clone of PfFabG. Table 1: Comparison table of the Blast nucleotide analysis of the samples submitted to DNA sequencing. The last column indicates whether or not the sample was a positive or negative clone.
 * Summary of Week 3 & 4:** DNA sequencing request for Sample B2 was analyzed and it was confirmed that the plasmid was a positive clone of PfFabG. Plasmid was transformed in DH5alpha and BL21(DE3) cells. Transformation in DH5alpha cells are necessary to increase the stock plasmid available. First attempt was unsuccessful. Second attempt was successful only for the transformation of PfFabG in BL21(DE3) cells. BL21 (DE3) cells underwent large scale protein expression. The cells were centrifuged, the supernatant disposed of, and resuspended in lysis buffer. The sample was placed in the -80 degrees Celsius freezer.

Week 1 & 2: 8/28/2013 - 09/07/2013 Melissa - do you have more wet lab work here? Dr. B 090913
 * Summary of Week 1 & 2:** Blast analysis of the received DNA sequence from the Core Lab was conducted to determine if there was a match to a positive clone. Sample B2 showed 99% query coverage and 99% identities but the reverse pLIC sequenced request did not show as much success. The reverse and forward sequences were resubmitted for DNA sequencing.

SUMMER 2013 Week 8: 7/20/2013 - 7/27/2013
 * Summary of Week 8:** After more pNIC-Bsa4 plasmid was recollected, pNIC was restriction enzyme digested using Bsa1 to prepare the plasmid as an accepting vector again. Cohesive end generation was reconducted. Essentially the first cloning attempt was reconducted except the ratios between the accepting vector and the cohesive inserts were modified.

Week 7: 7/15/2013 - 7/19/2013
 * Summary of Week 7:** Pnic-Bsa4 plasmid was restriction enzyme digested using BsaI to prepare the plasmid as an accepting vector. Then the cohesive end generation was conducted on PCR inserts and the accepting vector. Then the inserts and vector were annealed and transformed in DH5alpha cells and placed on LB+Kan+Sucrose (5%) plates. The first cloning attempt was unsuccessful so more pnic-Bsa4 was grown overnight and midi-prepped. Because the concentration of the purified plasmid was so low, more pnic-Bsa4 will be grown and midiprepped.

Week 6: 7/8/2013 - 7/12/2013 Good work Melissa - Dr. B 071713 Because a distinct band did not appear at the correct location (935 base pairs), secondary PCR was re-conducted.
 * Summary of Week 6**: PfDXR was FPLC filtrated to further purify the sample. The DNA sequence request for pnic-Bsa4 was received and the DNA sequence was analyzed. On 7/10/13, secondary PCR was re-conducted using a different annealing temperature of 66 degrees Celsius and a gel was run. After determining that secondary PCR was successful, PCR squared was conducted, yielding a total sample of 200 uL. Then the plasmid solution was cleaned up using the Sigma PCR Clean Up kit. After collecting the sample, the total average concentration of the solution was 160.95 ng/uL.

Week 5: 7/1/2013 - 7/5/2013
 * Summary of Week 5:** The tail primers for FabG for P. falciparum were designed and ordered on 7/1/2013. On Tuesday, 7/2/2013, the primers were diluted from 100 uM to 1 uM in the master mix of the 24 oligonucleotides necessary to synthesize the gene. Overnight cultures of pnic-Bsa4 in DH5alpha cells were grown and midiprepped. On 7/3/2013, primary PCR on the oligo mix was conducted. Because the tail primers were not available on Wednesday, secondary PCR was conducted using the first and last oligonucleotide as the primers. The identity of the primary and secondary PCR were confirmed using an agarose gel (shown in Fig 18). On 7/5/2013, PCR samples were prepared for secondary PCR using tail primers designed on 7/1/2013.

Lane 1: 1 kb ladder Lane 2: Primary PCR sample for FabG Lane 3: Secondary PCR sample for FabG Lane 4: N/A Lane 5-7: Oscar V.'s samples

WEEK 4: 6/24/2013 - 6/28/2013 Lane 1: N/A Lane 2: PAGE gel ruler Lane 3: Cell lysate before induction Lane 4: Cell lysate after induction Lane 5: Soluble fraction Lane 6: Flow through Lane 7: Wash Lane 8: Elution 1 Lane 9: Elution 2 Lane 10: N/A Lane 1: 1 kb DNA ladder Lane 2: Uncut plasmid Lane 3: EcoRI - HF Lane 4: PvuII Lane 5: EcoRI-HF + PvuII
 * Summary of Week 4:** A homemade SDS-PAGE gel was made in lab on 6/25/2013. Then on 6/26/2013, the sample of PfDXR in BL21(DE3) cells were sonicated to release the protein from the cell and then the protein was purified using a Ni-NTA column. Samples were collected from each step (from cell lysate to Elution 2) and were characterized using the SDS-PAGE gel made. On 6/28/2013, a sample of pGBR22 underwent an restriction enzyme digest.


 * WEEK 3: 6/17/2013 - 6/21/2013**
 * Summary of Week 3:** The DNA sequence submitted to the DNA sequencing core during Week 2 for FrTUHP in pnic-Bsa4 was analyzed in order to confirm the presence of the correct plasmid. On 6/17/2013, a gel was run on the GFP PCR conducted on 6/13/2013 to confirm the identity of the plasmid. on 6/18/2013, a PCR was run on pnic-Bsa4 and a gel was run. A large scale protein expression of PfDXR in pnic-Bsa4 in BL21(DE3) E. coli cells. was conducted on 6/18/2013. A growth curve was calculated in order to measure the concentration of the bacteria in media up to 0.5 OD and then 263 uL IPTG was added. The 3.37 g of cells were harvested by centrifugation, re-suspended in re-suspension buffer, and then stored at -80 degree Celsius. pNIC-Bsa4 was amplified using PCR on 6/19/2013 but when the gel was run on 6/20/2013, no DNA was present and thus the PCR was uneffective.

Fig 10: DNA sequence received for FrTUHP in pnic-Bsa4
 * NNNNNNNNTNGNTTACTTTAGNNGAGANATACATATGCACCATCATCATCATCAT TCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGTGGGTT ACTCTTCTAAGCTGATCTTCGTGTCTATGATCACCCGTCACGGCGATCGTGCGCC GTTTGCGAACATCGAAAACGCGAACTACTCTTGGGGTACCGAACTGTCTGAGCTG ACCCCAATCGGTATGAACCAGGAATACAATCTGGGTCTCCAGCTGCGTAAACGTT ACATCGACAAATTCGGTCTGCTGCCGGAGCACTACGTTGACCAATCTATCTACGT CCTGTCTTCTCACACGAATCGTACCGTTGTTTCTGCGCAATCTCTGCTGATGGGT CTGTACCCAGCGGGTACGGGTCCGCTGATCGGTGACGGCGACCCGGCAATCAAAG ACCGTTTCCAGCCGATCCCGATCATGACCCTGTCTGCGGACTCTCGTCTGATCCA GTTCCCGTACGAACAGTACCTGGCGGTTCTGAAAAAGTACGTGTACAACAGCCCG GAGTGGCAAAACAAAACCAAAGAAGCGGCTCCGAACTTTGCCAAATGGCAGCAAA TCCTGGGTAATCGTATCTCTGGTCTGAACGACGTCATCACCGTTGGTGACGTTCT GATCGTGGCCCAGGCGCATGGCAAGCCGCTGCCGAAAGGTCTGTCTCAAGAAGAC GCGGACCAGATCATCGCGCTGACCGACTGGGGTCTGGCGCAGCAGTTCAAATCTC AGAAAGTTTCTTACATCATGGGTGGTAAACTGACCAACCGTATGATCGAAGACCT CAACAACGCGGTTAATGGTAAATCTAAATACAAAATGACCTACTACTCCNGTCNC GACCTCACCCTGCTCGAAGTTATGGGTACTCTGGGTGNNNCGCTGGACACCGCCC CAGGTTACGCGAGCAACCTGGAAATGGAAANNGNNCNNNGATGGTGACATCTACN NCGTNNAACTGCGNTANNACNGNCAAGTANNNNAAANNTGCCNNNNNNNGANAAA AANACTNTNNNNNNTGGACNNNCTGACANNNNNNCNNNNNNNNANNAANANTCNN ANTAANNNNANGNNNGANNCGGATCCNAATTNNAGNNNNNNNNNNANNNN ||



The source of the nucleotide sequence used in the comparison was Joey and Christina's primer design sequence used in their research last summer. Their nucleotide sequence was optimized to for E. coli so the sequences in the NCBI database were not appropriate for comparison.



A gel was run on GFP PCR that was conducted last Thursday, 6/13/2013.

The samples in each lane are as followed: 1: 100 bp DNA ladder 2: 0.011 ng/ul GFP with VDS forward and reverse primers 3: 0.11 ng/ul GFP with VDS forward and reverse primers 4: 1.1 ng/ul GFP with VDS forward and reverse primers 5: NO GFP with VDS forward and reverse primers 6: 100 bp DNA ladder 7: 0.011 ng/ul GFP with M13 forward and reverse primers 8: 0.11 ng/ul GFP with M13 forward and reverse primers 9: 1.1 ng/ul GFP with M13 forward and reverse primers 10: ??? 11.1 ng/ul GFP with M13 forward and reverse primers

In the protocol provided for this PCR run, there was supposed to be no DNA present in Lane 10 which would be used as a control. Because there is a band for lane 10 and the band is more florescent than Lane 7-9, it is possible that the 11.1 ng/uL (1:100 dilution) was placed in the sample.



WEEK 2: 6/10/2013 - 6/14/2013 Melissa - excellent work! Specify which side of the PCR gel is yours. -- Dr. B CCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATAT CATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGA CACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGT AAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGT CTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCA GTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGT ACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGG TGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAAC ACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTT GGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGA GGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTA CACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCA CCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGC CCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGNGTAATAGC GAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGNGAATGGNCN NNNCCTGTAGCGGCGCATTAAGCGCGGCNGGGNGNGGNNGNTACNNNCAGCGNGACNGC NNNCNCTTNCNGCNCCNANCGNCCGCTCNTTNNNNTTCNTNCNNNNTTCTNNCNNCNTNNNN NNTTNCCNNNAGNNNTAANNNGGGNNCCNTTNNNNNCNANTTNNNNTTNNGNNNCNNNNNN NNNNNNNANNNNNNANNNNNNNNNNGGNNTNNCCNNNNNNANNNNNNNNTNNNNNNCCN || Fig 6: DNA sequence of pGBR22 plasmid
 * NNNNNNNNNNNNNNNGNNCTATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCT









Average concentration was 70.1 ng/uL.

WEEK 1: 6/3/2013 - 6/7/2013



The average concentration of the pGBR22 plasmid was 185.05 ng/ul. The average ratio between the purity in relation to the protein is 1.9. The average ratio between the purity in relation to other contaminants is 2.665. The average absorbance at 230 nm is 1.391. The purity of the protein in comparison to other contaminants is low because the ideal ratio value is 2.1 but the ratio determined was 2.665.







Because the plasmid is a low count plasmid, the number of colonies on the plate were much lower than was to be expected. Because E. coli competes for space, it was expected that the higher the concentration of DNA plasmid, the less amount of E. coli colonies per plate.