Andrei+L.+(RP+Summ+14)

**Weeks 14-15**
//Analysis// 300mL of overnight culture was prepared for re-expression of BL21 E. Coli cells. 1500mL of large culture was made for preparation of protein purification next semester. These cultures were spun down in the large centrifuge at 6K G's and re-suspended in lysis buffer. 500mL of culture were suspended in 10mL of buffer each, in 15mL conical tubes. These tubes were stored at -80 degrees Celsius for the Christmas break.

//Conclusion// Enzyme assays completely failed, so we hypothesize that either our protein or substrate is nonfunctional due to improper storage/preparation. Different methods of substrate preparation will be tried next semester. If all else fails, we will order our protein pre-designed and go from there. If our protein is functional an assays work, we will move on to inhibition specific assays and X-ray crystallography.

Weeks 11-13
Fig 1: Trial 1 of enzyme assays using first glycerol sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 30uL of NADPH and 30uL of biopterin. Final volume of enzyme added was around 100uL. Fig 2: Trial 2 of enzyme assays using second glycerol sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 180uL of NADPH and 180uL of biopterin. Two increments of 50uL of enzyme were added. Drops in absorbance were due to increased final volume diluting the concentration of reagents. Fig 3: Trial 3 of enzyme assays using second glycerol sample again. 200uL of 0.2M sodium acetate buffer was added initially, along with 150uL of NADPH and 200uL of biopterin. A second increment of 50uL of NADPH was added to bring the absorbance between 1-1.5. Final volume of enzyme added was 100uL. Fig 4: Trial 4 of enzyme assays using the first glycerol sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 180uL of NADPH and 180uL of biopterin. Two increments of 50uL of enzyme were added. Drops in absorbance were due to increased final volume diluting the concentration of reagents. Fig 5: Trial 5 of enzyme assays using the second liquid N2 sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 180uL of NADPH and 180uL of biopterin. Two increments of 50uL of enzyme were added. Drops in absorbance were due to increased final volume diluting the concentration of reagents. Fig 6: Trial 6 of enzyme assays using the first glycerol sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 30uL of NADPH and 30uL of biopterin. Two increments of 50uL of enzyme were added. Drops in absorbance were due to increased final volume diluting the concentration of reagents. Final volume was 400uL as the final concentration of the working dilutions was changed. Fig 7: Trial 7 of enzyme assays using the first glycerol sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 30uL of NADPH and 30uL of biopterin. Around 200uL of enzyme were finally added, with no change in absorbance. This might be due to spectrophotometer problems, as proper detection isn't reliable around 2. Fig 8: Trial 8 of enzyme assays using the most recent Elution 2 sample. 200uL of 0.2M sodium acetate buffer was added initially, along with 20uL of NADPH and 20uL of biopterin. Enzyme was added, respectively: 10,10,10,20,20,30,50,50uL. Absorbance only changed with regard to volume.

//Analysis// No enzyme assays worked. At first, assay procedures were followed according to literature, and then changed accordingly. Finally, when no FPLC samples were working, we decided to use an Elution to conduct an assay with. Throughout conducting trials, it was discovered that if enzyme is added in incremental small amounts, then changes in absorbance wouldn't be due to volume changes.

//Conclusion// Because all trials were unsuccessful, we will scrap all existing samples and re-express for further assays next semester. If assays continue not to work, then we will order our designed enzyme and test that instead.

Weeks 7-10
Fig 1: FPLC results from second round of expression. Tubes 41-54 were collected and showed a molecular weight of around 70 kDa. This indicates that PTR1 protein dimerized in solution.The resulting sample was saved as 1mL in 20% glycerol and another 1mL was snap frozen with liquid nitrogen. Fig 2: Nanodrop results of Elution 1 (third round of expression) measured at A280nm. The yield was 4.20 mg/mL. Fig 3: Nanodrop results of Elution 2 (third round of expression) measured at A280nm. The yield was 0.45 mg/mL.

A third round of expression was begun due to the dimerization of PTR1 shown by the above FPLC graph. A single gradient of 900mM NaCl was used, and salt was added before and after column purification. We hypothesized that nickel purification removed the salts post filtration and thus the sample is left stored improperly. If this round of expression does not dimerize, our enzyme assays will show if PTR1 is active in both the dimer and normal forms.

Virtual screening was completed on the control library, and the first 30K ligands from the ChemBridge library were screened using the NADP active site. A second round of screening will have to be done using the DVP active site, since targeting the NADP active site might cause side effects due to its widespread biochemical use. Ligands from both runs will be selected and compiled based on highest scoring.

We will set up enzyme assays this following week. The spectrophotometric absorbance of NADP will be measured at 520 nm using 6-biopterin as a substrate. If PTR1 is active, then the absorbance will decrease as the reagents are used up. We will test both glycerol and liquid nitrogen stored proteins. The Km value for our enzyme and NADP is 0.012 mM.

9232014- Great work

Weeks 5 and 6
Fig 1: SDS PAGE gel characterization of tube C post purification. Salt concentration was 900 mM NaCl. Lanes 5C and 6C (Elutions 1 and 2 respectively) show banding around 35kDa. This indicates no dimerization of protein PTR1 and contamination is minimal.

//From right to left:// 1kDa ladder 0 - Cell lysate before induction 1 - Cell lysate after induction 2C - Soluble Fraction 3D - Flow through sample D* 4C - Wash 5C - Elution 1 6C - Elution 2 3C - Flow through sample C*


 * Error was made in loading lanes. Sample 3C loaded in the remaining well for consistency.

Fig 2: SDS PAGE gel characterization of tube D post purification. Salt concentration was 400 mM NaCl. Lanes 5D and 6D (Elutions 1 and 2 respectively) show banding around 35kDa. This indicates no dimerization of protein PTR1 and contamination is minimal.

//From right to left:// 1kDa ladder 0 - Cell lysate before induction 1 - Cell lysate after induction 2D - Soluble Fraction 3D - Flow through 4D - Wash 5D - Elution 1 6D - Elution 2

Fig 3: BL21 competent cells transformed with 5ng of pNIC+B1 plasmid on Kan+Sucrose plates.

Fig 4: BL21 competent cells transformed with 25ng of pNIC+B1 plasmid on Kan+Sucrose plates.

Fig 5: BL21 competent cells transformed with 50ng of pNIC+B1 plasmid on Kan+Sucrose plates.

__Analysis__ Because the old plates were faulty with contamination and/or sucrose build up, we re-transformed BL21 E. Coli using B1 plasmid. Different concentrations of added DNA were used. Surprisingly the medium amount of ~25ng produced the most and largest colonies. Those were picked and used in overnight culture. Large culture volumes were quadrupled in hopes of obtaining a larger bacterial/protein yield. There was not enough overnight culture left to bring the OD600 values to 0.5 fast enough, so bacteria could have exited log phase growth when induction started, leading to error. Haley spun down the resulting cultures, lysing the bacteria to extract PTR1 protein. Salt gradients ranging from 400-900 mM NaCl were used in hopes of keeping the protein from dimerizing. Salt discourage polar interactions and thus dimerization. These salt gradient samples were purified via nickel column filtration. Tubes C and D were characterized because they are the upper and lower gradients (900, 400 mM). So if they are good, the salt concentrations between those values are good as well. Characterization showed relatively uncontaminated banding around 35 kDa in Elutions 1 and 2. This is good because the weight corresponds to the undimerized form of our protein.

__Conclusion__ The next step would be to FPLC Elutions 1 and 2 for all salt gradients. This will result in a more pure protein, although we are not sure if it will dimerize in this step. Last time this same procedure was performed, characterization shoed banding around 35 kDa, but FPLC yielded proteins only in the 70 kDa range. If no dimerization occurs, then the protein will be stored via glycerol and snap freeze, with salt helping the preservation process.

Weeks 3 and 4
Protein expression was performed using BL21 culture cells from previous weeks. The resulting miniprep samples were run through column purification using Nickel Resin. Samples throughout the procedure were loaded onto a SDS page gel. Fig 1: Nanodrop graph of Elution 1 after column purification with a concentration of 1.40 mg/mL measured at A280nm.

Fig 2: SDS page gel run using P. Reductase Samples. From left to right, respectively: Well 1 - Pageruler ladder Well 2 - Sample 1 (Cell Lysate after induction) Well 3 - Sample 2 (Soluble Fraction) Well 4 - Sample 3 (Flow Through) Well 5 - Sample 4 (Wash) Well 6 - Sample 5 (Elution 1) Well 7 - Sample 6 (Elution 2) Fig 3: Pageruler ladder with kDa sizes visible. Elution 1 from above shows heavy banding around 30kDa, in range for our protein. Elution 2 confirms this result but with lower concentration.
 * There was no Sample 0 (Cell Lysate before induction).

Fig 4: FPLC results using Elution 1 from column filtration. Samples collected ranged from tubes 28-33. Even though P. reductase is around 30 kDa, the machine registered proteins in the 50-60 kDa range. This could be due to dimerization, or the fact that our protein has trans-membrane regions.

Fig 5: First attempt at post-FPLC concentration. Collected samples 28-33 were combined and concentrated to 4mL. The resulting concentration was 0.22 mg/mL measured at A280nm.

Fig 6: Second attempt at post-FPLC concentration. Sample from Figure 5 was concentrated again to 1mL. The resulting concentration was 0.34 mg/mL measured at A280nm.

__Summary and Analysis__ When the sample was reduced to a proper concentration, 0.5mL were stored in 20% glycerol at -20 Celcius. The other 0.5mL was snap-frozen with liquid nitrogen and stored at -80 Celcius. Next week we will perform enzyme assays to determine which storage method works best. Even though the concentrations are lower than expected, we hope enzyme assays will still work. Further steps include virtual screening of our protein and new rounds of protein expression. To compensate for low yields, more bacteria will be grown using 2L of LB media. This change in procedure should yield more P. reductase protein and thus make assays more reliable.

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Fig 3: BLAST pairwise alignment of B1 with T7 forward (Query) compared with known sequence of pLIC plasmid (Subject). There was a 98% match that was then confirmed to be 100% after N's were replaced with proper bases according to cromatograph.=====

Fig 4: Color key alignment for pairwise alignment in figure 3. Red line indicates a high match probability.


Fig 5: Nanodrop result of sample D1 with a concentration of 56.5 ng/uL blanked with 5mM Tris-HCl pH 7.5.



Fig 8: Nanodrop result of sample B1 after Midi-Prep with a 58.6 ng/uL.
Fig 9: Transformed BL21 cells with 1ng of clone B1 DNA. There is only 1 colony growth.

Fig 10: Transformed BL21 cells with 5ng of clone B1 DNA. There is only ~1 colony growth. Fig 11: Transformed BL21 cells with 25ng of clone B1 DNA. There are multiple colony growths.

__ Summary and Analysis __ Hailey's master plates were miniprepped for DNA sequence analysis. Out of all the samples, only B1 returned a positive clone using T7 forward primer. The master plate B1 sample was then transferred to DH5alpha large culture for midiprepping. After a large amount of B1 clone was collected, most was stored. Transformation was then started using BL21 competent cells and confirmed B1 clone plasmid. Only the plate with 25ng of DNA added had any growth, suggesting that plasmid efficiency is low. Next week we will complete culturing and then move on to protein purification. The B1 midiprepped sample will need to be sequenced again to ensure that it is the proper gene, otherwise any further procedures will be futile.

Week 4
__PAGE Gels__ Homemade gels were made for characterization of FtHap and loaded with the following: Well 1: PageRuler Ladder Well 2: Cell lysate before induction Well 3: Cell lysate after induction Well 4: Flow Through Well 5: Wash Well 6: Elution 1 Well 7: Elution 2

The gel did not run as expected and instead resulted in smeared, indistinguishable bands. It can be attributed to the fact that the gel was made improperly and there was too much 5% stacking gel added on top of the 12% separating gel.


 * Figure 1:** Failed PAGE gel of FtHap characterization. Bands are not distinguishable and ladder shows no weight discrimination. Procedure will be repeated next week.

__My First PCR__ Attempt at PCR was successful. Agarose gels showed proper banding and no contamination. Well 1: 1kb DNA Ladder Well 2: 0.3 ng of pGBR22 Well 3: 3 ng of pGBR22 Well 4: 30 ng of pGBR22 Well 5: Control - no DNA present Well 6-9: Empty Well 10: 100bp DNA Ladder
 * Figure 2:** PCR agarose gels. Wells are as follows:

__DNA Analysis__ pGBR22 sequence was analyzed and inserted into pGEM vector backbone. The same procedure was used to analyze Midi-Prep pGBR22 plasmid sequence. It was determined to match the accepted sequence and thus the sample was transferred to the verified plasmids box.

Week 3
Transformation efficiencies calculated for last week's pGBR22 plates. Yields were estimated due to large over-growths.

Results: Efficiencies grow with increasing amounts of plasmid added. This is true up to a point, usually where adding excess plasmid no longer has an effect. There was an error in Plate C where the TE was calculated to be less than plates A&B. This is probably due to the fact that all colony numbers were estimated since the agar plates were overgrown with contamination.

Colonies were picked and grown in small culture for MidiPrep. Note - agar plate colonies were purple whereas the small culture was beige and cloudy. Dr. B explained that DH5 alpha cells are good for expressing DNA rather than protein (pGBR22 protein is purple whereas DNA is colorless).

Results:
 * Fig 1:** pGBR22 sequencing with M13 forward primer. 1292 nucleotides present. Next step will be sequence confirmation.
 * Fig 2:** pGBR22 sequencing with M13 reverse primer. 1260 nucleotides present.


 * Fig 3:** Plasmid pGBR22 nanodrop results for Trial 1 at 230nm for a concentration of 30.2 ng/uL.
 * Fig 4:** Plasmid pGBR22 nanodrop results for Trial 2 at 230nm for a concentration of 28.6 ng/uL. Both results averaged to 29.4 ng/uL.

Concentrations of plasmid thought to be too low and therefore worthless. However, sequencing yielded promising results that will be verified next week.

Overnight culture performed with Luis using FtHap+pNIC+Bsa4 BL21(D3) cells.The spectrophotometer "Chipper" was used to reach 0.5 absorbance before IPTG buffer was added and culture was left to incubate. Once done, centrifugation yielded a wet pellet wet of 9.33 grams. Re-suspension and sonication were performed.

I then helped him with expression so he would have a proper culture to MidiPrep for his plasmid...

Week 1 and 2

 * Figure 1:** Plate A with 1ng of plasmid pGBR22 in competent DH5alpha cells grown in SOC coated ampicillin agar plate. Small purple colonies are present among contamination.


 * Figure 2:** Plate B with 5ng of plasmid pGBR22 in competent DH5alpha cells grown in SOC coated ampicillin agar plate. Larger, more prolific colonies are present among contamination.


 * Figure 3:** Plate C with 25ng of plasmid pGBR22 in competent DH5alpha cells grown in SOC coated ampicillin agar plate. This plate yielded the most colonies, indicating that greater amounts of plasmid leads to a higher transformation efficiency.


 * Figure 4:** Plate D with 0ng of plasmid pGBR22 in competent DH5alpha cells grown in SOC coated ampicillin agar plate. No colonies grew on this plate due to lack of plasmid and therefore lack of ampicillin resistance.


 * Figure 5:** Sequence fail for the pGFP plasmid using M13 reverse primer. Procedure will be repeated using SP6 promoter next week.