User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06

Purpose

• Protein was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography.
• Adenosine deaminase plasmid with the mutation E34K was transformed into DH5α-T1 E.coli cells with a second attempt.
• UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on 2012/10/31 to measure absorbance of supernatant of Au/Lysozyme samples

Procedure for Protein Extraction and Purification from BL21(DE3) Cells

• Frozen BL21(DE3) E.coli cells from 2012/11/04 were taken out from the freezer with gloves.
• The frozen cells were placed into a 500mL beaker filled with ~30°C of warm tap water for 10 minutes. After 10 minutes, cells are completely thawed and appeared in liquid form.
• On ice bath, the cells were sonicated in three 1 minute intervals with three repeated cycles of procedures below:
  • Cells are sonicated under 8mV for 30 seconds with ______Sonicator.
  • Cells are immediately placed on ice for 30 seconds
• After sonication, the E.coli cells were transferred from a 25mL falcon tube into a 30mL centrifuge tube. Another 30mL centrifuge tube was filled with tap water with the mass equal to the mass of 30mL centrifuge tube containing cellular sample.
• The sample and water weight for balance were centrifuged in _______instrument with the following conditions:

    Speed: 18,000rpm
    Temperature: 4°C
    Time elapsed: 2 hours
    Rotor used:_____

• After centrifugation, supernatants and pellet form. The pellet was discarded, and about 100mL supernatant yielded and was kept in a 500mL beaker.
• The supernatant was filtered through Supor®-450 47mm membrane filter by pouring the supernatant through filter with membrane attached. Filter was attached to pressure suction that draws the supernatant through the membrane filter. The supernatant was filtered into a 800mL Erlenmeyer flask, then poured into a 25mL falcon tube.
• The filtered protein supernatant was injected into the GE Pharmacia® AKTA Purifier 100 Fast Protein Liquid Chromatography to separate ADA proteins from the rest of proteins in sample through the following procedure:
  • The column was ran using elution buffer with _____ at a speed of ___mL/s
  • Protein samples were injected into FPLC with a 10mL syringe and let run through the column at a speed of _____
  • Binding buffer was run through the column with _____at a speed of_____
  • UV-vis spectrum was taken at the eluted product over time and chromatogram was yielded. Because the chromatograph was not saved, the data cannot be displayed here.
  • Fractions of protein was collected based on the UV-vis spectrum, and three separate fractions are formed as a result of affinity binding and competitive binding via imidazole.
  • Fraction 1 and 3 from the column were collected and transferred into a 15mL falcon tube
  • Fraction 2 from the column, which were suspected to obtain the highest concentration of ADA protein, were transferred into a separate 15mL falcon tube.

Result for Protein Extraction and Purification from BL21(DE3) Cells

• Because the UV-vis spectrum from FPLC was not saved, data cannot be displayed. However, there was a clear peak at 280nm that indicates presence of ADA protein.
• The absorbance peak at 280nm was the highest in fraction 2 of elution, fraction 2 was suspected to have the highest concentration of ADA proteins. Furthermore, it indicated that ADA protein started to unbind from the column via competitive binding with imidazole in fraction 1. Both fraction 1 and 3 also yielded an absorbance peak at 280nm but not as high as the ones from fraction 2. As a result, fraction 1 and 3 were also collected for maximum yield of ADA proteins in the same falcon tube.

Procedure for E.coli cell Transformation

• DH5α-T1 Competent E. coli cells was obtained from -80°C freezer and was thawed on ice bath after 30 minutes.
• PCR products with the mutation E34K in ADA plasmid strand obtained from 2012/10/16 was thawed on ice bath.
• 40μL of E.coli cells was added into a 1mL microcentrifuge tube, 10μL of ADA E34K plasmid was added into bacteria without mix.
• The culture was placed on heat block for heat shock for 30 seconds. After heat shock, the culture was placed back on ice.
• 250μL of SOC media obtained from New England Biolabs® was added to bacteria culture. The microcentrifuge tube was taped and attached to a small test tube. Sample was placed in orbital shaker under the following conditions:

    225rpm
    37°C
    1 hour

• Cell culture was taken out of shaker, and plated on LB agar plates made with LB, agar, and ampicillin. The LB plates were made in the following way:

    Following reagents were weighted out:
       20g of LB Agar powder from Teknova® 
       5g of tryptone from Teknova®
       2.5g of yeast extract from BD®
       5g of NaCl from Fisher Scientific®
       7.5g agar from BD®
    Above were placed into a 800mL Erlenmeyer flask.
    500mL distilled water was added to reagents.
    The solution was autoclaved under liquid cycle (see 2012/11/03 for protocol for liquid cycle)
    After cooling to ~50°C, 50mg ampicillin was poured into autoclaved solution, swirled to mix
    Under sterile condition, the solution was poured into separate petri dish when temperature reaches ~40°C
    Plates were left under sterile condition to solidify

• 2 plates were yielded: one with 50μL of transformed cell culture, the other with 200μL of transformed cell culture.
• Plates were placed in incubator for 15 hours at 37°C upside down for cellular growth.

Results for E.coli cell Transformation

• After 15 hours in incubator, the plates appeared to have no colonies.
• This indicates that the transformation did not work. This procedure is on hold for trouble shooting.

Procedure for UV-vis on Au/Lysozyme samples

• Au/Lysozyme samples made on 2012/10/31 were used to take spectrum of all samples via UV-vis spectrometer. The below ratios of Au/Lysozyme sample were run:

    20 - 30 - 40 - 50 - 60 - 70 - 80 - 120 - 130

• The appearance of Au/Lysozyme samples were noted and displayed in results section below.
• The UV-vis spectrometer was set to spectrum method and wavelength from 200nm to 800nm was ran for all samples.
• Samples were run with distilled water as baseline standards. 2mL of samples were placed in quartz cuvette for each sample run.
• Absorbance at each wavelength for the above ratios were recorded and plotted on graph, shown in results section below.

Results for UV-vis on Au/Lysozyme samples

• The Au/Lysozyme solutions have various appearances based on the ratio of gold to lysozyme. For Au/Lysozyme with mole ratio of 20 to 60, the supernatant appeared purple in color and clear. No fibers were observed. For Au/Lysozyme with mole ratio of 70 to 120, the supernatant appeared clear with purple fibers formed and collected at the bottom of test tube.
• Detailed observation for each mole ratio of Au/Lysozyme were recorded in table below:

• The supernatant were used to ran UV-vis spectrometry. A graph with absorbance versus wavelength from 200nm to 800nm were plotted and shown below:

• From the graph above, it is shown that Au/Lysozyme with mole ratio of 40 yielded the greatest absorbance, and hence indicated the greatest number of gold nanoparticles in solution. Followed by 40 is mole ratio of 50, then mole ratio of 60 that yields decreasing concentration of gold nanoparticles in solution. This indicates that the concentration of gold nanoparticles decrease after the maximum mole ratio of 40.
• It was expected for mole ratio of 130 Au/Lysozyme sample to have an absorbance around 0.00. But according to graph, the absorbance for sample came out to be 0.04. This might caused by accidental resuspension of purple fibers into supernatant when withdrawing the supernatant for sampling.
• The peak at wavelength 525nm was looked at separately based on Bakshi, et al, which stated that gold nanoparticles can be indicated through a peak at 530nm when gold nanoparticles are present in samples. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below:

• Based on the table, a graph of absorbance versus increasing mole ratios of Au/Lysozymes were made to better depict relationship between concentration of gold nanoparticles in supernatant and mole ratios of Au/Lysozymes. The graph is shown below:

• From the graph above, it can be shown that the concentration of gold nanoparticles in supernatant increases as the mole ratio of Au/Lysozyme goes from 20 to 40. The concentration of gold nanoparticles peaks at mole ratio of 40, then decreases from there on as ratio of Au/Lysozyme increases further. It seems like that the concentration of gold nanoparticles in supernatant increases from mole ratio of 120 to 130, but it might be caused by accident resuspension of fibers when absorbance of sample 130 Au/Lysozyme was taken.
• The 40 mole ratio of Au/Lysozyme can be further investigated from 20 to 50 to determine an exact ratio of gold to lysozyme that encouraged protein aggregation.