Ecoli Cells And Plasmid Dna Biology Essay

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E. coli cells were grown up on LB agar plates. The LB medium is provided by my task supervisor, Dr. Biagong Yue. The LB medium was covered with foil and autoclaved immediately to prevent the growth of bacteria from the atmosphere. Ampicillin (100µg/ml) and Chloramphenicol (34mg/ml) were added to the cooled LB medium under sterile conditions prior to use. The solid LB agar was melted in microwave oven. The molten LB agar was allowed to cool to about 50-60oC on the bench and the appropriate antibiotics were added. The molten agar was poured into the sterile petri dishes, while taking good care of not to introduce air bubbles into the agar. The petri dishes were then allowed to set at room temperature. The plates were stored up-side down at 4oC until needed. The E. coli strain, BL21, B834, CODON+ and Rosetta, was used for amplifying plasmid DNA in the transformation experiments.

Plasmid DNA:

Transformation of competent cells

Plasmid DNA used in this experiment is; 8082: p300 (1096-1721) and 8075: CBP (1098-1858), which is provided by Dr. Biagong Yue.

For internalization of plasmid DNA (GST-p300/CBP-HAT) into the four different BL21, B834, CODON+ and Rosetta cells, all of the competent cells were first thawed slowly on ice (approximately 1 hour) and then the plasmid DNA was added under sterile conditions. 1µL of CBP (0.89µg) and 2µL of p300 (0.08µg) was added to a 100µL aliquot of cells. Each tube was flicked gently to mix the cells and DNA and then stored on ice for 30mins. To induce internalization of the DNA into the cells, the tubes were placed at 42oC for 2 minutes (heat shock) and then the tubes were placed immediately on ice for another 2 minutes. LB (900 µL) was added to each aliquot of cells to promote the growth of the cells, which are then incubated at 37oC for 1 hour in a horizontal shaking incubator (Innova 4000 incubator shaker). Cells were recovered from the LB media by centrifugation in a microfuge (Biofuge Pico Heraeus) for 2 minutes and the supernatant was removed and discarded from each tube. The pellet was resuspended in the remaining supernatant and 0.1mL and 1mL of the cell suspension pipette onto a LB agar plate with the correct antibiotics. LB agar plates with ampicillin were used for BL21 and B834 competent cells and LB agar plates with ampicillin (Amp) and chloramphenicol (Chl) was used for CODON+ and Rosetta. The plates were inverted and incubated at 37oC for overnight. If the transformation was successful, the plates will have many thousands of colonies.

Mini-inductions

After successful transformation, a mini-induction to check protein expression is carried out. A single colony is picked from the plates and transferred into 2.5mL LB media with the appropriate antibiotics. The culture is incubated at 37oC with vigorous shaking for 2 hours. 1mL of the culture is places in two separate eppendorf tubes and to on the 1mL, 25µL 20mM IPTG (0.5mM final concentration) is added. And 25µL of water is added to the other 1mL of culture, as a negative control. The balance 0.5mL of the culture is stored in the 40C fridge. This culture was grown at 37OC with vigorous shaking for another 2 hours. SDS-PAGE analysis (see _______) was done to identify a good inducer. 10µL aliquot from the culture was then transferred aseptically into a glycerol stock. The tubes were then stored at -80oC for long-term storage.

Large scale expression (Bacterial expression of GST-fusion proteins)

Cultures of cells bearing the p300 and CBP constructs were prepared by inoculating 20mL LB+Amp+Chl in a 50mL centrifuge tube with 10µL from the frozen glycerol stock. A negative control culture was also prepared with 20mL LB+Amp+Chl with 10µL of water. The tubes were subjected to vigorous shaking (250 rpm) at 37oC overnight. Cells were recovered from the overnight culture by centrifugation at 3000rpm for 5 minutes at 4oC (Eppendorf Centrifuge 5810 R). The supernatant was carefully poured off and the pellet resuspended in the residual liquid. The constructs were aerate well with shaking at 37oC until OD at 600mm reaches 0.5 ~ 0.6. It normally takes about 4 hours. When the OD600 reached 0.6, protein expression was induced by the addition of 0.5mL and 1mL IPTG (to obtain final concentration of 0.5mM and 1mM respectively). Cells were grown at 200C for an additional 16 hours and 0.5mL samples were removed at every 1h, 2h, 4h and overnight to check for protein expression. SDS-PAGE analysis was done to analyze fractions containing GST-p300&CBP-HAT domains.

Purifying protein from inclusion bodies

Cells were recovered from the overnight large culture by centrifugation at 8000rpm in Sorvall SLL-6000 rotor for 15 minutes at 4oC (Thermo Scientific Sorvall Evolution RC). The supernatant was carefully poured off and the pellet was weighed out to determine the amount of grams of cells we have. The pellet was resuspended in 10mL of Buffer A (50mM Tris-HCl ph 8.0, 5mM EDTA and 10Mm NaCl). The suspended pellet was transferred to 50mL conical centrifuge tubes and 160µL of 50mg/ml lysozyme was added. The tubes were placed in 37oC water bath until solution becomes viscous. The solution was subjected for 3Ã-2 minutes sonication at 4oC to chop up the DNA and reduce the viscosity. The solution was centrifuged at 20,000rpm in Sorvall SS-34 rotor at 4oC for 30 minutes.

into a new clean 50mL centrifuge tube, the supernatant was removed and transfer (called Supernatant A) and stored in -80oC, which is later on purified with glutathione beads. The pellet was resuspended in 10mL of Buffer B (20mM Na2HPO4 ph 7.2, 20mM NaCl, 5mM EDTA and 25% (w/v) sucrose) and centrifuged at 20,000rpm in Sorvall SS-34 rotor at 4oC for 30 minutes. The supernatant was carefully removed and transferred into a new clean 50mL centrifuge tubes and stored in -80oC (called Supernatant B). The remaining pellet will be the inclusion bodies and it is resuspended in 6mL of 8M urea (called Urea). 10µL of samples were removed from Supernatant A, Supernatant B and Urea for SDS-PAGE analysis.

Purifying GST (glutathione-S-transferase) fusion proteins

Supernatant A is removed from the freezer and filtered using the 0.45µm syringe filter. Then it is added to 3mL of washed Glutathione beads (Glutathione Sepharose 4 Fast Flow). The beads are pre-washed in GST binding buffer [PBS pH 7.3 (140Mm NaCl, 2.7mM KCl, 10mM Na2HPO4, I.8Mm KH2PO4, pH 7.3)] and resuspended to about 50% slurry. Supernatant with beads is rocked on a roller mixer at 4oC for 1 hour. The beads are then centrifuge at 500rpm for 5 minutes at 4oC using the Sorvall SS-34 centrifuge rotor. 20µL is saved for SDS-PAGE analysis. The beads are resuspended with 14mL GST binding buffer and then centrifuge at 500rpm for 5minutes (Eppendorf Centrifuge 5810 R) and the supernatant is saved for analysis. This step is repeated twice. After transferring the bead slurry to 2mL eppendorf tube, the beads were spun in a microfuge (Biofuge Pico Heraeus) at speed 3.0 for 5 minutes and supernatant by pipetting. 1mL of elution buffer [50mM Tris pH 8.0, 10mM DTT (dithiothreitol), O.5mM NaCl, 10mM reduced glutathione and 1 tablet of protease inhibitor (Protease Inhibitor Cocktail Tablets, 1 tablet for 50mL Ref: 11873580001, Lot14975900)] is added to the bead pellet to elute the GST proteins. The beads are carefully mixed by inverting the eppendorf tubes three times and then incubate tubes on a roller mixer at room temperature for 15 minutes. The beads were centrifuged in a microfuge (Biofuge Pico Heraeus) at speed 3.0 for 5 minutes. The supernatant was carefully removed and transferred into a clean eppendorf tube (which is called eluate 1, which contains the protein) while being careful not to carry over any beads. Elution step is repeated to get eluate 2 and 3. Dispense eluate 1, 2 and 3 into microcentrifuge tubes in aliquots of 20µL in each tube. These tubes are froze in liquid nitrogen and stored in -80oC. 20µL was removed from all the samples for SDS-PAGE analysis.

Purifying GST fusion protein with Sarkosyl

Pellet containing expressed protein were resuspended in 10mL ice-cold STE (10mM Tris pH 8.0, 10mM NaCl, 1mM EDTA) and 16µL of 50mg/ml of lysozyme by repeat pipetting and then left on ice for 15 minutes. 100µL 5mM DTT, 1Ml 0.5% Sarkosyl and 1/2 tablet of protease inhibitor (Protease Inhibitor Cocktail Tablets, 1 tablet for 50mL Ref: 11873580001, Lot14975900) was added to the cells. The cells were than subjected for 3Ã-2 minutes sonication at 4oC. The solution was centrifuged at 20,000rcf in Sorvall SS-34 rotor at 4oC for 30 minutes. Triton X-100 was added to the supernatant until concentration 0.5% is achieved and filtered using the 0.45µm syringe filter. Then it is added to 3mL of washed Glutathione beads (Glutathione Sepharose 4 Fast Flow). The beads are pre-washed in cold GST binding buffer [PBS pH 7.3 (140Mm NaCl, 2.7mM KCl, 10mM Na2HPO4, I.8Mm KH2PO4, pH 7.3)] and is incubated with the supernatant on a roller mixer at 4oC for 30 minutes. The beads are spun down and supernatant is kept in a separate eppendorf tube for analysis. The beads are resuspended with 12mL cold PBS and then centrifuge at 2500rpcf for 53minutes (Eppendorf Centrifuge 5810 R) and the supernatant is saved for analysis. This step is repeated twice. After transferring the bead slurry to 2mL eppendorf tube, the beads were spun in a microfuge (Biofuge Pico Heraeus) at speed 3.0 for 5 minutes and supernatant by pipetting. 1mL of elution buffer [50mM Tris pH 8.0, 10mM DTT (dithiothreitol), O.5mM NaCl, 10mM reduced glutathione and 1 tablet of protease inhibitor (Protease Inhibitor Cocktail Tablets, 1 tablet for 50mL Ref: 11873580001, Lot14975900)] is added to the bead pellet to elute the GST proteins. The beads are carefully mixed by inverting the eppendorf tubes three times and then incubate tubes on a roller mixer at 4oC for 20 minutes. The beads were centrifuged in a microfuge (Biofuge Pico Heraeus) at speed 13.0 for 1 minute. The supernatant was carefully removed to a clean eppendorf tube (which is called eluate 1, which contains the protein) while being careful not to carry over any beads. Elution step is repeated to get eluate 2 and 3. Dispense eluate 1, 2 and 3 into microcentrifuge tubes in aliquots of 20µL in each tube. These tubes are froze in liquid nitrogen and stored in -80oC. 50µL was removed from all the samples for SDS-PAGE analysis.

Bradford protein assay

Bradford protein assay is used to estimate the protein concentration and BSA was used as a standard. BioRad protein assay dye reagent was diluted 20% in water. 5mL Bio-Rad dye reagent was dissolved in 20mL double distilled water. Test sample for blank, BSA (Bovine serum albumin) standards and protein sample to be tested were prepeared in disposable cuvettes according to Table 1. All cuvettes were incubated with 1mL Bio-Rad protein assay dye reagent for 5 minutes at room temperature. Optical density readings against a blank were taken at 595nm using Eppendorf Bio Photometer. A standard curve was generated by plotting the optical density readings against concentrations. The concentration of protein present was obtained by using the slope of the standard curve.

Test sample

Sample Volume (µL)

Water volume (µL)

Bradford reagent volume (mL)

Blank

0

10

1

BSA Standard 2µg/mL

1

-

1

BSA Standard 4µg/mL

2

-

1

BSA Standard 6µg/mL

3

-

1

Before Flow-through

2

-

1

After Flow-through

2

-

1

Wash 1

20

-

1

Wash 2

20

-

1

Wash 3

20

-

1

Eluate 1

10

-

1

Eluate 2

10

-

1

Eluate 3

10

-

1

Beads

10

-

1

Table 1: Preparation of test samples for the Bradford protein assay.

SDS-PAGE

Gel preparation

Plates were assembled in the apparatus and separating gel is made up, adding TEMED last. Three 12% polyacrylamide separating gels which are made up using the following components; 14ml separating gel buffer (1.5M Tris pH 8.8, 10% SDS and 30% acrylamide), 130µL 10% APS (10% w/v ammonium persulfate) and 6.5µL of TEMED (N, N, N´, N´-Tetramethylethylenediamine). The gel mixture was poured between two glass plates leaving about 1 centimeter below the bottom of the comb for the stacking gel. Bubbles are removed and 0.2% SDS gel (2mL of 10% SDS is diluted in 100mL double distilled water) is slowly poured to seal the plate. The gel is left to polymerise at room temperature for at least 30 minutes. The stacking gel, again adding TEMED last, was poured on top of the separating gel and the comb was inserted, leaving the gel to polymerise at room temperature for 1 hour. Three 5% polyacrylamide stacking which are made up using the following components: 6ml stacking gel buffer (30% acrylamide, 1.0M Tris pH 6.8), 6.0µL 10% APS and 5µL of TEMED.

Sample preparation

Aliquots of samples are centrifuged for 5 minutes at 13.0 speed using the microfuge (Biofuge Pico Heraeus). Supernatant is removed and the pellet is resuspended with either 4Ã-SDS Gel Loading Buffer (200mM Tris pH 6.8, 400mM DTT, 8% SDS, 0.2% Bromophenol Blue and 40% Glycerol) or 2Ã-SDS Loading Gel (100mM Tric-Cl pH 6.8, 200mM DTTD, 4% SDS, 0.2% Bromophenol Blue and 20% Glycerol). The tubes are then put in 90oC water bath for 5 minutes and are centrifuge for 30 seconds at 13.0 speed to remove condensation that is formed on the tube cover.

Gel electrophoresis

The apparatus was set-up. The plate was clamped in and 1 Ã- SDS running buffer was poured till full between the plates and half filled in the surrounding reservoir. 5Ã-SDS running buffer was prepared by mixing 94g glycine, 15.1g Tris base, 5g SDS and distilled water up to 1 litre. Thus, 200mL of 5Ã-SDS running buffer was added to 800mL of distilled water to make 1 Ã- SDS running buffer. The comb was removed and the marker (BioRad Percision Plus Protein, Catalog no: 161-0373) and samples were loaded into the gel. The gels were run at 170V for 1 hour.

Stain and De-stain

The gels were stained in a staining tray (___________________________________) and kept on the rocker for 30 minutes at room temperature. The gels were than rinsed with distilled water. Sufficient amount of de-stain (_______________) was added, enough to cover the gels and kept on the rocker for 30 minutes at room temperature. After 30 minutes, the de-stain was discard and the gels were rinsed with distilled water. De-stain was added again and the gels were kept on the rocker for another 30 minutes at room temperature. After 30 minutes, de-stain was discarded and the gels were placed in distilled water for another hour or overnight before drying them out.

Drying

The gels were analyzed using the Fujifilm LAS-4000 Imager and dried on BioRad Model 583 Gel Dryer using a chromatographic paper (3MM Whatman Paper).

2.0 HAT Assay

In this experiment, fluorescence-based assay is rapid and non-radioactive assay that measures the production of CoASH was used. The first step that was carried out is to plan the assay and the reaction is carried out in 96-well plate. Each well had different amounts of reagents (by varying different amount of substrates) added to it and 3 replicates were done for each reaction. And then the reagents were prepared:

HAT assay buffer (100mM HEPES pH 7.5, containing 0.8% Triton X-100)

Histone- it was bought commercially from Roche (Catalogue No: 223565)

HAT Acetyl CoA- The tube contains 0.5mM Acetyl CoA and kept in -80oC, which is prepared by Dr. Biagong Yue

Histone acteyltransferase (p300/CBP)- The tube contains 20µL recombinant p300/CBP-HAT (Eluate 1)

HAT stopper- Isopropanol

HAT developer- CPM (7-diethylamino-3-(4´-maleimidylphenyl)-4-methylcoumarin), which is made of 88µL DMSO and 10.5mL HAT assay buffer.

HAT inhibitors- Curcumin and Garcinol is purchased commercially. Curcumin is purchased from

Different concentrations of HAT inhibitors were added to the 96-well plate and placed on a shaker for 1 minute and incubated for 10 minutes at room temperature. HAT assay buffer, histones and Ac-CoA was added to each of the well. The plate was placed on a shaker for a minute and then incubated for 10 minutes at room temperature. The first reading (background reading) was taken using the fluorescence microplate reader (Biotek Synergy HT Reader). 35µL of isopropanol was added into each well plate and the plate was mixed on the shaker for 1 minute. 70µL of CPM was added to each well and the plate was incubated for 20 minutes at room temperature. The plate was read using the fluorescence microplate reader and two readings were taken by placing the plate forward and backward (forward and backward reading). Absorbance for individual reactions were determined by getting the average reading for the forward and backward reading and then subtracting that with the background reading.

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