Team:Victoria Australia/Project/Materials and Methods

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Materials & Methods

1 Materials & Methods
2 Transformation of fluorescent cells
3 Protein expression
4 Preparation of reagents
5 Inoculation and purification of GFP and BFP
6 Preparation of yellow plasmid with E coli bacteria
7 Purification of yellow fluorescent plasmid
8 Preparation of the E.coli cell free system
9 Expression and purification of yellow and green fluorescent protein
10 Electrophoresis of YFP
11 Mutagenesis of YFP

Transformation of fluorescent cells

To begin with, reagent solutions were made up. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L.

The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawedon ice (4ºC).

Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h.

Protein expression

Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.

Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made

up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).

Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above.

Preparation of reagents

Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer.

100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water.

Inoculation and purification of GFP and BFP

1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin.

This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The supernatant formed was the GFP and BFP and this was removed.

Preparation of yellow plasmid with E coli bacteria

A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml.

4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC.

After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.

Purification of yellow fluorescent plasmid

The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.

While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.

Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and

50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).

Preparation of the E.coli cell free system

The E.Coli cell free system was constructed via the following method:

Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol). The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes.

These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes.

1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed = our E.coli cell free system.

Expression and purification of yellow and green fluorescent protein

An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.

An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000.

Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.

The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C.

Electrophoresis of YFP

In preparation for the electrophoresis run of the YFP protein to measure its purity, reagents were made being: 6X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue. This wade up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel).

The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel. The plate was put into the tank that was filled with TAE buffer. Electrophoresis was run at 120V for 30 min.

Mutagenesis of YFP

Mutant Strand Synthesis Reaction

The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:

The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.

Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters:

Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng.

Following the dilutions the following was carried out:

5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL.

Segments	Cycles	Temperature	Time
1	1	95°C	1 minute
2	18	95°C	50 seconds
		60°C	50 seconds
		68°C	1 minute
3	1	68°C	7 minutes

Dpn 1 Digestion of the Amplification Product

1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged

for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.

@@ Line 86: / Line 86: @@
 Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000.
 |width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.
-The proteins were then induced by adding 147µL of 1 PTG into each conical flask containing the YFP and GFP. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C.
+The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C.
 |}