GFP Project

Daniel McKay

Introduction

We were tasked by Actavis, a local company to carry out a method used in every day industry, the production and purification of a protein. In this case, we attempted to purify the Green Florescent Protein (GFP) gene from jelly fish, given to us by Sheffield University. The purification of proteins is very important in the medical industry, with such uses of producing pure insulin for diabetes suffers.

GFP traditionally refers to the protein first isolated from the jellyfish ‘Aequorea Victoria’, otherwise known as the ‘Crystal Jelly’, native to the West Coast of North America. The GFP gene is made up of 238 amino acids which emit a bright green light when shone under ultra violet light. However, Aequorea Victoria actually releases a blue light (caused by Ca2+ ions) which in turn reacts with aequorin (a photoprotein) and then GFP. For this extraordinary work into the field of GFP, Osamu Shimomura was awarded the 2008 Nobel Prize for Chemistry.

The GFP gene to us however was not pure, as it had been inserted into the E.Coli bacterium cell. Inside the simple bacterium there is the genome and plasmid. This plasmid contains the DNA base of the cell. This is where, using an enzyme, the plasmid was ‘sliced’ open and the GFP gene inserted. This is now known as a recombinant DNA. Hence, when mitosis takes place, the GFP gene will be contained inside all the E.Coli bacteria. Once multiplied, it is now known as a recombinant protein.

This experiment involved a number of different processes, from microbiology techniques to the extraction and purification. There were a number of ways for many of the methods, however we kept the methods as simple as possible to avoid error.

Each method had its own specific hypothesis. The overall objective and hypothesis of this experiment was however, that in the final stage of a sodiumdodecylsulphate polyacrylamide gel electrophoresis (SDS Page), we would see a pure layer of GFP. This method was chosen as it 100% proved if we had made pure GFP, or if impurities were still present due to the number of layers.

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Methods

Microbiology Techniques

When dealing with microbiological cultures, a practical process is needed when using them. A degree of care had to be taken with the E.Coli bacteria as even though it was not lethal, could still cause health problems. We used 2 different techniques for dealing with the microbiology techniques, both with their own separate hypothesis.

Petri Dishes & Solid Medium

The Solid Medium experiment was the first experiment we did. This was to grow a batch of E.Coli strain, containing GFP, inside a petri dish. The hypothesis in this situation was to produce a high concentration of agar ‘streaks’ along the dish, proving that inoculation had taken place.

EquipmentLarge Petri Dish (Sterile)Permanent MarkerInoculation Loops (Sterile) Liquid Agar (Stored at 55°C)Bacteria Culture (E.Coli)Incubator

Method1. Liquid Agar will be stored in a water bath at 55°C. This is to ensure that the agar will stay in liquid

state and not solidify. 2. Collect a sterile large petri dish, which will be empty, and ensure it is kept closed until needed. This is

to ensure a bacterial or viral contamination of the agar is kept to a minimum. To reduce this risk even further, the experiment could be taken place inside of a fume cupboard.

3. Take the liquid agar and pour a 1-2cm layer into the dish. Do this by loosening the liquid agar lid, and removing only when needed. Only open the petri dish slightly. Both of these are to reduce the risk of contamination to the cultures and ourselves. This is to ensure the proper growth of the bacterial culture, a layer too thin or too think may cause the cultures not to grow normally.

4. Let the agar set by leaving to stand for around 10-15 minutes. This is to ensure that the agar sets as an even layer. If the agar is not a smooth even layer, it may cause problems during growth.

5. Once set, using an inoculation loop, inoculate the petri dish with the E.Coli culture in a basic 4 streak pattern [FIG 1.]. This technique will show clearly where bacterial cultures have grown.

6. Place in an incubator overnight at a 37°C temperature. This is an optimum temperature for the E.Coli Bacteria to grow at.

ResultsAfter 1 night in the incubator at 37°C, bacterial cultures have developed as expected. These cultures have developed in the areas in which I spread them on the 25th November. The E.Coli bacteria has a slimy yellow appearance, lighter than that of the agar. Colonies cover around 40-60% in one continuous colony where the cultures were spread. A strong odour can be smelt. A volatile E.Coli strain which gives a musky aromatic smell, suggests this bacteria is also airborne. No other bacterial colonies are present.

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Luria Broth & Liquid Medium

We did this experiment in conjunction with the solid medium. Both the solid medium experiment and liquid medium experiment was to show grow rates in the E.Coli bacteria. The hypothesis is that we would create E.Coli colonies if we inoculated correctly.

Equipment

Falcon Tube (Sterile)Permanent MarkerInoculation Loop (Sterile)1000µl [1ml] Gilson Pipette1000µl [1ml] Gilson Pipette Tips (Sterile)Luria Broth [LB] (Sterile)Bacterial Culture (E.Coli)Incubator

Method

1. Collect a sterile falcon tube, a 1000µl Gilson Pipette with tips and Luria Broth. Leave the containers and tubes closed till needed. This is to ensure contamination is kept to a minimum.

2. Set the Gilson to the correct volume [1ml in our case] and put a tip on the end. Once collecting the tip, close the container. This is to prevent any contamination and to keep the tips as sterile as possible.

3. Add 1ml of Luria Broth to the Falcon Tube. Press down on the Gilson to the 1 st pressure point to bring up the liquid. Press down on the Gilson till the 2nd stop to excrete the liquid. This is done by unscrewing the lid. Leave the lid closed until having the insert the LB then immediately replace the lid. This is again to ensure minimum contamination.

4. Using the waste bins provided, waste the tip. This is to ensure contamination of the lab is kept to a minimum.

5. Collect a sterile inoculation loop and inoculate by using prepared E.Coli. Using this, inoculate the LB by placing the loop inside and swilling in the liquid. Once done, seal the falcon tube by re-screwing the lid. This is to prevent any spillage or contamination.

6. Place the Falcon Tube in a moving incubator at 37°C. This is an optimum temperature for the E.Coli Bacteria to grow at.

Results

Once incubated overnight at the same temperature as the petri dish, it has a similar smell to the solid medium. A white solid culture has formed at the bottom of the falcon tube and luria broth. Towards the top of the tube, condensation has started to collect.

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Fig 1. – Streaking plate pattern. (www.homepages.wmich.edu/~rossbach/bios312/LabProcedures/Streak%20plate%20procedure.html, n.d.)

Growth Rates of E.Coli

As a class, we were tasked to investigate the different growth rates of the E.Coli bacteria. We did this by using a temperature variable. To minimise error, a number of different samples were taken at the 2 separate temperatures [30°C and 37°C]. We then used spectroscopy to measure light levels. The hypothesis was that as the growth rate of the E.Coli increased, the light levels and hence spectroscopy levels would decrease.

Equipment

Falcon Tube (Sterile)Permanent Marker10µl Gilson Pipette1000µl Gilson Pipette10µl Gilson Pipette Tips (Sterile)1000µl Gilson Pipette Tips (Sterile)Luria Broth [LB] (Sterile)Bacterial Culture (E.Coli)Timer (If alarm is present, set at 20 minute intervals)CuvettesEppendorfsLab spectrometerDistilled WaterBench Top Vortex (optional)Incubator

Method

1. Collect a clean falcon tube and add 5ml of luria broth. Add to this 10µl of the E.Coli bacteria solution to the tube. Use practical techniques from the previous part of “Microbiology Techniques”.

2. Mix this solution well by either shaking or using a bench top vortex. [This step should be done as quickly as possible as the bacteria will have already started mitosis]. This is to ensure the LB and bacteria is well mixed to reduce error in the experiment.

3. The new LB/Bacteria Solution will now need to be diluted at a ratio of 1:10 [100µl E.Coli solution:900µl LB]. Dilute this in an eppendorf then vortex. This is to ensure the bacteria is as well evenly spread as possible to give fair results.

4. Extract the new 1000µl solution from the eppendorf and place in a cuvette. Zero the spectrometer using a cuvette of distilled water then collect readings from your solution 3 times. Take an average. This is to reduce the risk of an anomalous point.

5. Store the rest of the E.Coli solution in a moving incubator at a set temperature [30°C or 37°C] until needed again. This is to ensure that the mitosis process continues at a set pace. Remember to label tubes before incubation with name, temperature, and what is inside.

6. After an incubation period of 20 minutes, repeat from Step 3 and take a new average. Continue this over a set time period (usually 48-72 hours). This is to ensure a wide range of results are taken.

Results

A mean result was found for the entire class across the times (Shown on next page as [Fig 2. & 3.]). It shows that at both temperatures, there was a general trend of increasing values from start to finish. This however ‘dropped off’ at places during the experiment and towards the end of the higher temperature.

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Absorbance Values (Mean)

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Time (min) 30°C 37°C0 0.09453 0.02528

20 0.08947 -0.05335100 0.13629 0.10173120 0.12837 0.13383225 0.07647 0.12850245 0.13263 0.15561335 0.17341 0.07739355 0.13049 0.14780375 0.16431 0.17183

1665 0.35924 0.366941685 0.35740 0.271541805 0.41050 0.283461825 0.49413 0.395001950 0.52757 0.465671970 0.56323 0.503882100 0.50022 0.545632120 0.50211 0.551003410 0.63000 0.500223420 0.63860 0.48580

Fig 2. & 3. – Table and graph of mean values for class

0 20 100 120 225 245 335 355 375 1665 1685 1805 1825 1950 1970 2100 2120 3410 3420

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Class Mean for Growth Rates of GFP at 30°C & 37°C

30°C 37°C

Time (Min)

Spec

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Extracting, purifying and activating the GFP gene

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Once the growth rate had been observed, we had an optimum temperature at which to grow the E.Coli GFP at. We now wanted to try and extract, purify and then activate the GFP gene for the final stage.

Extraction & Purification of the GFP

Before being able to prove we had GFP, we had to use a number of processes to extract the GFP from the solution. However, this was then in a non-pure solution of cells, hence the purification using a distillation/fractionation column. To prove we then had pure GFP, we used an SDS page.

Equipment

Biomass Universal Tubes (25ml; Sterile)Luria BrothInoculation Culture (E.Coli)Permanent MarkerIncubatorPBS BufferNH4SO4 SolutionDistilled WaterCentrifugeLyzbasomeIce Bucket (With Ice)Chromatography Column ResinEppendorfsBench VortexInodazole100µl Gilson Pipette100µl Gilson Pipette TipsLoading BufferHot BlockSDS PAGE

Method

1. Take 2 separate, sterile, 25 ml universal tubes. Insert to these 5ml of Luria Broth. Add to this 100µl of your own inoculation cultures.

2. Label these tubes with your name, date, bacterium solution inside and finally ‘N’ and ‘D’ on opposing tubes. Store these tubes at 30°C. Inside the universal tubes is now a mixture of LB and E.Coli. This is to show other unknown people the contents of the tubes, aswell as differing the day and night tubes. This temperature is proved to be the optimum temperature for E.Coli growth.

3. Make up 2 separate solutions; one of PBS Buffer and one of NH4SO4 solution. The buffer is created was a ½ dilution of ½ a PBS Buffer tablet along with 500ml of distilled water. The NH4SO4 solution was a 4M concentration worked out by using its molar mass. Both can be kept at room temperature.

4. After being left overnight, collect 2 separate eppendorfs. Transfer 1500µl of the different incubated cultures into opposing tubes. Label respectively.

5. Centrifuge both eppendorfs at full speed for 5 minutes. This is an optimum time for efficiency and product yield.

6. Remove from centrifuge. Remove all the supernatant (liquid) from the top of the pellet and discarded. After this, add more of the E.Coli culture. Repeat from Step 5 and respin. Do this until all the E.Coli Culture in the separate universal tubes are used and a solid pellet is created.

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7. Add 400µl of PBS Buffer and remix thoroughly till nearly dissolved. This needs to then be respun in a centrifuge.

8. Remove the supernatant once again. Add 400µl of PBS Buffer once more. However do not spin this time.

9. Add to this 100 µl of lyzbasome. This enzyme will break down the GFP cells and release the GFP into the soluble solution.

10. Store samples overnight at 40°C. This will enhance the ‘break up’ process. 11. To start the purification process via chromatography, respin the Eppendorf. This is to move

any cells. As the protein is less dense than the cells, it will ‘float’ on top. 12. Once spun, remove the supernatant into a clean Eppendorf. Place all tubes on ice. 13. Add the resin into two separate, clean eppendorfs. Add the two separate supernatants with

the resin. Vortex to mix. Add this solution into two separate, clean chromatography columns.

14. Add to the column PBS Buffer and collect 3 200µl samples. This is to wash the samples any remove any unwanted cells left.

15. Once ran dry, add inodazole. Collect 3 200µl samples. This will release the GFP. These are the most important samples.

16. Finally, run through 5M NaCl. This will wash and clear the column of any waste.

This purified GFP is now ready from Step 15. However, proof is needed if it is pure. We did this by using an SDS page.

17. Add 50µl of each sample into different eppendorfs. Add to this 50 µl of loading buffer. This loading buffer contains SDS and biphenyl blue dye.

18. Heat in a hot block at the minimum of 80°C and leave for 15 minutes. The heat and SDS will denature the proteins.

19. After heating, insert the samples into separate lanes of the SDS PAGE.

Results

After the SDS PAGE was completed, our results were shown to us (see results section). It shows that the samples collected of GFP were 100% pure and we were very successful with the experiment.

Induction

To activate the GFP gene, we needed an inducer. The inducer in this case took form in ITPG. The E.Coli bacterium naturally metabolises the sugars of glucose and lactose. However, E.Coli only metabolises lactose as a secondary sugar, if no primary sugar (glucose) is available. Hence, due to this, E.Coli constantly releases an enzyme to metabolise the primary sugar glucose. However, E.Coli has an inducer to produce enzymes for when lactose is present. By inserting the GFP gene and this particular gene within the E.Coli, when lactose was added as an inducer, theoretically the inducer gene will switch on!

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Results

Overall, the results were very successful. With the microbiology techniques, we successfully made agar plates as well as growing a bacterial culture.

On top of this, the growth rates of E.Coli also provided very good results. The results however were slightly unexpected. The final drop off at for the 37°C temperature was unusual. However, this can be explained through the 4 Bacterial Growth Stages: Lag Phase, Log Phase, Stationary Phase and Death Phase. We would have observed the start of the last stage, which is surprising at this length of time.

The final extraction and purification of the GFP gene was very successful as seen in [Fig 4.]. You can see by our results (circled in red) that one, thick band was seen. One band is extremely good news. This means that there were no impurities present and that the chromatography worked. Furthermore, the thicker the line, the more GFP was present! This was almost a perfect experiment, with no known errors.

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Fig 4. – GFP results showing a single, thick layer.

Analysis

Within the first experiment using the petri dishes, no other bacterial cultures were found. This is because the agar and luria broth both contained ampicillin. This prevents other bacterial cultures from growing in the same way penicillin kills them off. However, this particular E.Coli strain has a resistance to ampicillin, hence can grow freely. This was the best decision to make.

Within the second experiment with liquid broth, condensation was observed at the top of the falcon tube. This can be assumed as a sign of aerobic respiration from the E.Coli bacteria.

When studying the growth rate of E.Coli, an unexpected dip was seen at the final stage of the 37°C growth. Many reasons could have caused this. Once could be the amount of time samples spent outside the incubator could have caused this. However, the theory I believe to be true is that the start of the 4th stage in bacterial growth (Death Phase) could have started hence the decrease. This shows that 30°C is an optimum temperature over this time period and hence should be used for future reference.

The final result was excellent. A large, pure layer was seen proving the experiment had worked fully. No other recommendations can be made.

Conclusion

In conclusion, the experiment was a total success! Not only did we find along the way the most optimum temperature at which to grow the E.Coli bacteria, but also how to get the best yield from a time/temperature relation. This experiment was a small scale version of what happens in medical industry.

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Bibliography

Online

Green Fluorescent Protein, n.d. Wikipedia [online]. Available at:<http://en.wikipedia.org/wiki/Green_fluorescent_protein> [Accessed 06/01/15]

Aequorea Victoria Jelly Fish, n.d. Wikipedia [online]. Available at:<http://en.wikipedia.org/wiki/Aequorea_victoria> [Accessed 08/01/15]

Streak Plate Techniques, n.d. West Michigan University [online]. Available at: <https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Streak%20plate%20procedure.html> [Accessed 11/01/15]

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