- The document summarizes Daniel McKay's project to purify the Green Fluorescent Protein (GFP) gene from jellyfish. It involved growing E. coli bacteria containing the GFP gene, then extracting and purifying the GFP protein through techniques like centrifugation and chromatography. - Experiments tested the growth rates of E. coli at different temperatures, finding 37°C yielded the fastest growth. This optimized temperature was used for subsequent culturing. - The final purification step involved running protein samples on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) which confirmed a pure layer of GFP had been successfully isolated, proving the goals of the project were achieved.

1. Daniel McKay
PHYSICAL SCIENECES PATHWAY
GFP PROJECT

2. 1
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. Thepurification of proteins isvery importantin themedical industry,
with such uses of producingpure insulin for diabetes suffers.
GFP traditionally refers to the protein first isolated from the jellyfish ‘Aequorea Victoria’, otherwise known as
the ‘Crystal Jelly’,nativeto the West Coastof North America. The GFP gene is madeup 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 Prizefor Chemistry.
The GFP gene to us however was not pure, as it had been inserted into the E.Coli bacterium cell. Inside the
simplebacteriumthere is the genome and plasmid.This plasmid contains theDNA baseof the cell.This is where,
usingan enzyme, the plasmid was ‘sliced’open and the GFP gene inserted. This is nowknown as a recombinant
DNA. Hence, when mitosis takes place, the GFP gene will be contained inside all the E.Coli bacteria. Once
multiplied,itis now known as a recombinantprotein.
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 possibleto avoid error.
Each method had its own specific hypothesis. The overall objective and hypothesis of this experiment was
however, that in the final stageof a sodiumdodecylsulphate polyacrylamidegel 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.

3. 2
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 itwas not lethal, could still causehealth problems. We
used 2 different techniques for dealing with the microbiology techniques, both with their own separate
hypothesis.
Petri Dishes&SolidMedium
The Solid Medium experiment was the first experiment we did. This was to grow a batch of E.Coli strain,
containingGFP, insidea petri dish.The hypothesis in this situation was to produce a high concentration of agar
‘streaks’alongthe dish,provingthat inoculation had taken place.
Equipment
Large Petri Dish (Sterile)
Permanent Marker
Inoculation Loops (Sterile)
Liquid Agar (Stored at 55°C)
Bacteria Culture(E.Coli)
Incubator
Method
1. Liquid Agar will bestored in a water bath at 55°C. This is to ensure that the agar will stay in liquid state
and not solidify.
2. Collecta sterilelarge 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 looseningthe 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 leavingto 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.
Results
After 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 yellowappearance,lighter than thatof the agar.Colonies cover around 40-60%in onecontinuous
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 isalso airborne.No other bacterial colonies arepresent.

4. 3
Luria Broth& LiquidMedium
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 Marker
Inoculation Loop (Sterile)
1000µl [1ml] Gilson Pipette
1000µl [1ml] Gilson Pipette Tips (Sterile)
Luria Broth [LB] (Sterile)
Bacterial Culture(E.Coli)
Incubator
Method
1. Collecta sterilefalcon tube, a 1000µl Gilson Pipettewith 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 correctvolume [1ml in our case] and put a tip on the end. Once collectingthe tip,
closethe 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 1st pressurepoint to bring
up the liquid.Press down on the Gilson till the2nd stop to excrete the liquid.This isdoneby unscrewing
the lid.Leave the lid closed until havingtheinsertthe LB then immediately replacethe 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. Collecta sterileinoculation loop and inoculateby usingprepared E.Coli. Usingthis, inoculate the LB by
placingtheloop insideand swillingin theliquid.Oncedone, 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.
Fig 1. – Streaking plate pattern. (www.homepages.wmich.edu/~rossbach/bios312/LabProcedures/Streak%20plate%20procedure.html, n.d.)

5. 4
Growth Rates of E.Coli
As a class,wewere tasked to investigatethe different growth rates of the E.Coli bacteria.We did this by usinga
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 lightlevels. The hypothesis was that as
the growth rate of the E.Coli increased,the lightlevels and hence spectroscopy levels would decrease.
Equipment
Falcon Tube (Sterile)
Permanent Marker
10µl Gilson Pipette
1000µl Gilson Pipette
10µl Gilson Pipette Tips (Sterile)
1000µl Gilson PipetteTips (Sterile)
Luria Broth [LB] (Sterile)
Bacterial Culture(E.Coli)
Timer (If alarmis present,set at 20 minute intervals)
Cuvettes
Eppendorfs
Lab spectrometer
Distilled Water
Bench 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 ata set temperature [30°C or 37°C]
until needed again. This isto ensure that the mitosis process continues ata set pace. Remember
to label tubes before incubation with name, temperature, and what is inside.
6. After an incubation period of 20 minutes,repeatfromStep 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 increasingvalues fromstart
to finish.This however ‘dropped off’ at places duringthe experiment and towards the end of the
higher temperature.

6. 5
Absorbance Values (Mean)
Time (min) 30°C 37°C
0 0.09453 0.02528
20 0.08947 -0.05335
100 0.13629 0.10173
120 0.12837 0.13383
225 0.07647 0.12850
245 0.13263 0.15561
335 0.17341 0.07739
355 0.13049 0.14780
375 0.16431 0.17183
1665 0.35924 0.36694
1685 0.35740 0.27154
1805 0.41050 0.28346
1825 0.49413 0.39500
1950 0.52757 0.46567
1970 0.56323 0.50388
2100 0.50022 0.54563
2120 0.50211 0.55100
3410 0.63000 0.50022
3420 0.63860 0.48580
Fig 2. & 3. – Table and graph of mean values for class
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 100 120 225 245 335 355 375 1665 1685 1805 1825 1950 1970 2100 2120 3410 3420
SpectroscopyValues(Mean)
Time (Min)
Class Mean for Growth Rates of GFP at 30°C & 37°C
30°C 37°C

7. 6
Extracting, purifying and activating the GFP gene
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 activatethe GFP gene for the final stage.
Extraction& Purificationof 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 Broth
Inoculation Culture(E.Coli)
Permanent Marker
Incubator
PBS Buffer
NH4SO4 Solution
Distilled Water
Centrifuge
Lyzbasome
Ice Bucket (With Ice)
Chromatography Column
Resin
Eppendorfs
Bench Vortex
Inodazole
100µl Gilson Pipette
100µl Gilson Pipette Tips
LoadingBuffer
Hot Block
SDS 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 opposingtubes. 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 theE.Coli Culture in the separateuniversal tubes areused and a solid pelletis created.

8. 7
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 overnightat 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 supernatantinto a clean Eppendorf. Placeall 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.Collect3 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 usingan 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, insertthe samples into separatelanes 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 activatethe GFP gene, we needed an inducer.The inducer in this casetook form in ITPG. The E.Coli bacterium
naturally metabolises thesugars of glucoseand lactose.However, E.Coli only metabolises lactoseas 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 lactoseis
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!

9. 8
Results
Overall, the results were very successful. With the microbiologytechniques, we successfullymade
agar platesas well asgrowinga bacterial culture.
On top of this, the growth rates of E.Coli also provided very good results. The results however were
slightlyunexpected.The final dropoff atforthe 37°C temperature was unusual.However,thiscanbe
explained through the 4 Bacterial Growth Stages: Lag Phase, Log Phase, StationaryPhase and Death
Phase.We wouldhave observedthe startof the laststage,whichissurprisingatthis lengthof 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.
Thismeansthatthere werenoimpuritiespresentandthatthe chromatographyworked.Furthermore,
the thickerthe line,the more GFPwaspresent!Thiswasalmostaperfectexperiment,withnoknown
errors.
Fig 4. – GFP results showing a single, thick layer.

10. 9
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
particularE.Colistrainhas a resistance toampicillin,hencecangrow freely. Thiswasthe best
decisiontomake.
 Within the second experiment with liquid broth, condensation was observed at the top of
the falcontube.Thiscanbe assumedasa signof aerobicrespirationfromthe E.Colibacteria.
 Whenstudyingthe growthrate of E.Coli,anunexpecteddipwasseenatthe final stage of the
37°C growth. Many reasons could have caused this. Once could be the amount of time
samplesspentoutsidethe incubatorcouldhave causedthis.However,the theoryIbelieveto
be true isthat the start of the 4th
stage in bacterial growth(DeathPhase) couldhave started
hence the decrease.Thisshowsthat 30°C isan optimumtemperature overthistime period
and hence shouldbe usedforfuture reference.
 The final result was excellent. A large, pure layer was seen proving the experiment had
workedfully.Nootherrecommendationscanbe made.
Conclusion
In conclusion,the experimentwasatotal success!Notonlydidwe findalongthe waythe most
optimumtemperature atwhichtogrowthe E.Coli bacteria,butalsohow to get the bestyieldfroma
time/temperaturerelation.
Thisexperimentwasasmall scale versionof whathappensinmedical industry.

11. 10
Bibliography
Online
Green Fluorescent Protein, n.d. Wikipedia [online].Availableat:
<http://en.wikipedia.org/wiki/Green_fluorescent_protein> [Accessed 06/01/15]
Aequorea Victoria Jelly Fish,n.d. Wikipedia [online].Availableat:
<http://en.wikipedia.org/wiki/Aequorea_victoria> [Accessed 08/01/15]
Streak Plate Techniques, n.d. West Michigan University [online].Availableat:
<https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Streak%20plate%20procedure.html >
[Accessed 11/01/15]