Title: Understanding How roGFP Works in Biosynthesis
Background:
In this research, the protein GFP (Green Fluorescent Protein) is specifically being looked
at. Roger Y. Tsien talks about how there is a chromophore that is inside the barrel like structure of
GFP, which is responsible for the fluorescent property of the protein in his paper “The Green
Fluorescent.” Tsien in “The Green Fluorescent Protein” continues by saying that there are two
forms to the chromophore, a neutral form with a HO part and an anionic form with an O- part
(Tsien 2004). The pKa intrinsic property of chromophore tells the proportion of the abundance of
each forms of chromophore. Gero Miesenbock further relates pH to the pKa property of
chromophore in his article “Visualizing Secretion and Synaptic Transmission with pH-sensitive
Green Fluorescent Proteins.” If the pH is higher than the pKa of the chromophore, more of the
anionic form is present (Miesenbock 1998). If the pH is lower than the pKa, more of the neutral
form of the chromophore is present (Miesenbock 1998). Tsien explains in “The Green
Fluorescent” that the different forms of chromophore have different fluorescent emission, which is
related to their unique excitation peak. The neutral chromophore has an excitation peak at 395 nm
while the anionic chromophore has an excitation peak of 490 nm wavelengths (Tsien 2004). Tsien
also adds in his paper that the chromophore has an emission peak of around 515 nm.
George Hanson writes about how scientists are able to engineer the GFP so that it reacts to
oxidation stress in an environment in his paper “Investigating Mitochondrial Redox Potential with
Redox-Sensitive Green Fluorescent Protein Indicators.” First attach two cysteine amino acids in
respective area that are close to the chromophore (Hanson 2008). The structure of the engineered
chromophore responds to the oxidative stress introduced. There are two structures of the
engineered chromophore that have two different pKa intrinsic properties (Hanson 2008). Hanson
differentiates between the two structures by saying that in the oxidative form, the cysteine amino
acids have separate SH molecules while in the reduced form the cysteine amino acids form
disulfide bridges between the two. When the cysteine amino acids go from their oxidative
structure to their reduced form, the pKa of the chromophore is shifted (Hanson 2008). Now the
comparison of the values of the pH and the chromophore’s pKa is different, thus there is a change
in the fluorescence property (Hanson 2008). There are many suggested causes of this process.
Hanson goes into further detail about a specific suggested cause in “Investigating Mitochondrial
Redox Potential with Redox-Sensitive Green Fluorescent Protein Indicators” dealing with the
change in charges in the chromophore environment. There is electrostatic repulse. When oxidative
stress is introduced, oxidation occurs on the cysteine amino acids and they form disulfide bridges
(Hanson 2008). When the bridges are created, the cysteine amino acids shift a molecule with a
negative charge close to the O- atom of the anionic chromophore (Hanson 2008). When negative
charges become close, they tend to repel from one another. However, the O- atom in chromophore
cannot move in this situation. So the way it deals with this negative molecule’s presence is that the
O- atom grabs an H to attach (Hanson 2008). Therefore, the O- turns into a HO. Thus the
chromophore changes forms from the anionic form to the neutral form, which means that more
chromophores are in the neutral form (Hanson 2008). The chemical structure of a molecule
determines their pKa property, and mutagenesis can change the chemical structure. This alters the
pKa. The shifted pKa property changes the fluorescent property to have an excitation peak at 395
nm instead of at 490 nm (Hanson 2008). pKa is chromophore’s intrinsic property. The pH is the
independent (experimental) variable, and the fluorescence property is the dependent (measured)
variable. This is a tool that can let researchers observe oxidative stress in live cells by the
fluorescence property of the cells.
The goals of this research was to have this more of a library project in which the concepts
were more looked at, the results were more theorized, and to learn and use molecular biology to
work with this protein and bacteria with excitation and emission spectra.
Experimental Set Up – Method:
First the sample of plasmid DNA would be prepared. The plasmid DNA had the specific protein
that was labeled as pQE60 – Grx1 – roGFP2. The plasmid DNA was prepped by using a method
known as Gene JET Plasmid Mini-Prep. First the bacteria would be resuspended by adding 250
microliters of resuspension solution (RNAse) to the bacteria. The content would be spun for one
minute. Next 250 microliter would be added along with 350 microliters of alkaline. The content
would then be spun for five minutes. After spinning, transport 800 microliters of the solution to a
column that is inside of a tube. Spin the 800-microliter solution inside the column, which is inside
a tube for one minute. After spinning, there should be waste that left through the column at the
bottom of the tube. Dispose of this waste through the sink. Wash the column with 500 microliters
of ethanol buffer and spin for one minute. After spinning there should be more waste at the bottom
of the tube. Dispose of the waste. Do this step with ethanol again. Add 50 microliters of elution
buffer. Now place the column in a new tube. Incubate for two minutes and then spin for two
minutes. There will be liquid at the bottom of the new tube from the column. Dispose of the
column. Incubate the new liquid in the new tube in -20 C. The sample of E.Coli bacteria is then
prepared. Transform bacteria with plasmid DNA (that encodes for engineered GFP). Mix plasmid
DNA that codes for roGFP and ampicillin resistant genes with E.Coli. Heat shock the E.Coli.
Place E.Coli into media to let it sit for around 60 minutes. Plate transformed bacteria on agar
containing ampicillin. Then prepare liquid colonies of the transformed bacteria. These liquid
colonies were prepared by adding four microliters of carboncillin and four milliliters of standard
LB broth into six different tubes. Add bacteria from the plate colony to the tubes. For getting
bacteria from a plate, first stroke a clean area on the agar with the ring tool to make sure the agar
doesn’t sizzle because the ring was too hot (the ring was flamed to be more sterile). After, touch a
visible colony on the plate with the ring. Put the ring with bacteria on it in the solutions of
carboncillin and standard broth. Incubate these test tubes. Observe for fluorescence from the
bacteria by using a Synergy Micro plate reader.
Results:
The research succeeded in filling the goals for the project. How the roGFP reacts to oxidative
stress and biosynthesis was understood through reading and researching different articles and
journals that discussed this specific enzyme. Also excitation and emission graphs of the bacteria
were collected at the end and analyzed for peaks.
Fluorescence Intensity
Excitation Spectrum
40000
30000
20000
10000
0
350
450
550
Wavelength (nm)
Relative Flourescence
Intensity
Emission Spectrum
4000
3000
2000
1000
0
500
600
700
Wavelength (nm)
The excitation and emission spectra from the experimental research bacteria are shown on the left
while the expected excitation and emission spectra from the pure protein (roGFP) from Hanson’s
paper “Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent
Protein Indicators” and Tsien’s paper “The Green Fluorescent Protein” are shown on the right.
The excitation and emission spectra from the bacteria from the experiment is pretty similar to their
respective expected spectra from the pure protein. For the excitation spectrum, both the
experimental bacteria and the pure protein have a peak at around 390 nm. However, the peak on
the experimental bacteria is broader than the sharp peak on the pure protein’s expected excitation
spectrum. There is also another smaller peak at around 480 nm on the expected excitation
spectrum belonging to the pure protein that is not found in the excitation spectrum of the
experimental bacteria. In the expected emission spectrum of the pure protein, the dotted graph is
the one being specifically looked at. For the emission spectrum of the experimental bacteria, there
is a peak around 530 nm, which is also found in the expected emission spectrum of the pure
protein. However, this emission peak is much broader than the peak from the pure protein.
Discussion/Conclusion:
These broad peak and the missing peak in the excitation peak are due to the many other
factors in the bacteria influencing the fluorescence. The experimental bacteria’s spectra would be
somewhat different from the pure protein’s since there are many other parts of the bacteria that
would react differently with the wavelengths and with fluorescence such as different organelles.
There was not a peak at 480 nm on the experimental bacteria’s excitation peak because there
might have been other factors in the whole bacteria that overshadowed the fluorescence of the
protein. The excitation and emission spectra are different between the two because the right graphs
show the expected excitation and emission graphs from the pure protein while the graphs on the
left show excitation and emission from not only the protein but also the whole bacteria.
The reason why the experimental bacteria’s excitation and emission spectra were used to
compare with other sources’ pure protein spectra is because the protein was not separated from the
bacteria. The reason why this step was not done was because the wrong enzyme was used to
prepare the plasmid DNA. The enzyme that was used was a DNase that cleaved DNA; however,
the enzyme that was needed was an enzyme that cleaved the cell wall so that the roGFP would be
expressed more easily. Since the wrong enzyme was used, the step of separating the specific
protein was not done. If the correct enzyme were used, then the roGFP would be extracted from
the experimental bacteria. Thus the experimental roGFP will be analyzed for fluorescence (the
excitation and emission spectra from these samples would be more similar to the expected graphs
of the pure protein). Once the excitation and emission spectra was collected for the experimental
roGFP, oxidative stress would be introduced to both the roGFP samples and the experimental
bacteria sample. The excitation and emission graphs would then be analyzed and compared
between the samples of roGFP and bacteria.
References:
Hanson, G. (2004). Investigating Mitochondrial Redox Potential with Redox-Sensitive Green
Fluorescent Protein Indicators. The Journal of Biological Chemistry, 279(13), 13044-13053.
Miesenbock, G., De Angelis, D., & Rothman, J. (1998). Visualizing Secretion and Synaptic
Transmission with pH-sensitive Green Fluorescent Proteins. Letters to Nature, 394, 192-195.
Tsien, R. (1998). The Green Fluorescent Protein. Annual Review of Biochemistry, 67, 509-544.
Summary of Experience:
I personally enjoyed the entire experience! I was able to be part of a real research group filled with
undergraduate upperclassmen, graduate students, and a post-doctorate student! At first, it was
pretty hard getting started because a lot of the topics dealing with this research were mostly
complex organic chemistry concepts. It was hard understanding some of these concepts because I
barely had any prior organic chemistry experience. But Dr. Tantama really helped me go through
the concepts and understand them. Another intimidating part was walking into the research group
because all of them were either upperclassmen and graduate students. There was even a postdoctorate student! However, all of them were really nice and welcoming to the group! They taught
me many things and answered my questions thoroughly in lab. Working with these students made
my first year experience so much better because here I was just a undergrad freshman working
alongside upperclassmen and graduate students! Overall, the experience was amazing! I was able
to apply chemistry to an actual research. Before this my only experience with biochemistry was in
the classroom (and the few science fair projects I did in high school). However, this experience
allowed me to use biochemistry outside of the classroom and in a real lab. This was my first time
working in lab not for a class or for a grade, and it was a neat experience! I felt that I learned a lot
more through this real life application of biochemistry than learning about it in class. I was able to
use what I learned, and it made me feel intellectual! Dr. Tantama really pushed me and tested me
throughout this research experience, and I hope I can return and be part of the group again! I do
not think I am able to add recommendations to make this program better; it already is amazing!