FRET Characterisation of a Novel Peptide Sensor
Claire Walsh, CoMPLEx
Supervisors: Dr. Guy Moss & Dr. Angus Bain
Word count: 4500
May 2, 2012
A fluorescence resonance energy transfer sensor for dipeptides, using a construct consisting of
periplasmic binding protein (Dipeptide-binding protein DppA) and two GFP (green fluorescence protein) derivatives, CFP (cyan fluorescent protein) and Citrine has been produced and characterised. Successful production of the construct via transfection and lysis of HEK (human embryonic kidney) cells
has been demonstrated. FRET characterisation of the constructs and components has revealed lifetime
measurements for the Citrine and CFP individually that are compatible with the literature, however
within the construct lifetime changes have not shown a consistent effect with the addition of di-alanine
which we propose is due to complex decay background from control cells which are untransfected.
Contents
1
Introduction
1
2
Theory
2.1 FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Peptide binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 The complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1
2
3
3
Methods
3.1 Midi Preps . . . . . . . .
3.2 Cell culture preparation
3.3 Cell Transfection . . . . .
3.4 Cell Lysing . . . . . . . .
3.5 FRET . . . . . . . . . . .
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3
3
4
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5
4
Results and Discussion
4.1 Transfection . . . . . . . . . . .
4.2 Acceptor and donor Behaviour
4.3 Detection Window Analysis . .
4.4 Di-alanine response . . . . . . .
4.5 Count Rates . . . . . . . . . . .
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5
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9
5
Conclusions and Further Work
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9
1
Introduction
The use of the green fluorescent protein derivatives CFP and YFP as sensors in biological molecules
has become a vastly expanding area of research. The ability to genetically encode these proteins into
macromolecules and other proteins of interest combined with their FRET pair compatibility, has made
them subject of many FRET based biological sensors[20, 4]. This work aims to characterise a set of
constructs designed for dipeptide sensing made by Dr De Linz[13] using a technique created by [22, 23].
The construct consists of a periplasmic binding protein (Dipeptide-binding protein DppA) with CFP
inserted at the N-terminus and Citrine inserted at a selection of randomised locations within the CFPDppA construct. This has created a library of approximately 36 constructs distinguished by the position
of Citrine. Upon binding of the dipeptide DppA undergoes a conformational change which can be
measured using via the change in FRET rate and hence lifetime changes to the CFP chromophore. Not all
positions of Citrine within the construct will be conducive to this measurement without interfering with
either DppA folding or binding of the dipeptide. By using a randomised process to create the library of
constructs it is considered that there will be a number of promising candidates that, once characterised,
will provide a robust peptide sensor which may be easily adapted to bind other biologically common
peptides. In addition the sensor has the potential for in vitro use.
The this work used the pre-existing library of constructs stored as plasmid solution to produce and
purify the CFP-DppA-Citrine constructs. This was done by transfection of HEK cells with the plasmid
followed by lysing to obtain relatively pure protein constructs. These proteins were then characterised
by time resolved fluorescence lifetime analysis.
2
2.1
Theory
FRET
Fluorescence resonance energy transfer (FRET) was first physically described by Förster [9]. It is a process whereby energy is transferred nonradiatively from an excited donor to an acceptor chromophore
via dipole-dipole interaction. This transfer can only take place if the emission spectrum of the donor
sufficiently overlaps the absorption spectrum of the acceptor, the two are within a critical distance of
(typically no more than 100 Å), the quantum yield of the donor and absorption coefficient of acceptor
are sufficiently large and the transition dipoles of the two chromophores are appropriately oriented[26].
All this being the case energy transfer from donor to acceptor takes place as described by eqn. 1.
kT =
1 R0 6
( )
tD R
(1)
where k T is the rate constant of FRET, tD is the donor lifetime, R is the distance between donor and
acceptor and R0 is the Forster distance (the distance at which FRET efficiency is 50% of maximum).
As can be seen there is a 6th power relation between donor and acceptor separation and FRET rate,
making this technique a powerful tool for distance measurements on a nanometer scale[7]. This process can be used to provide information on dynamic protein interactions within living cells and other
process via a variety of instrumental setups, and techniques; for a comprehensive review of these see
[3]. I will briefly outline those that are used in this work. Time Correlated single photon counting is a
technique first pioneered by [2] and is now widely used in taking lifetime measurements. The lifetime
of a chromophore is a measure of the radiative decay from the S1 excited state the S0 ground state. This
lifetime is determined as being inversely proportional to the sum of the radiative and non radiative rate
constants.
t=
1
k f + k nr
(2)
From eqn.?? it can be seen that when a donor chromophore is FRETing lifetime will increase owing to the
non-radiative decay term. Using a pulsed laser to measure the time-dependent distribution of photons
emitted from a sample enables the determination of the donor and acceptor lifetimes to be calculated via
a least squares multi-exponential decay fitting. In this case both the lifetimes and the fractional contributions from each lifetime are calculated and provide information regarding the molecular conformation.
1
This can be done either with a single photon excitation at a wavelength within the of donor absorption
spectrum (l), or with two photon excitation at a wavelength of 2l is also possible. In the latter case two
photons may be absorbed simultaneously and via a virtual state combine their energies to produce a
similar electronic excitation as a the single photon of double the energy. This technique requires very
high laser intensity but due to the lower power and small focal point produces less overall photobleaching of the sample.
In addition the fluorescence anisotropy can also be obtained from the complex and used to provide
information as to the rotational dynamics of the construct. This is calculated as a difference between
fluorescence intensity measured parallel to the polarisation of incident light ( F|| ) and that which is perpendicular ( F? ).
r=
F||
F?
F|| + 2F?
(3)
Experimentally this is setup either as two separate detectors aligned parallel to and perpendicular to
the incident laser light, or a single detector which rotates (both systems are used in this work). These
results are similarly analysed using a least squares multi exponential fits, where the lifetimes correspond
to the rotational time for the various components of the construct. In the case of the constructs for this
work, it is expected that there would be a longer anisotropy lifetime corresponding to the rotation of the
entire construct and possible a shorter lifetime corresponding rotation of the chromophores. This will
be dependent to what extent if any the chromophores are able to rotate independently of the complex
as a whole.
2.2
Peptide binding
Peptide sensing is of particular interest to many groups owing to their wide spread involvement in
many biological processes. For example neuropeptides such as vassopressin and oxytocin are know to
affect processes as diverse as, regulation of water permeability of the kidneys [18], maternal bonding
during lactation [14], monogamous pair bonding in prairie voles, and male-male aggression [14]. The
behavioural affects of neuropeptides are of particular interest because of their potential application to
mental illness[17], and in general there is interest in understanding the biological mechanisms underlying secretion and regulation[14].
In other species peptides also play a crucial role. In bacterium, peptides and their associated binding
proteins are used in chemotaxis and uptake of solute. The sensor molecule used in this work is the
dipeptide binding protein of Escherichia coli. It is a member of a super-family of periplasmic binding
proteins produced by the bacteria, which have become increasingly well exploited in conjunction with
fluorescent probes to create peptide sensors[6]. Periplasmic binding proteins are large proteins located
between the inner and outer gram membranes of the bacteria which are characterised by a distinctive
venus-fly trap structure [8]. A highly simplified structure of two lobes with the hinge region between is
shown in Figure 1. As seen the protein has two conformations, the open unbound state and the closed
bound state. These two conformational states are common to the whole superfamily and the movement
of the two lobes provides a large enough distance change for measurement by FRET. As seen in Figure
1. there are a number of different options for placement of the fluorophores, the crucial factor being that
the presence of the fluorophore must not affect the binding affinity for the peptide or disrupt the folding
of DppA.
Figure 1: simplified representation of a periplasmic binding protein with two sets of fluorescent tags (1
and 2). A) shows the open state, B) the closed state.
Others have already constructed sensors from this superfamily: The maltose binding protein has been
2
investigated by [15]. In their work they outline a basic two possible general positions for the fluorophores to be placed, either they are placed either side of the ’jaws’ or placed on the hinge. The relative
distance of the fluorophores will change upon conformational change and hence FRET rate will be affected. However results reported on the creation of such complexes by [15] have not provided conclusive
results and it may be considered that this idea of placement is an oversimplification. It is notoriously
difficult to predict optimum places for fluorophore insertion that will not disrupt protein function but
will provide good fluorescence signal and undergo FRET changes during conformational change[10].
As such the randomised method of insertion employed here enables an wide range of candidates to be
constructed and tested in a more unbiased fashion.
2.3
The complex
The chromophores used as a FRET pair in this work are two variants of Green Fluorescent Protein (GFP),
Cyan Fluorescent Protein and Citrine. Citrine is a mutant of Yellow Fluorescent Protien, (YFP) (mutation
in Q69M) which make Citrine less susceptible to acid quenching, more easily expressed at 37 degrees
and less sensitive to chloride than its original version [11, 16]. It has a reported lifetimes of 3.61ns and
800ps [11, 3]. The absorption and emission spectra and the two photon cross section for both CFP and
Citrine are shown in Figure 2. The absorption emission spectra shows that there is a good spectral
window for investigation of donor lifetime in which there is minimal acceptor bleed through. CFP has
been shown to have two exponential lifetimes corresponding to two conformational states of the chromophore [12, 4], these lifetimes are reported by [4] to be 1.03ns and 3.57ns however many sources report
only one life time 2.6nm which appears to be the average of these two[3, 19]. It seems likely that this
averaging would distort lifetime changes as a result of FRET interactions.
Figure 2: (Left) single photon absorption and emission data taken from [25]. (Right)two photon cross
section data taken from [1]. Black lines indicate the excitation wavelengths used in this work.
The complex library was created by Dr De Linz[13] using a technique developed by Hughes and Sheridan [22, 23]. From this library, work by De Linz and Robert Stanley[24] has highlighted some promising
candidate constructs, 2E10, 5C3 and 7G7. The crystal structure of DppA with the positions of the CFP
and Citrine for each constructs can be seen in Figure 3. Based on this previous work these constructs
were the focus of this work. In addition the construct p-disp was also used extensively, this construct
contains only DppA and CFP.
3
3.1
Methods
Midi Preps
Midi Preps were carried out to provide ample plasmid for transfection. A selection of the constructs
from the original library were selected including 2E10, p-disp, 5C3 and 7G7. Using plasmid from mini
preps agar plates were seeded with transformed JM109 bacterium. Transformation was carried out by
the addition of 1µl of plasmid to 50µl of JM109 solution; this was left for 10mins, before submerging
3
(a)
(b)
(c)
Figure 3: Showing the three different constructs investigated in this work with the positions of the two
chromophores make in bright yellow (a) Complex 2E10, the CFP in the left hand of the two markers the
Citrine is shown in the top right yellow mark. (b) Complex 5C3 with CFP on the left and Citrine on the
right.(c) Complex 7G7 with CFP the upper yellow marker and Citrine the lower. Structures taken from
[5]
in water at 42 C for 45 secs, then put on ice for 2 mins. 1ml of NZY was then added to each tube
and these were incubated for 1hr. During this time 10cm petri-dishes were prepared with a solution of
1µlof ampicillin and 10ml of Agar jelly which was allowed to set. Bacterium were then taken from the
incubator and 50µl was spread thinly over the agar plates using a bent glass pipette tip. These were
then incubated overnight.
The following day a single colony from each agar plate was added to a solution containing 3ml of LB
media and 3µl ampicillin. These were then left in the shaker incubator for 4-5hrs. 100µl of this solution
was then added to a conical flask containing 75µl of ampicillin and 75ml of LB medium, and reincubated
overnight. The remainder of the process was then carried out as per the PureYieldTM Plasmid Midiprep
System technical manual[21] using the Pure Yield Clearing Column and Pure Yield Binding Column
(Promega). The concentration of the products from the midi prep were measured using a SmartSpec Plus
Spectrophotometer (Bio-Rad).
3.2
Cell culture preparation
Human embryonic kidney cells (HEK 293) obtained from Dr Benton were used throughout the experiments, these were cultured in 10ml of solution containing 500ml of Dulbecco’s Modified Eagle Medium
(DMEM) (Invitrogen), 50ml of Fetal Bovine Serum (Invitrogen), 1000 units/ml penicillin and 1% 1000
µl/ml streptomycin. Every two to three days one flask of cells was split, this was done by draining the
current medium of the flask, adding 10ml of magnesium and calcium free Hank’s Buffer Salt Solution
(HBSS) (Invitrogen) which was then also drained. Next 1ml of 0.05% Tripsin (Gibco) was added to the
flask in order to break intercellular bonds and prevent large rafts of cells forming, the flask was then
incubated for approximately 30seconds. On removal from the incubator the flask was hit to remove the
cells from the base. 10ml of DMEM was then added and the solution transferred to a centrifuge. The solution was centrifuged for 2 mins at 1000rpm in order to pellet the cells from the solution. The medium
was then poured away leaving behind the pelleted cells. These were then shaken to re-suspend them
and 10ml of DMEM was added and mixed with the suspension via drawing the solution into the pipetted several times. This solution was then divided between large (10cm) and small (2cm) petri dishes
for experimentation, and 25cm3 flasks to be keep and further split, in a ratio of 1:5 of cell suspension
solution and DMEM.
3.3
Cell Transfection
Cell transfection was carried out 1 or 2 days after cell culture depending on the confluence of the dish.
Two centrifuge tubes per sample were prepared with 250 or 500µl of OPTI-MEM (Invitogen) solution
depending on size cell culture dish. To one of the tubes Liprofectamine reagent (Invitrogen) was then
added and to the other a solution containing the plasmid solution, in a ratio of 1:1 by mass respectively.
This was then left for 5 mins before the contents of the two tubes was combined and mixed. This was
then left for a further 20mins before the solution was added to the cultured cells and mixed via gentle
4
agitation of the dish.
To ascertain whether transfection had been successful cells were imaged using fluorescent excitation by
mercury lamp in the live cell imager the following day prior to lysing.
3.4
Cell Lysing
Cells were lysed on the day following transfection to ensure the highest possible concentration of construct. The culture medium of transfected cells was removed from dishes by washed 3 times with washing solution containing 150mM NaCl and 10mM HEPES. Cell lysis buffer at pH 7.5 containing 50mM
HEPES (Ultograde, Calbiochem), 150mM NaCl (Anala R Normapur), 5mM EDTA (Sigma), 1% Triton, 0.1%
Sodium dodecyl sulfate (SDS (Sigma)) and 1 per 10ml protease inhibitor cocktail tablet (Complete mini)
was then added. Cells were removed from the base of the dish using a cell scraper. The solution was
then drawn into an 18 gauge needle and 2ml syringe before being transferred to a 1.5ml eppendorf. This
was then rotated in a cold room for 15mins. Following this the sample was centrifuged for 5mins at
140000rpm to pellet nuclear material. 1ml of supernatant was then transferred to a fresh eppendorf and
this was then frozen until used in FRET experiments.
3.5
FRET
Time resolved fluorescence microscopy was carried out using a mode-locked frequency doubled Ti/Sapphire
laser with LiBO (lithium borate) crystal circa 100fm pulse (Coherent). Excitation wavelength was at
400nm and 3.8MHz. Excitation at 460nm was carried out using the mode-locked Regeneratively amplified Ti/Sapphire laser and optical parametric amplifier at 250kHz (Coherent). Excitation at 490nm
was carried out using the PicoTa pulse diode laser within a FLIM (Fluorescence Lifetime Imaging Microscope) at excitation frequency of 4MHz (Picoquant). TCSPC was used as the detection technique as
discussed above (Ortech). The samples were prepared in lysis buffer described above and 200µl was
transferred to a 1ml cuvette that had been irradiated with UV light to remove contaminants. In the case
of the microscope setup, 10µl of sample was placed on a coverslip. Di-Alanine sensitivity experiments
were done via the addition of 20µl of 10µM solution of di-Alanine to the existing 200µl of cell solution in
a cuvette, the solutions were mixed by titration before measurements were taken. Filters used throughout the experimental were either interference filters Semrock or Schott Glass filters.
Analysis was carried out using Origin pro 6.0 (Microcal) for fits of single data sets and Fluro Fit (Picoquant) was used for global analysis. Both packages use least squares fitting approach.
4
4.1
Results and Discussion
Transfection
(a)
(b)
(c)
(d)
Figure 4: Showing HEK cells after transfection with the four different constructs (a) 2E10, Under YFP
filter set (b) 5C3 under YFP filter set.(c) and (d) P-disp under GPF filters.
The initial aim of the work was to transfect HEK cells with a plasmid containing the construct of
choice, and ensure a high enough rate of successful transfection for FRET studies to be carried out. A
number of transfections were imaged in the Live cell imager to investigate the success of transfection,
the images can be seen in Figure 4. As can be see there was successful transfection of all the constructs of interest. The construct 10E5 was also originally of interest as in this complex the Citrine had
5
inserted within the CFP and it was hoped that this might result in a functioning Citrine unit and a nonfunctioning CFP, providing a means for investigating acceptor characteristics only. Live cell imaging of
this complex however revealed that there there was no fluorescence present suggesting that both the
CFP and Citrine were rendered non-functioning by the insertion.
4.2
Acceptor and donor Behaviour
Owing to the nontrivial form of the decays for each chromophore it was considered that a characterisation of each separately should be done before lifetime changes were looked for. As previously stated
the constructs with Citrine only were not viable, however, by excitation at 490nm and detection in a 510
long pass window, direct excitation of and emission from Citrine were possible. The results as shown
in Figure 4.2. can be fitted with a bi-exponential decay with lifetimes of 1.0ns and 3.5ns. The longer of
these two agrees well with the Citrine lifetime as found by [11, 3]. The shorter of the two is not obviously a Citrine lifetime but it could be a CFP lifetimes as 490nm will also excite a small amount of CFP
as seen in Figure 2. The fractional contributions of the two lifetimes also support this hypothesis A1
and A2 are 2566 and 8971 suggesting approximately 3.5 times the contribution from the Citrine lifetime
as from the possible CFP lifetime. Investigation of the CFP only was done using the p-disp construct,
Figure 5: Decay from construct 2E10 excited at 490nm and detected with a 510nm long pass filter, life
times are found to be 1.02ns and 3.51ns with fractional contributions of 2566 and 8972 respectively
which contained CFP and DppA only. This was excited at both 400nm and 460nm following preliminary
measurements which showed an unexpectedly complex decay of p-display at 400nm. It was thought
that this couplexity may be due to excitation of transitions other than the S0 to S1 or that non-biological
molecules present in the solutions were excited. The results of these two can be seen in Figure 6. Curve
fitting for the the two excitation wavelengths provided a tri-exponential decay with slightly differing
lifetime values (red lines) in both cases. A bi-exponential decay (green line) is also fitted to the 460nm
sample with the lifetimes fixed to those found by [4] to provide a comparison. Table 1 summarises the
lifetimes and fractional contributions.
Excitation
490nm
400nm
460nm
460nm
t1
1.02±0.03
1.73±0.37
4.01±0.07
1.03±0
A1
2566±47
44171±506
18296±237
57018±424
t2
3.50±0.013
0.38±0.01
0.37±0.02
3.57±0
A2
8972±60
39146±587
22629±519
74037±100
t3
4.37±0.04
1.78±0.09
-
A3
35184±833
18190±611
-
c2
1.59
2.60
1.90
1.99
Table 1: Lifetimes and fractional contributions of construct p-disp and direct excitation of Citrine.
6
(a)
(b)
Figure 6: showing the decays for the CFP only construct (p-disp) for (a) a 400nm excitation with a 462485nm detection window and (b) a 460nm excitation with a 484-505nm detection window. In the 460nm
the green line is a bi-exponential fit with lifetimes fixed at 1.09ns and 3.57ns as in [4].
The two lifetime fit of [4] fits the data well however the slightly higher c2 value suggests that a
three lifetime fit better represents the sample. However the lifetimes do not correspond chromophore
lifetimes, it was thought that they could be caused either by background from the lysate buffer or were
the result of contaminant. Repeated measurements with new solutions and thorough cleaning proved
contaminants not to be the cause. Measurements of untransfected cells, lysed according to the protocol,
showed a similar three lifetime decay. The results of these lifetimes for 400nm and 460nm are shown
in Table 2. From what source the three lifetimes in the untransfected cells come from is unclear, it is
Excitation
400nm
460nm
t1
1.46±0.03
0.030±0.006
A1
69617±1036
102389±2293
t2
0.289±0.008
4.04±0.04
A2
168732±5079
25634±588
t3
5.76±0.07
1.250.037
A3
27505±499
37011±694
c2
3.60938
5.07239
Table 2: Table of the lifetimes and fractional contributions for the untransfected cells
possible that they are a non-biological fluorescence from molecules in the buffer, or that molecules from
the cell cytoplasm or membrane, not removed during the lysate procedure, contribute. Regardless of
the source the lifetimes presented significant problems in interpreting responses to di-Alanine as it is
not clear which of the lifetimes corresponded to CFP and hence would be modulated by the peptide
presence.
4.3
Detection Window Analysis
To further understand these three lifetimes a detection window dependence experiment was done for
all three constructs of interest (2E10, 7G7 and 5C3). A selection of detections windows were investigated using the 400nm excitation. The filters used for these windows were 430nm long pass, 474/23nm,
513/17nm, 556/20nm. The results from these were subjected to a global fit analysis which found that no
3 lifetime global fit was possible for any construct. For complexes 5C3 and 7G7 a two global fit where
the mid length lifetime varied was possible as was a fit where the longer lifetime varied. In the case
of 2E10 the only global fit possible was by allowing the middle lifetime to vary and globalisation the
longer and shorter lifetimes. The globalised lifetimes were calculated to be 0.466ns and 5.032ns. Figure
7. shows how the middle lifetime varies with detection wavelength as well as the fractional contributions. As can be seen for the filters 474/23nm, 513,17nm and 556/20nm there is an increase in lifetime.
This shift implies that the mid-length lifetime is the weighted average of two lifetimes of which more of
the longer lifetime is present in yellower windows, however for an individual detection window, this
fitting algorithm is not able to discriminate between the two. The 430nm long pass as can be seen by
7
Figure 7: Showing the lifetime dependence on detection window and fractional contributions. The
windows used are 430nm long pass, 474/23nm, 513/17nm, 556/20nm. The 430nm long pass is expected
to have a lifetime which is the weighted average of the other windows, this is demonstrated by the black
line.
Figure 2. allows the entire emission spectrum to be collected and hence would be expected to results in
a slightly blue shifted average of the other filter windows as shown in Figure 7.
The result from this experiment suggests that there are more than 3 decays in the constructs, the deconvolution of which is not possible with the current measurements.
4.4
Di-alanine response
The effect of di-alanine on the three constructs, 7G7, 5C3 and 2E10 was then investigated. At 400nm only
2E10 appeared to show a lifetime change that could not be attributed to the addition of plain di-Alanine
solution see appendix for details of 5C3 and 7G7. Hence this construct was measured at both 400nm
and 460nm and a concentration study was performed at 460nm. The results of these are summarised in
Figures 8 and 9
Figure 8: Showing the effect of additional components of the construct on fluorescence decay at
(a)400nm excitation, and (b) 460nm
As seen by Figures 8. excitation at 400nm appears to demonstrate a lifetime change with addition of
di-alanine the values for this can be seen in Table 3.
This change however is not present in Figure 8(b). where the addition of di-alanine appears to have no
affect of the lifetimes of the construct. To investigate whether this was genuinely the case the concentration study shown in Figure 9 was done.
8
Excitation
400nm
400nm
460nm
460nm
t1
5.35±0.09
5.69±0.07
4.01±0.07
1.95±0.08
A1
10042±331
10942±4177
18297±237
35269±1210
t2
1.45±0.04
1.64±0.03
0.37±0.02
4.13±0.07
A2
52281±2337
32610±602
22629±519
32366±1763
t3
0.580±0.03
0.483±0.01
1.78±0.09
0.418±0.01
A3
35090±2289
34635±630
18190±612
36149±702
c2
1.967
2.21
1.90
1.94
Table 3: Table of the lifetime changes due to di-alanine in each case the lower row is the lifetimes with
di-alanine addition
Figure 9: Showing the effect of varying dialanine concentration on decay of construct 2E10 at 460nm
Figure 9. shows that there appears to be no correlation between the concentration of di-alanine and
changes in the decay of construct 2E10. This could be due to the construct not FRETting, or to there
being an insufficient change in donor lifetime, or that the change caused cannot be deconvoluted from
the background of other decays. The last of these possibilites is also supported by observations of the
count rates.
4.5
Count Rates
One overriding problem with all the constructs was a very low count rate. Counts of less than 1000 s
were often seen for laser powers in the order of magnitude of several µW. The difference between count
rates of untransfected samples and constructs was not more than 1 order of magnitude at any point
and for several samples was considerably less. As these two are of similar magnitude and the changes
expected as a result of di-alanine addition are small it becomes highly problematic to deconvolute the
lifetimes of interest. Another problem was the variation of this count rate between samples, the same
constructs made in different batches showed significant variation in count rate.
The only occasion on which count rate was significantly higher was for the case of the p-disp shown in
Figure 6. For this sample using laser power of 4µW achieved count rates of 2500 s and as shown the
decay is compatible with that of other works. However this was not the case for any constructs. The
2E10 measured on the same day and made in the same batch required laser powers of 50 60µW to
achieve counts of the same magnitude. As seen in Figure 4. pre lysing it appears that both 2E10 and
p-disp are well transfected with a similar proportion of cells taking up the plasmid. This being the case
it suggests that the lysing stage of the process may damage the constructs containing CFP and Citrine
more than those containing only CFP. With this in mind an experiment on unlysed cells cultured on
glass bottomed dishes was set up, however due to equipment failure could not be completed.
9
5
Conclusions and Further Work
This work has shown that adequate transfection of HEK cells can be achieved with good expression levels of the construct of choice. FRET lifetime results from these constructs are difficult to interpret owing
to the number of lifetimes that appear to be present in the decays of all samples, including untransfected
cells. Lifetime dependence on detection window has been shown for all constructs suggesting that there
are possibly more than three decay lifetimes present. Of the constructs investigated none appear to show
a constant di-alanine response however it is suspected that this is due to low concentrations of construct
which result in low count rates which have many complex decays that cannot be de-convoluted. It has
been possible to show that for constructs containing only CFP and DppA there is a high initial count
rate and decay lifetime comparable with work in the literature. Excitation of Citrine produces a decay
with a characteristic lifetime of 3.51ns again comparable with literature. The poor count rate is the single largest hinderance to analysis.That all CFP-DppA-Citrine constructs suffer from a poor count rate
by comparison to the CFP-DppA only sample suggests that these constructs are disrupted more during
the lysing process, possibly due to poor protein folding.
In future work this must be the most important area to address. There are several ways in which a
greater concentration of construct could be achieved. Stable cell lines of constructs could be cultured
prior to lysing. Lysis technique could be altered to be less aggressive in order to ensure constructs are
not damaged. In vitro scanning of cells expressing constructs as attempted in this work would be of
double benefit, as it would both provide ample concentration of chromophore as well as exclude the
need for lysing potentially eliminating the background decays. In addition two photon excitation could
also provide a means for excluding the background decay lifetime, this was attempted in the course of
this work and looked to be a promising avenue of inquiry however due to equipment failure this was not
completed. The further natural progression of this work is to continue to screen remaining constructs
from the library to investigate their potential as a more optimised sensor.
10
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