Promoter Characterization Experiments
Braff JC, Conboy CC, Endy DE
Abstract
The rational design of integrated biological systems requires a set of
characterized, compatible components. Here we attempt to improve the characterization
of a set of transcriptional promoters contained in the registry of standard biological parts.
We measure the relative strength of thirty-two promoters by flow cytometry, via
expression of a YFP reporter, measured by flow cytometry.
Intro
The following series of experiments was intended to characterize the relative
strengths of the various promoters in the Registry of Standard Biological Parts. This work
is part of the effort to measure the relevant parameters of biobrick parts in order to
facilitate the rational design of more complex devices and systems. Promoter strength is a
relevant parameter for both system design and stability. Previous construction of
synthetic systems including flip-flops (Collins) and oscillators (Elowitz) indicated that
having well-matched promoters was important for function in these systems.
Additionally, the inclusion of strong promoters in a system may increase the load on the
cell, leading to a selective pressure to mutate the system, thus decreasing stability.
Ideal promoter characterization experiments would directly measure PoPs
(polymerase per second, the rate of transcription initiation at a given point on a piece of
DNA). However, as no such method is currently available, we used detection of a
fluorescent reporter with flow cytometry to infer relative levels of transcription initiation.
Constructs
In order to measure relative amounts of transcription generated by the biobrick
promoters, a construct was assembled for each with the promoter to be characterized
upstream of a YFP output devices on a high-copy plasmid, pSB1A2. Constructs were
characterized in MC4100, a lacI- strain.
Promoter
(variable)
BBa_Prefix
RBS (B0034)
EYFP
EYFP
(E0030)
p(BLA)
AmpR
APr
Promoter
pSB1A2
Characterization
30 19 bp
Construct
(on pSB1A2)
Terminator
(B0015)
BBa_Suffix
rep (pMB1)
rep (pMB1)
Fig. 1: Schematic of a promoter characterization construct.
Table 1: Promoter Characterization Constructs.
Construct
Promoter
Promoter Description
BBa_I6030
R0010
LacI regulated
BBa_I6031
R0011
Hybrid promoter based on lambda pL, repressed by LacI
BBa_I6032
R0040
Hybrid promoter based on cI, repressed by TetR
BBa_I6034
R0050
HK022 cI regulated
BBa_I6036
R0051
Lambda cI regulated
BBa_I6038
R0052
434 cI regulated
BBa_I6040
R0053
p22 cII regulated
BBa_I6042
R0062
lux pR from V.fischeri, activated by HSL-bound LuxR
BBa_I6044
R0063
lux pL from V.fischeri, repressed by HSL-bound LuxR
BBa_I6046
R0065
Hybrid promoter, repressed by lambda cI, activated by HSL-bound LuxR
BBa_I6048
R1050
Standard promoter, repressed by HK022 cI
BBa_I6050
R1051
Standard promoter, repressed by lambda cI
BBa_I6052
R1052
Standard promoter, repressed by 434 cI
BBa_I6054
R1053
Standard promoter, repressed by p22 cII
BBa_I6056
R1062
Standard promoter, activated by HSL-bound LuxR
BBa_I6058
I0500
pBad/AraC, arabinose-inducible promoter
Methods
Promoter activity time course: 5mL cultures of LB with 50ug/mL Amp were
inoculated from fresh plates of MC4100 containing one of a select set of promoter
characterization constructs. Cultures were grown overnight to saturation at 37C, then
diluted 1:500 into 50mL fresh LB/amp at the beginning of the time course. Samples were
collected for flow cytometry from the saturated overnights (2mL), at the time of dilution
(10mL), and every 45 min thereafter for six hours. (10mL at t=0.75 hr; 5mL at t=1.25 and
2 hr; 2mL thereafter.) An additional sample was drawn at the tenth hour. Samples were
spun down, resuspended in 0.5 or 1mL of PBS (according to cell yield) and stored on ice
until all samples were collected. A control experiment showed that reserving samples on
ice for up to 12 hours did not affect the level of YFP detected by flow cytometry
significantly. Variation of +/- 5% was observed. (Data not shown.)
Promoter activity “snapshots”: Based on the curves generated by the promoter
activity time course experiments, OD600=1.0 was selected as the condition for making a
single measurement of YFP output for the complete set of promoter characterization
constructs. This 32-sample set includes all promoters available in the Registry as of 0701-04. Overnight cultures were prepared as above, diluted 1:100 into 5mL fresh LB/amp,
and grown for 2.5 hours at 37C before sampling. The average OD600 at the time of
sampling was 1.12, with a standard deviation of 0.16, excluding one sample (I6058)
which exhibited a severe growth delay. I6058 was sampled at 4.5 hrs post-dilution when
it reached OD600=0.91. (See discussion.) Samples were prepared as above from 2mL of
culture.
Analysis of flow data: A standard gate for live cells, determined by forward and
side-scatter, was used to acquire flow data. This gate centered on the majority of the cell
population for cultures above OD600~0.5, but was a poor fit for earlier time points,
perhaps due to changes in average cell size at these times. In order to analyze average
cells and gate out outliers for each sample, a second, tighter FSC/SSC gate was
established on a sample-by-sample basis.
Plate Reader Promoter Characterization: Inoculated 5mL cultures of LB (50
ug/mL ampicillin) from single colonies on fresh plates. Grew overnight at 37°C. Diluted
saturated cultures 1:100 into M9 minimal media, supplemented with 0.4% glycerol, 0.1%
casamino acids, and 50 ug/mL. Two samples were diluted from I6058 (pBad
characterization construct) and one was induced with 0.2% arabinose. Grew cultures in
supplemented M9 for 4 (7?) hours. Rediluted samples1:100 into 200 uL of fresh
supplemented M9 in individual wells of a 98-well plate for analysis with the Victor 3
plate reader. Samples were covered with ~2 drops of mineral oil to prevent sample
evaporation during the experiment.* [Details of the plate reader program.]
Samples were pulled for flow analysis from the 5 mL culture at the beginning and
end of the plate reader run.
*This technique also reduces oxygen diffusion into the media. Subsequent experiments
have used the alternative method of continually adding water to the samples in proportion
to the rate of evaporation.
Fluorimetry Experiments
For the initial flourimetry experiment (3/25/04), in which we hoped to establish
baseline promoter strength for the 2003 IAP promoter set, (almost) all constructs were in
high copy plasmids transformed into MC4100 cells. For each promoter, we had two test
constructs, one for each of our standard YFP output devices (E0432/32 +/- LVA). Test
constructs with LVA-tagged YFP will not be discussed in detail in this report. Saturated
overnight cultures were diluted back 50 uL into 5 mL of LB with the appropriate
selection (50 ug/mL AMP), grown at 37 C, and measurements were taken at 2.5 and 4.5
hours post-dilution. Samples were generally around OD 600 0.75 at the first time point
and 1.6 at the second time point. Although all samples were diluted back at the same
time, the OD 600’s varied considerably. For all samples with fluorescence well above
baseline, fluorescence declined between 2.4 and 4.5 hours post-dilution
Most promoters contained in the 2003 IAP systems and the quad part inverters
currently being characterized fell into the mid to strong range, with the exception of
R0050, which yielded signal barely above baseline (Fig.2). R0062 and R0065 gave very
low signal, as expected, in the absence of the LuxR-hsl regulatory complex. There
function in the presence of LuxR-hsl has not yet been characterized. The test constructs
for R0052 and R1052 were not yet complete at the time of these experiments, but, based
on the E0432 test constructs, high signal was expected, especially for the wild type
version. The ’10 series’ promoters did not exhibit equal strength as we had hoped,
though they were perhaps better matched that the others. R1062 appears to be moderately
strong in the absence of LuxR-hsl. R1053 was a bit weaker than R0053, and R1051 a bit
weaker than R0051. The data from the E0432 constructs indicates that R1052 is also
reduced in strength, in comparison to the wild type promoter. The wide range of
promoter strength observed suggests that care will have to be taken to allow plausible
device-device matching.
1.4E+06
1.2E+06
1.0E+06
8.0E+05
2 hr
4 hr
6.0E+05
4.0E+05
2.0E+05
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0
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0.0E+00
co
nt
ro
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Baseline Adjusted YFP (counts/sec/OD)
Promoter Characterization: YFP Output (-LVA)
Construct
Figure 1: 3-25-04 Flourimetry data, corrected for OD600 and background fluorescence.
Characterization of promoter strength in log phase growth, LVA- constructs.
Flow Experiments: Relative Strength in log phase
These experiments were repeated with flow cytometry (4/1/04) in order to verify
the relative promoter strength observed via flourimetry and to give us a sense of the
degree of phenotype variation across a given population. As above, saturated overnight
cultures were diluted back 50 uL into 5 mL LB and allowed to grow for 2.5 hrs. before 1
mL samples were taken and resuspended in 1 mL of PBS. Samples were kept on ice for
the duration of the experiment. OD 600s ranged from 0.62 to 1.24. The LVAcharacterization constructs verified the relative promoter strengths determined on the
flourimeter. (Fig.3) However, the flow data revealed that in some cases the population
exhibited either bifurcation or a rather wide range of fluorescence, a fact obscured by the
bulk measurements obtained on the flourimeter. R0010, the wild type Lac promoter,
showed some bifurcation within the population, with peaks at approximately 500 and
2000 ‘flow units.’ Of the LVA- constructs, Pbad (I0500) grown (o/n or not???) with
0.2% arabinose, showed a very dramatic split population, with about 40% of the
population looking like the negative control and the remaining 60% forming a distinct
peak around 150 ‘flow units.’
900
0.72
800
700
0.75
600
500
1.03
1.01
400
0.99
300
0.86
0.63
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51
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1.05
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OD600=
1.26
51
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100
1.03
1.01
62
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0
200
ne
g.
YFP Fluorescence (Flow Units- GMC, all)
Promoter Characterization:
LVA- constructs
Construct (All in pSB1A2, MC4100)
Figure 2: 4-1-04 Flow data, characterizing relative strength of Biobrick promoters in log phase (2.5
hrs post-dilution) with an LVA- YFP output device.
Flow Experiments: Time series
During previous characterization experiments where samples were taken at
different time points, we observed that, as expected, YFP fluorescence for a given culture
varied significantly with cell density. As a result, we could not be sure that the relative
promoter strengths we measured were not distorted by variation in cell density/growth
phase between cultures. To address this question, we did a series of flow cytometry time
course experiments using select constructs from our previous promoter characterization
experiments. For our first time course experiment we chose to look at R0010 (wild type
Lac), R0040 (Tet), R0051 (Lambda cI), and R0063 (Luc pL) with both E0430 and
E0432, on pSB1A2 in MC4100 cells as before. Saturated overnights were diluted back
500 uL into 50 mL LB with 50 ug/mL AMP and were grown at 37 C with shaking.
Samples were taken in duplicate starting at two hours post-dilution and continuing until
12 hours post-dilution, when the samples were well into stationary phase (OD 600 4.46.0) Both sets were prepared for flow cytometry as before; one sample was run
immediately and the other was packed on ice at 4 C and run at 10 hours post-dilution to
determine if holding samples on ice and then running all samples together at the end of
the time course would be a viable alternative to performing the time course analysis in
real time.
Qualitatively, the results of this experiment indicated R0063 and R0010 are
stronger promoters and R0040 and R0051 are weaker. However, the exact ranking varied
at different points in culture growth. This was also inconsistent with our earlier results,
which indicated that R0010 and R0051 would be stronger and R0040 and R0063 would
be weaker. The samples all gave moderately strong fluorescence (200-1000) at two hours
post-dilution. (Fig.6) Signal climbed steadily until about eight ours post-dilution when
our stronger samples had reached the upper limit of our flow cytometer instrument
settings.
Initially we had used a standard gate based on our negative control to gate our
sample population. This gate centered on the majority of our cell population in many
cases, especially at higher OD’s, but at the earlier time point it did not give such a good
fit. We analyzed our data both with this original standard gate for all samples, and went
back and re-gated each sample with a smaller gate around the core population. This
reduced our mean fluorescence across the board, perhaps by gating out very large cells
(?), but did not change the overall rankings. When the samples stored on ice until 10
hours post-dilution were run and plotted against the samples run in real time, they looked
very similar, +/- about 5%. This was true for both the E0432 and E0430 samples.
Promoter Timecourse: YFP Expression vs. Time
(G3 gated population, LVA-Constructs. *scale)
YFP Fluorescence (Flow Units, GMC)
6000
5.55 (0.70)
5.2 (0.57)
4.42 (0.13)
5000
4.75 (0.40)
4000
2.19 (0.79)
3000
2000
AveOD600 (SD)=
0.54 (0.21)
1000
0
0
2
4
6
8
10
12
14
Time (hours post-dilution)
R0010.E0430 (Lac)
R0040.E0430 (Tet)
R0051.E0430 (Lambda cI)
R0063.E0430 (Lux pL)
Figure 3: 5-1-04 Flow Data. Promoter characterization time series, LVA- constructs.
At two hours post-dilution the cultures were already in mid-log so this experiment
yielded very little information about the stationary to log phase growth transition. To get
better resolution across this transition and during early-mid log phase growth, we
performed a similar experiment (5/08/04) taking samples every 30 minutes starting at 0
hours post-dilution. Samples were diluted back 100 uL into 5 mL LB (rather than 500 uL
as we did on 5/1/04). Since storage on ice did not seem to adversely affect fluorescence
data, all samples were held on ice until the end of the experiment and then run on the
flow cytometry machine used for the previous experiments. We looked at R0040.E0430
and E0432 and R0063.E0430 and E0432. We also ran a plain MC4100 negative control
at all time points to verify that autofluorescence was not an issue at the higher OD’s.
Before data analysis, all samples were re-gated with a tight gate around the central
population.
Promoter Characterization:
Tet and Lux pL, LVA- timeseries
3500
AveOD600=
4.7
YFP Fluorescence
3000
2500
2000
5.30
1500
0.01
1000
4.80
0.03
500
0.80
2.25
3
4
3.70
0.20
0
-1
0
1
2
5
6
7
8
Time (hours)
R0040.E0430 (Median_R3)
MC4100 only (Median_R3)
R0063.E0430 (Median_R3)
Figure 4: 5-8-04 Flow Data. Promoter output time series, LVA- constructs.
The saturated overnight cultures of the E0430 samples were both highly
fluorescent, but fluorescence was dramatically lower at time point ‘0,’ which was taken
within minutes of diluting back the cells. (Fig.8) We are not sure how to interpret this,
except to hypothesize that the cells divide almost immediately upon dilution. Shifts in
forward and side scatter support this theory. R0040.E0430 shows a slight peak in
fluorescence around OD 0.02, but both samples show reduced fluorescence during midlog phase growth, with fluorescence climbing between OD 2 and 5 as growth slows, and
the cells transition back into stationary phase. Many factors may contribute to reduced
fluorescence during exponential growth, including lower cell volume and rapid dilution,
competition for transcription and translation machinery, and lagging plasmid copy count.
Based on the –LVA set, this experiment shows R0040 to be stronger than R0063 during
early log phase, with the samples running quite parallel during late log into stationary
phase. The previous time course had also shown R0040 to be stronger at the log phase
time point (2 hours post dilution), but R0063 had seemed considerable stronger at the
later time points.
The data for R0040.E0432 and R0063.E0432 is a little puzzling. The
R0063.E0432 saturated overnight culture exhibited a moderately YFP signal, which
dropped off immediately upon dilution and remained very low for the duration of the
experiment, rising just slightly above base line as the culture moved into stationary phase.
The higher signal in the saturated overnight suggests that the output device on the high
copy plasmid may have maxed out the available degradation machinery in the cells and
allowed YFP to accumulate after the overnight cultures reached stationary phase.
However, we do not have enough data on either transcription from these promoters or
clpX/P mediated degradation during stationary phase to have a good sense of what may
be going on here. R0040.E0432 is of particular interest to us, as this is the output device
contained in our current versions of the Dualator and the Synchronator. Unlike with
R0063, the saturated overnight of this culture was just barely above the negative control
(40 vs. 3). Fluorescence increased with a peak of around 450 at OD 0.4, which is still
relatively low though easily detectable. By OD 0.2 fluorescence had dropped back down
to baseline and remained at this low level until the culture had transitioned back into
stationary phase (around OD 5).
Although this time course data is very preliminary, it reinforces our sense that
system behavior varies considerably with culture growth phase. This leaves us with two
general options, either to build more robust systems that behave in a more consistent
manner or to pick clearly defined standard operating conditions in which to characterize
our systems. Both strategies will likely be key to creating functional biological systems,
but out primary approach for now is to define our standard operating conditions. We are
currently considering three distinct SOCs:
1) Chemostat: Log phase growth (OD600= 0.5-1.5)
2) Agarose pad on microscope (log phase?)
3) Liquid culture (saturated o/n or log phase growth)
Time Course Density
10
OD600
1
0.1
0
2
4
6
0.01
Time Post Dilution (hr)
8
10
12
Time Course Activity
3500
3000
YFP Fluorescence (GMC)
2500
MC4100
R0011.E0430 (lac)
R0040.E0430 (Tet)
R0051.E0430 (lambda cI)
R0063.E0430 (Lux)
R1051.E0430 (Standard)
R1052.E0430 (Standard 434 cI)
I0500.E0430 (pBad)
4.75
Ave OD600=
3.75
5.03
2000
0.02
0.07
0.01
1500
3.45
0.29
2.61
1000
0.65
0.81
500
0
-2
0
2
4
6
8
10
12
Time Post Dilution (hr)
6/23/04 Promoter Snapshots
1.80
1.40
1.20
1.00
0.80
0.60
0.40
0.20
1
R0 1.E
04 04
R0 0.E 30
05 04
R0 1.E 30
05 04
R0 2.E 30
05 04
R0 3.E 30
06 04
R0 2.E 30
06 04
R0 3.E 30
06 04
R1 5.E 30
05 04
R1 0.E 30
05 04
R1 1.E 30
05 04
R1 2.E 30
05 04
R1 3.E 30
06 04
I0 2.E 30
50 04
R0 0.E 30
06 04
R0 1.E 30
07 04
R0 1.E 30
07 04
R0 3.E 30
07 04
R0 4.E 30
07 04
R0 5.E 30
07 04
R0 7.E 30
07 04
R0 8.E 30
07 04
R0 9.E 30
08 04
R0 0.E 30
08 04
R0 1.E 30
08 04
R0 2.E 30
08 04
R0 3.E 30
08 04
R2 4.E 30
00 04
R2 0.E 30
00 04
M 1.E 30
C
41 04
00 30
on
ly
0.00
R0
0
Flow units normalized
1.60
Promoter
Discussion issues:
a. R0052 as example of really strong promoter that causes a load problem/is
selected against (?) Didn’t mutate, but showed abnormal histogram/ weird
population distribution.
b. MC4100 not a good chassis for pBad.
c. Address the curves seen in the time course experiments, which show an
initial climb in fluorescence during the lag-phase coming out of stationary,
as much as a four-fold drop in YFP fluorescence in early log, followed by
a symmetric increase in fluorescence in late log to stationary. Comment on
Jason’s model. Argument for doing snapshots later than OD~1, in order to
see a wider split in strengths.
d. Bifurcating populations (on non-inducible and inducible promoters.) (?)
e. Possible future work:
1. Same experiment in M9 minimal media supplemented with
0.1% casamino acids or other standard media.
2. Same expt in plate-reading fluorimeter (denser sampling,
time-course data for all promoters in parallel.)
3. (If #2 not possible, and maybe even if) repeat snapshots at a
higher OD.
4. Evaluate same constructs in different standard operating
conditions, i.e. chemostat to mitigate the effects of culture
growth state on promoter activity measurement.
5. mRNA measurements (rather than indirect protein
measurements) from similar constructs with an internal
standard using rtPCR or RNA Protection Assay to better
quantify the amount of transcription initiated at each
promoter.
f.
References
Collins flip-flop
Elowitz Repressilator