Team:UChicago/Experiments
Experiments and Protocols
Oscillator System
We plan oscillations using western blots and densitometry analysis
to track the different phosphorylation states of KaiC.
In order to synchronize a population of cells, we used M9 minimal media with no
carbon supplements in order to limit the ATP provided to the E.coli cells. This
shock was conducted for both one and six hours (See results).
In addition to examining oscillations of our KaiABC
biobrick, we also aimed to optimize these
oscillations by altering the concentrations of KaiA,
which is driven under an L-rhamnose inducible
promoter. The input to output ratio of KaiA was first
investigated using a 10 fold gradient of L-rhamnose
concentrations. We assayed the amount of KaiA
produced using Bradford assay and western blots. We estimated the correct
concentrations of L-rhamnose as well as the time of
induction data from the Paris-Bettencourt 2014 team. Induction was completed
for 10 hours. We additionally conducted a time-course of induction for 16 hours
in order to determine the saturation point of KaiA,
and to examine the degradation rate of KaiA after
removal of the inducer from the synchronization/shock in M9 minimal media.
Read-Out System
We plan to assay the read-out system by putting together pMC002 or
pMC003 with the KaiC phosphomimetics
pMC004-7. pMC004-7 exhibit different phosphorylation
states of KaiC, essentially portraying a snapshot of KaiC throughout its oscillations in the KaiABC
system. We will pair each of these phosphomimetics
with pMC002, containing the master regulator RpaA,
its activator SasA, and a response regulator RpaB (the function of this has not been clearly
characterized yet). We will also pair each of the phosphomimetics
with pMC003, exhibiting the same factors RpaA, RpaB, and SasA with the addition
of CikA, a negative regulator of RpaA.
We will assay the activation and deactivation of RpaA
by measuring the fluorescence of GFP under a plate reader. Given that the
LVA-Stop tagged GFP is directly downstream the circadian kaibc
promoter, we expect differences in activation/deactivation of RpaA to directly impact the amount of GFP expressed. As
such, we expect different fluorescence readings for each of the phosphomimetics. Furthemore, we
hypothesize that pMC003 will yield a greater contrast in fluorescence readings
between each phosphomimetic, given that it is a
negative regulator of RpaA.
Specific Assays:
Gibson Assembly
Gibson assembly was conducted using NEBs 2x HiFi
Assembly Master Mix. Volume of Gibson was halved in order to save reagent. 50
ng of the longest fragment in each reaction was added, and other fragments were
added in ratio relative to the longest fragment. 5uL of the master mix was
used, and H2O was added to bring whole volume of reaction to 10 uL. Reaction was incubated at 50C for 1 hour.
PCR
Phusion DNAP from Thermo
Fisher with HF buffer used for reactions. Each reaction was supplemented with
DMSO. Gradient PCRs, miniprep PCRs, and colony PCRs
were conducted with 10uL as the final reaction volume. Samples that would be
gel extracted were conducted with 50 uL as the final
reaction volume. 10 uM primers were added. 30 cycles
of PCR were conducted. See Notebook for details on cycle temperatures.
Gel Electrophoresis
Gel set up in TAE buffer. 1% agarose gel used for standard
electrophoresis. 0.8% gel used for smaller fragments. 0.7 uL
of EtBr added for imaging. Gels run for 30 mins at 120V. Invitrogen 1 kB ladder
used for standard protocols.
Gel Extraction
Machery-Nagel gel extraction kit used. See
notebook for complete protocol. Samples eluted in 15 uL
of elution buffer.
Transformation
Competent cells made by team using RbCl2 and CaCl2 chemically
competent protocols.
Efficiencies of competent cells were 4.00 x 10^5 transformants/ng and 1.15 x 10^6 transformants/ng respectively. Transformation was
conducted by incubating on ice for 30 mins and conducting a heat shock at 42 C
for 1 min. Colonies plated using glass beads method. Incubation at 37C
overnight.
Sequencing
Sequencing conducted by the University of Chicago’s sequencing
centers.
Growing cultures
Cultures grown for induction were incubated at 37C. OD600 for
initial assays were measured to be around 1.5. In latter assays, cells were
diluted to be OD 600 ~0.6-0.7. Initial cultures were always grown in LB.
Induction
Induction of rhamnose to drive KaiA expression conducted in M9 minimal media with
supplements: 0.4% glycerol and 0.1% casamino acids.
Induction solutions made with varying concentrations of rhamnose
in order to characterize the input-output ratio of rhamnose
to KaiA. This was significant for us to be able to
vary the concentrations of KaiA and to develop more
robust oscillations. Induction conducted for 10 hours at 30C. Induction
protocol followed from Silver Lab (Chen, A. H., Lubkowicz,
D., Yeong, V., Chang, R. L.,
& Silver, P. a. (2015a). Transplantability of a
circadian clock to a noncircadian organism. Science
Advance, 1(5), 1–6. http://doi.org/10.1126/sciadv.1500358) .
Synchronization
Synchronization to synchronize the KaiABC
clock was conducted by incubating induced cells in M9 minimal media only (no
supplements). Synchronization conducted for both 1 hour and 6 hours. Silver lab
reported 1 hour synchronization while Rust lab has seen 6 hours to be adequate
for cyanobacteria.
Oscillation Time Course
Following synchronization, cells were reintroduced in M9 media
with 1mM leucine and 0.5% succinate supplements to allow for slow growth. Oscillations
were tracked by freezing 2 mL cell samples every 6 hours in a -80C freezer. The
time duration for tracking oscillations was set at 3 days.
Western Blot
In order to track expression of KaiA,B,C, and phosphorylation states of KaiC western blots were conducted. For simple expression
assays, such as when characterizing the pRha promoter
and quantifying KaiA expression, a 14-20% BioRad precast protein gel was used during SDS page gel
run. For tracking different phoshphorylation states
of KaiC, a 4-7% BioRad
precast protein gel was used. Bradford assays were conducted before SDS-page in
order to standardize the amount of protein added per sample. A positive control
of cyanobacterial lysate, and a negative control of E.coli cells containing an
empty Cam circular backbone were run. Purified protein samples provided by the
Rust lab were run to quantify amount of KaiA, B, and
C expressed by transformed E.coli cells. Transfer accomplished using PVDF
membranes. Membrane sandwich apparatus was prepared under Towbin
transfer buffer. Blocking to prevent non-specific binding of antibodies was
conducted using TBST+2% dry milk. This was followed by a stain of primary
anti-rabbit antibodies for the Kai proteins provided by the Rust Lab. The
membranes were washed using wash buffer then stained with anti-goat HRP
secondary antibody allowing us to visualize the primary antibody stain. The
membranes were washed then imaged using chemilumenescnece
imaging systems.
Imaging and Quantification
Densitometry of images was conducted in ImageJ.
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