Protein Degradation in Unstable Variants of mCherry
Formal Report: William Lynch
Mentor?: Yashna Thappeta
Principal Investigator: Christine Jacobs-Wagner
July 26, 2019
CJW Lab | Yale MSI
In this experiment, the degradation rate of
unique variations of the red fluorescent protein,
mCherry, were observed. This was done in order
to aid Yashna Thappeta in her research of being
able to determine if the bacteria within the gut of
a mouse is actively growing or not. In order for
her vision to be fulfilled, she desires a variant of
mCherry to be degraded as quickly as possible.
The principal behind this is that if the protein’s C-
terminal is tagged with certain assortments of
amino acids, then the protein will in turn become
a target for the ClpX system (a cellular protease).
According to Bo Andersen, et. al., different tags
will cause the proteins to be degraded at different
rates. In Andersen’s experiment, they observed
the rate at which the green fluorescent protein
(gfp), degraded based on an addition of ssRA
tags. They determined that the relative stability of
gfp would vary as they manipulated the last three
amino acids. In their experiment, they used C-
terminal extension sequences of
RPAANDENYALAA, RPAANDENYALVA,
RPAANDENYAAAV, and RPAANDENYA-
ASV. The only variations among the sequences
were the last three amino acids. Thus, they
characterized their samples as gfpmut, gfp(LAA),
gfp(LVA), gfp(AAV), and gfp(ASV).
In a similar way, mCherry was tagged
with the same C-terminal sequences in this
experiment. However, the amino acid sequence
RPAANDENYAASV was not tested due to the
aim of the experiment In Bo Andersen et. al.
gfp(ASV) had the slowest degradation time. A
similar degradation time was expected for an
mCherry protein containing the ASV ssRA tag,
thus testing was not done for it. All of the other
previously mentioned ssRA tags were tested.
Stains of Escherichia coli containing a
plasmid with the mCherry gene and different
ssRA tags, and a Chloramphenicol resistance
gene, to assert that when introduced to media
containing Chloramphenicol, only bacteria
containing the plasmid, and subsequently their
unique mCherry gene, would be present. In the
experiment, strains were categorized YT34B(mut),
YT35D(LAA), YT36D(AAV), and YT37B(LVA).
Since the goal of the experiment was to
track how quickly the protein mCherry would
degrade, Isopropyl β-D-1-thiogalactopyranoside
(IPTG) was used to induce expression within the
bacteria. The mCherry gene uses a Lac repressor
(LacI), so that when lactose is absent, the gene
will not be expressed, and thus the associated
protein will not be produced. However, when
IPTG is present, lactose is not needed to induce
this gene, yet expression will still occur due to
LacI more favorably binging to IPTG than the
repressor. Furthermore, IPTG is a desirable
substitute for lactose because IPTG is not
degraded by E. coli.
Since the relative fluorescence of
mCherry was tracked over time, Ortho-
nitrophenyl-Beta-D-Fucopyranoside (ONPF) was
used to aid in stopping the production of the
protein mCherry. ONPF works as an anti-inducer,
so that even in the presence of lactose, ONPF will
bind to the LacI-repressor complex, affirming that
transcription will not occur, and the associated
protein will not be produced.
Materials and Methods
E. coli was the bacteria used in this experiment
that expressed fluorescence.
1x M9 GluCAAT was used primarily in the culti-
vation of the variations of E. coli.
1x M9 was used as a method of managing the cell
division, ensuring that the cells do not divide, and
thus depleting their protein mCherry in a way
other than degradation via proteases.
Plasmids were used that coded for both variations
of the mCherry gene and a Chloramphenicol
resistance gene.
Beta-scope was used to image the cells.
Oufti and Matlab were programs used to analyze.!
!
Determination of ONPF
ONPF is not necessary in the process of
degrading mCherry. It is however a factor in
stopping the production of proteins within the
LacI system. Therefore, there is expected to be
some difference between how much protein is YT34 IPTG-induced expression after .25 hours
produced with ONPF versus without. When
ONPF is present, it is expected to produce less of
the protein mCherry than when it is absent. Thus
YT34B, and YT35D were tested with and without
ONPF to determine the difference between
protein production warranted the use of ONPF or
not. Although the difference between the YT34 IPTG-induced expression after 3.75 hours
expressed fluorescence with ONPF versus without Figure 2 E. coli that was imaged of YT34 over the
is relatively small compared to the expression experiment.
when induced with IPTG (see Figure 1), ONPF
was decidedly used in the experiment due to a
noticeable impact in protein production.

Figure 3 The fluorescence over time of YT34 in

Figure 1 The average fluorescence of YT35D
after a period of time inoculated in IPTG,
ONPF, and neither.
Results and Discussion
Much of this experiment was done using
the same methods as Bo Anderson et. al.
Therefore, the expected rates of degradation
among variants of mCherry were similar to the
observed degradation rates of gfp. Using this
hypothesis, YT34B was expected to show no
degradation, YT35D was expected to show rapid
degradation, YT36D was expected to show
moderate degradation, and YT37B was expected
to show rapid degradation. The fluorescence over
time is displayed in Figure 3
the presence of IPTG and ONPF.
Conclusion
In total, analyzing and quantification was
only accomplished for YT34B(mut). While the
bacteria was induced with IPTG, no surmountable
degradation was observed. However, when ONPF
was introduced to YT34, degradation was
observed at an exponential rate. This was not
expected because YT34 should not contain a
plasmid with an ssRa tag that will cause mCherry
to be degraded. A potential explanation may be
cell division, thus cleaving the relative number of
proteins in the population.