Cell Systems, Volume
15
Supplemental information
SEMPER: Stoichiometric expression of mRNA
polycistrons by eukaryotic ribosomes
for compact, ratio-tunabl
e multi-gene expression
Mengtong Duan, Ishaan Dev, Andrew Lu, Goar Ayrapetyan, Mei Yi You, and Mikhail G.
Shapiro
1
SUPPLEMENTARY INFORMATION
Figure S1: mCherry distribution plots for 2
-
ORF and 3
-
ORF
f
luorescent
p
rotein
SEMPER libraries.
Acting at the level
of each mRNA, t
his IRES mCherry normalization strategy aim
ed
to account for variability in transfection and transcription that
could
occur between tested constructs.
A
) mCherry distributions for 2
-
ORF SEMPER constructs (for expression of
msfGFP[r5M] and mEBFP2) after transfection in HEK293T cells.
B
) mCherry distributions for 2
-
ORF SEMPER constructs (for
expression of msfGFP[r5M] and mEBFP2) after transfection in CHO
-
K1 cells.
C
) mCherry distributions for 3
-
ORF SEMPER
constructs (for expression of msfBFP[r5M], msfGFP[r5M], and emiRFP670) after transient transfection in HEK293T cells.
D
)
mCherry distributions for 3
-
ORF SEMPER constr
ucts (for expression of msfBFP[r5M], msfGFP[r5M], and emiRFP670) after
transient transfection in CHO
-
K1 cells. mCherry distributions for CHO
-
K1 cells vary less than those
for
HEK293
T
, suggesting
differences in transfection efficiency and/or burden
.
However,
despite this variation
,
the rank orders of
ORF
expression ratios
and other
trends of
mCherry
-
normalized
expression titration
were
consistent
between the two cell types
in
Figure
s 1, 2, S5,
and S9
for various TIS combinations tested
.
Each violin plot cont
ains data from four combined
biological
replicates of the
depicted TIS combination.
The numbers of cells in each distribution are reported in
Table S1
A
-
D.
The
median
and quartiles
of the distribution are represented by the solid and dotted lines respectively
.
2
Figure S2: HEK293T 2
-
ORF compensation controls.
Related to Figure 1.
A
) Flow cytometry plots for single
-
color control
plasmids after transient transfection and compensation. Compensation was conducted using FlowJo. Depicted are cells that
have been gated for by size and then doublet
-
discriminated. Comp
-
BFP
-
A, Comp
-
GFP
-
A, and Comp
-
Cherry
-
A, represent the
channels used for mEBFP2, msfGFP[r5M], and mCherry
respectively
. mCherry and msfGFP[r5M] single
-
color controls
showed some, yet not substantial, fluorescenc
e crossover into the Comp
-
BFP
-
A channel. Measurements were taken using a
MACSQuant VYB cytometer
(
n
=
1).
B
) Flow cytometry plots for pUC19 plasmid transfections containing no fluorescent
marker. Minimal fluorescence was observed in all channels
(
n
=
1)
.
3
Figure S3: CHO
-
K1 2
-
ORF compensation controls.
Related to Figures 1 and S5.
A
) Flow cytometry plots for single
-
color
control plasmids after transient transfection and compensation. Compensation was conducted using FlowJo. Depicted are
cells that have been gated for by size and then doublet
-
discriminated. Comp
-
BFP
-
A, Comp
-
GFP
-
A, and Comp
-
Cherry
-
A,
represent the channels used for
m
EBFP2,
m
sfGFP[r5M], and mCherry
respectively
. Minimal to no fluorescence spillover was
observed in the Comp
-
BFP
-
A channel for cells transfected with
m
sfGFP[r5M] or mCherry sing
le
-
color controls. Measurements
were taken using a MACSQuant VYB cytometer
(
n
=
1)
.
B
) Flow cytometry plots for pUC19 plasmid transfections containing
no fluorescent marker
(
n
=
1)
.