Article
SEMPER: Stoichiometric expression of mRNA
polycistrons by eukaryotic ribosomes for compact, ratio-
tunable multi-gene expression
Graphical abstract
Highlights
d
SEMPER enables the tunable expression of multiple proteins
from a single transcript
d
Up to three fluorescent proteins expressed in various
cell lines
d
Single-transcript expression of gas vesicles and recombinant
monoclonal antibodies
d
Probabilistic modeling supports SEMPER’s underlying
mechanisms
Authors
Mengtong Duan, Ishaan Dev,
Andrew Lu, Goar Ayrapetyan,
Mei Yi You, Mikhail G. Shapiro
Correspondence
mikhail@caltech.edu
In brief
Discover SEMPER, a novel method for
precise, user-defined multi-protein
expression from a single transcript.
Utilizing the leaky scanning model,
SEMPER efficiently controls protein
ratios, proven in applications for
fluorescent protein, gas vesicle, and
antibody expression. Its design and
probabilistic model optimize complex
protein assemblies in eukaryotic
systems.
Duan, Dev, et al., 2024, Cell Systems
15
, 1–13
July 17, 2024
ª
2024 The Author(s). Published by Elsevier Inc.
https://doi.org/10.1016/j.cels.2024.06.001
ll
Article
SEMPER: Stoichiometric expression of mRNA
polycistrons by eukaryotic ribosomes
for compact, ratio-tunable multi-gene expression
Mengtong Duan,
1
,
6
Ishaan Dev,
2
,
6
Andrew Lu,
1
,
3
Goar Ayrapetyan,
2
Mei Yi You,
1
and Mikhail G. Shapiro
2
,
4
,
5
,
7
,
*
1
Division of Biology and Biological Engineering, Caltech, Pasadena, CA 91125, USA
2
Division of Chemistry and Chemical Engineering, Caltech, Pasadena, CA 91125, USA
3
UCLA-Caltech Medical Scientist Training Program, UCLA, Los Angeles, CA 90095, USA
4
Andrew and Peggy Cherng Department of Medical Engineering, Caltech, Pasadena, CA 91125, USA
5
Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
6
These authors contributed equally
7
Lead contact
*Correspondence:
mikhail@caltech.edu
https://doi.org/10.1016/j.cels.2024.06.001
SUMMARY
Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using
the leaky scanning model of translation initiation. In this approach termed ‘‘stoichiometric expression of
mRNA polycistrons by eukaryotic ribosomes’’ (SEMPER), adjacent ORFs are translated from a single
mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initi-
ation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two
different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas
vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing
cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant mono-
clonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through
in vitro
transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A
record of this paper’s transparent peer review process is included in the supplemental information.
INTRODUCTION
Mammalian cell engineering promises to enable the treatment of
age-related degeneration, reversal of genetic diseases, and
even transformation of cells into living therapeutics and sen-
sors.
1
Realizing this promise requires the development of ge-
netic circuits capable of finely tuning the relative expression stoi-
chiometries of multiple proteins to produce functional multimeric
protein assemblies, multi-component signaling systems, or
multi-enzyme biosynthetic pathways.
2–6
Current approaches to
doing so at the DNA level (e.g., varying promoter strength
7
,
8
or
titrating copy numbers of each gene
2
,
9
) yield lengthy DNA con-
structs that often must be packaged into multiple delivery vec-
tors and lead to undesirable cell-to-cell variability due to both
stochastic gene delivery and integration across the population.
Additionally, attainable protein expression stoichiometries are
limited by the transcriptional strengths of a relatively small
set of curated promoters that often demonstrate cell-to-cell
variability.
7
,
8
,
10
Post-transcriptionally, sequence motifs, such as internal ribo-
some entry sites (IRESs) or 2A self-cleaving peptides, may be
used to encode multiple open reading frames (ORFs) into a sin-
gle transcript and tune protein stoichiometries using relative
translation levels.
11
,
12
Such post-transcriptional mechanisms
are also useful for mRNA-based, multi-gene expression sys-
tems, which are of relevance to mRNA vaccine and therapeutic
development.
13
Although powerful tools, these genetic parts
have their disadvantages. For instance, IRES sequences have
a considerable genetic footprint (
200–600 bps),
14
leading to
lengthier genetic constructs that may reduce viral packaging ef-
ficiency. Likewise, 2A self-cleaving peptides leave peptide scars
and can yield undesired fusion proteins, both of which can be
detrimental to protein function.
15
In addition, when employed
in bicistronic vectors, these genetic parts can strongly attenuate
the expression of the second ORF relative to the expression of
the first ORF.
12
,
16
Here, we present an alternative approach
that enables compact and robustly tunable polycistronic expres-
sion in mammalian cells using plasmid- and mRNA-based
expression systems. We call this approach ‘‘stoichiometric
expression of mRNA polycistrons by eukaryotic ribosomes’’
(SEMPER).
SEMPER uses the canonical cap-dependent ribosome
recruitment and translation mechanism in mammalian systems,
which begins when the 43S preinitiation complex (PIC) of the
Cell Systems
15
, 1–13, July 17, 2024
ª
2024 The Author(s). Published by Elsevier Inc.
1
This is an open access article under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
).
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Please cite this article in press as: Duan, Dev, et al., SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact,
ratio-tunable multi-gene expression, Cell Systems (2024), https://doi.org/10.1016/j.cels.2024.06.001
ribosome is loaded onto the 5
0
end of mRNA.
17
,
18
This complex
then scans the 5
0
untranslated region (5
0
UTR) until it encounters
a translation initiation site (TIS), consisting of the start codon
(AUG) and
3–10 neighboring nucleotides.
19
The TIS sets the
translational reading frame and initiates translation by engaging
with the 60S ribosomal subunit. The full ribosome then translates
the mRNA into protein until it encounters a stop codon, where it
terminates translation and disengages the transcript. With some
frequency, the 43S PIC may scan through the first TIS and initiate
translation from a downstream ORF starting at another TIS, a
phenomenon called leaky ribosomal scanning (LRS).
20
As deter-
mined by its sequence, a strong TIS (e.g., the Kozak consensus
sequence) will reliably initiate translation, whereas weaker ones
will more frequently allow the 43S PIC to scan past.
21
By employing a short ORF (uORF) upstream of a gene of inter-
est (GOI), mammalian cells naturally use LRS and alternate TISs
to divert a portion of the ribosome flux away from a GOI, effec-
tively downregulating its translation.
22
Recently, Ferreira and
colleagues demonstrated that it is possible to use synthetic
uORFs to regulate the expression of a downstream recombinant
GOI.
23
They also empirically determined the strength of various
translation initiation sequences and showed that it is possible
to divert varying amounts of ribosomal flux away from the GOI
by varying the uORF TIS strength. uORFs have also been used
in synthetic systems to tune an endoribonuclease-based feed-
forward controller to manage resource limitation.
24
Others
have also utilized translation initiation strength variability to
tune the expression of a GOI in the context of modular genetic
design.
25
The SEMPER approach replaces non-protein-coding
uORFs with longer, functional coding sequences. In this study,
we show that by chaining together multiple ORFs while varying
the translation initiation strength of each ORF, this approach
achieves polycistronic expression of multiple proteins with
tunable translation rates. We further demonstrate that this frame-
work is functional in
in vitro
-transcribed (IVT) mRNA, paving the
way for advances in mRNA-based protein therapeutics of higher
complexity.
RESULTS
Tunable, plasmid-based SEMPER framework for
expressing two ORFs
To test tunable translation levels of two recombinant proteins
from single transcripts, we encoded fluorescent proteins (FPs)
with minimal spectral overlap into the first two ORFs of our
SEMPER plasmid vector (
Figure 1
A). We included 11 bps of dis-
tance between these ORFs to ensure that the read-through of
the stop codon of the first ORF would not result in a fusion con-
taining both proteins. We used the TIS sequence NNNAUG
Gto
initiate translation of our ORFs, where NNN represents one of the
following sequences: ACC, CCC, or TTT. These sequences were
described as having strong, medium, and weak translation initi-
ation efficiency respectively by Ferreira et al.
23
We engineered all
our FPs to contain a valine residue (GTG or GTT) following the
N-terminal methionine to ensure that changes in translation initi-
ation were due to the trinucleotide preceding the start codon and
not the dinucleotide succeeding it. TIS sequences will be
referred to by their variable NNN sequence. We used one other
sequence (TTTCCAT), referred to as ***, to scrub the TIS entirely
and prevent the ORF from being translated. For the first ORF,
we generated a methionine-less monomeric superfolder GFP
(msfGFP[r5M]) in which all methionines—except the N-terminal
one—were mutated to other amino acids.
26
,
27
In addition, out-
of-frame AUGs were removed from the msfGFP[r5M] coding
sequence using synonymous mutations. These mutations effec-
tively removed all internal TISs within ORF 1 that could reduce ri-
bosomal flux to downstream ORFs (
Figure 1
B). In the second
ORF, we encoded mEBFP2 with all its natural methionines.
Finally, downstream of the SEMPER ORFs, we included an
IRES followed by mCherry (IRES-mCherry) for normalization.
Because ribosomal binding to the IRES and subsequent transla-
tion of mCherry are conducted independently of ORF 1 and ORF
2 translation,
28
,
29
the mCherry allowed us to normalize single-
cell fluorescence measurements for msfGFP[r5M] and mEBFP2,
a strategy common to LRS-focused studies.
23
,
30
Because the
analyte ORFs and IRES-mCherry were encoded on the same
transcript, this normalization scheme accounted for variations
in transfection efficiency, transcription, and mRNA decay.
mCherry fluorescence also served as a proxy for transcript abun-
dance in each cell (
Figure S1
;
Tables S1
A–S1D).
After cloning various combinations of TISs in front of our two
ORFs, we transfected these ‘‘2-ORF SEMPER’’ constructs into
human HEK293T cells—a widely used research model for
mammalian cell biology. Three single-color control plasmids
with msfGFP[r5M], mEBFP2, and mCherry were also trans-
fected. We screened the transfected cells using flow cytometry,
utilizing the single-color control plasmids for compensation and
correction of fluorescence spillover emissions (
Figures S2
and
S3
). As hypothesized, our cell lines produced both msfGFP
[r5M] and mEBFP2 from single transcripts (
Figure 1
C). Further-
more, the tested TIS combinations (TIS for ORF 1/TIS for ORF
2) yielded unique relationships between the fluorescence of the
first ORF and that of the second ORF, with the relative expres-
sion of the former vs. the latter following the strength of the first
TIS. To analyze the 2-ORF SEMPER performance as a function
of transcript abundance or ‘‘copy number,’’ we binned cells
into three categories (low copy, medium copy, and high copy)
based on their mCherry fluorescence. This yielded distinct clus-
ters. We then calculated the means of msfGFP[r5M]/mCherry,
mEBFP2/mCherry, and msfGFP[r5M]/mEBFP2 for the cells in
each bin for each tested TIS combination. We conducted min-
max normalization for each ORF separately using two constructs
from our screen (***/ACC and ACC/***). We reasoned that
***/ACC would produce the maximum amount of mEBFP2 and
the minimum amount of msfGFP[r5M], as all 43S PIC flux should
bypass the first ORF and initiate translation at the second.
Conversely, we expected ACC/*** to produce the maximum
amount of msfGFP[r5M] and the minimum amount of mEBFP2.
Conducted for each mCherry bin independently, this analysis
enabled us to compare translation levels of a particular FP
across various TIS combinations relative to its hypothesized
maximum and minimum (
Figure 1
D). Pairwise statistical compar-
isons for these TIS combinations are provided in
Table S2
.
As we increased the TIS strength in front of msfGFP[r5M], we
observed increases in msfGFP[r5M] translation levels and de-
creases in mEBFP2 translation levels for all mCherry bins.
Across mCherry bins, we found that the rank order of relative
translation levels for mEBFP2 for our different TIS combinations
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Please cite this article in press as: Duan, Dev, et al., SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact,
ratio-tunable multi-gene expression, Cell Systems (2024), https://doi.org/10.1016/j.cels.2024.06.001
Low Bin
mEBF
P2/mCherr
y
msfGFP[r5M]/mCherr
y
10
-1
10
0
10
1
10
2
10
3
10
4
0.015
0.010
0.005
0
Unit Area
mEBFP2
msfGFP[r5M]
msfGFP
mCherry only
***/ACC
TTT/ACC
CCC/ACC
ACC/ACC
ACC/***
TTT/ACC
CCC/ACC
ACC/ACC
Medium Bin
High Bin
10
3
10
3
10
2
10
1
10
0
10
-1
10
3
10
2
10
1
10
0
10
-1
10
2
10
1
10
0
10
-1
10
3
10
2
10
1
10
0
10
-1
mEBFP2
mEBFP2
msfGFP[r5M]
msfGFP[r5M]
***/ACC
TTT/ACC
CCC/ACC
ACC/ACC
ACC/***
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative Translation
log10
( )
msfGFP[r5M]
mEBFP2
log10
( )
msfGFP[r5M]
mEBFP2
HEK293T
CHO-K1
B
A
C
E
D
Low
Bin
Medium
Bin
High
Bin
TIS Strength
ACC CCC
TTT ***
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
-0.2
Relative Translation
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative Translation
TIS Combination
TIS Combination
TIS Combination
TTT/ACC
CCC/ACC
ACC/ACC
-4
-3
-2
-1
0
1
2
3
4
5
p < 0.0001
p < 0.0001
p < 0.0001 p < 0.0001
p < 0.0001 p < 0.0001
TTT/ACC
CCC/ACC
ACC/ACC
-4
-3
-2
-1
0
1
2
3
4
5
Figure 1. 2-ORF SEMPER constructs demonstrate tunable, bicistronic expression
(A) Architecture of an mRNA transcript produced by transfected 2-ORF SEMPER plasmid DNA. Cap-dependent ribosomes translate ORF 1, msfGFP[r5M], or
ORF 2, mEBFP2, with frequencies dependent on the trinucleotide (NNN) upstream of each ORF. The relative strengths of the trinucleotides and TISs they
represent are depicted. *** (a.k.a. TTTCCAT) does not contain a start codon, preventing translation of the ORF. IRES-mCherry is included to normaliz
e fluo-
rescence measurements.
(B) mEBFP2 distribution plots for constructs containing different versions of monomeric superfolder GFP encoded in ORF 1. Removal of internal methi
onines from
the msfGFP leads to much stronger expression of the downstream mEBFP2. Both ORFs used the strong ACC TIS (n = 1, representative of four biological
replicates).
(C) Flow cytometry plots of mCherry-positive HEK293T cells transfected with 2-ORF SEMPER constructs. The legend contains the ORF 1 TIS and the ORF 2 TI
S
separated by a slash. (Left) Increasing the strength of the first TIS increases msfGFP[r5M] fluorescence relative to that of mEBFP2 (n = 1, representati
ve of four
biological replicates). (Right) Binning cells into three mCherry fluorescence ranges produces unique clusters of cells (n = 1, representative of fou
r biological
replicates).
(D) Min-max normalized fluorescence values for each GOI relative to ACC/*** and ***/ACC. Fluorescence measurements were first normalized by mCherry.
Cluster means were then calculated followed by min-max normalization. Error bars indicate mean ± SEM (n = 4 biological replicates). Welch’s ANOVA tes
ts were
conducted for blue and green bars independently (
p
< 0.0001 for all colors across all bins) followed by Dunnett’s T3 multiple comparisons tests. All pairwise
comparisons are provided in
Table S2
.
(E) Violin plots of log
10
(msfGFP[r5M]/mEBFP2) values for all mCherry-positive cells for four combined biological replicates of TTT/ACC, CCC/ACC, and ACC/ACC
plasmids transfected into HEK293T and CHO-K1 cell lines. Each distribution is produced from the measurements of at least 15,000 cells. The median and
quartiles of the distribution are represented by the solid and dotted lines, respectively. Statistical analysis was conducted using one-way Welch’
s ANOVA tests
(
p
< 0.0001 [left] and
p
< 0.0001 [right]) followed by Games-Howell’s multiple comparisons test to yield pairwise comparisons.
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Please cite this article in press as: Duan, Dev, et al., SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact,
ratio-tunable multi-gene expression, Cell Systems (2024), https://doi.org/10.1016/j.cels.2024.06.001
A
B
Low
Bin
Medium
Bin
High
Bin
msfBFP[r5M]/mCherry
msfGFP[r5M]/mCherry
/Ch
emiRFP670/mCherry
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Relative Translation
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Relative Translation
ACC/ACC/ACC
ACC/TTT/ACC
CCC/TT
T/ACC
TTT/ACC/ACC
TTT/CCC
/ACC
TTT/TTT/ACC
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Relative Translation
TIS Combination
TIS Combination
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Relative Translation
p = 0.0063
p = 0.0366
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Relative Translation
p = 0.0062
p < 0.0001
p < 0.0001
p = 0.0477
AC
C/ACC
/ACC
A
C
C/TTT/A
C
C
ACC/***/***
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Relative Translation
Figure 2. Scaling tunable polycistronic expression beyond two ORFs
(A) Architecture of an mRNA transcript produced by transfected 3-ORF SEMPER plasmid DNA.
(B) (Left) Min-max normalized relative translation levels for each fluorescent protein. Cells were first clustered into three mCherry fluorescence bi
ns (low, medium,
and high). Error bars indicate mean ± SEM (n = 4 biological replicates). Welch’s ANOVA tests were conducted for blue, green, and red bars independently
(legend continued on next page)
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was conserved. Additional TIS combinations were also tested in
HEK293T cells, showing that they can provide additional de-
grees of tuning in expression ratios (
Figure S4
).
To confirm generalizability across species and cell types, we
also demonstrated that the 2-ORF SEMPER constructs yielded
TIS combination-dependent relative translation levels of our
ORFs in CHO-K1 cells, a widely used Chinese hamster ovary
cell line for the production of biologics (
Figure S5
;
Table S3
).
31
Upon comparing the distributions of log
10
(msfGFP[r5M]/
mEBFP2) values between cell types (
Figure 1
E), we determined
that the three TIS combinations tested maintained the same rank
order in both HEK293T and CHO-K1 cell lines.
Scaling plasmid-based SEMPER constructs to
three ORFs
Enzymatic pathways and macromolecular assemblies are often
composed of more than two proteins or peptide subunits.
2
,
6
,
32
To determine if the SEMPER system could be scaled beyond
two ORFs to meet the needs of increasingly complex pathways
and assemblies, we screened a variety of ‘‘3-ORF SEMPER’’
constructs. Following a similar strategy to the 2-ORF system,
we first cloned and tested a methionine-less monomeric super-
folder BFP (msfBFP[r5M]).
We then encoded msfBFP[r5M], msfGFP[r5M], and
emiRFP670—a far-red FP—into a variety of 3-ORF SEMPER
plasmids, maintaining IRES-mCherry for normalization (
Fig-
ure 2
A). To conduct min-max normalization, we cloned three
plasmids where the starting TISs were removed from two of
the ORFs using the *** sequence, while maintaining a strong
TIS on the third: (1) ***/***/ACC, (2) ***/ACC/***, and (3)
ACC/***/*** (
Figure S6
). The emiRFP670 coding sequence
still encoded alternative TISs within it, yielding some observable
far-red fluorescence in constructs (2) and (3). Additionally,
we constructed and transiently transfected six other 3-ORF
SEMPER plasmids with unique TIS combinations into
HEK293T cells and measured their output using flow cytometry.
Four single-color controls (msfBFP[r5M], msfGFP[r5M],
emiRFP670, and mCherry) were used for fluorescence compen-
sation (
Figures S7
and
S8
). Following an mCherry binning strat-
egy similar to that employed for 2-ORF SEMPER constructs,
we analyzed the mCherry-normalized translation levels of our
three ORFs relative to their own theoretical maxima and minima
from (1), (2), and (3). With the tested TIS combinations, we
achieved a wide range of relative translation levels for each
ORF, demonstrating tunable co-expression of three genes
from a single transcriptional unit (
Figure 2
B, left).
Notably, two combinations, ACC/TTT/ACC and ACC/ACC/
ACC, yielded relative translation levels for msfBFP[r5M] greater
than 1.0 in HEK293T cells across all mCherry bins (
Figure 2
B,
right). We also observed relative translation values for msfBFP
[r5M] greater than 1.0 in CHO-K1 cells for TIS combinations in
both medium and high bins (
Figure S9
;
Table S5
). Consistent
with our observations, Wu et al. previously reported that the
presence of downstream ORFs can increase the translation of
upstream ones.
33
We hypothesize that constructs containing
multiple ACC TISs have a higher prevalence of translating ribo-
somes, allowing these ribosomes and subunits—with their docu-
mented helicase activity—to maintain the mRNA secondary
structure in an open conformation that is more amenable for ri-
bosomal scanning, translation initiation, and subsequent protein
expression.
34
However, more work is required to uncover the
exact mechanisms that elicit this observed phenotype. For mul-
tiple TIS combinations, we found that the second and third ORFs
achieved relative translation levels greater than 50% while still
producing substantial levels of upstream ORF products. These
results suggest that there may be sufficient ribosomal flux to
effectively scale the SEMPER paradigm to 4+ ORFs.
Based on the SEMPER mechanism, the relative expression of
ORFs is expected to be tunable regardless of their order in the
construct, although the exact expression levels could be
affected by any resulting differences in RNA structure and stabil-
ity. To examine this possibility, we interchanged the first and sec-
ond ORFs (both of which lack methionines) in our 3-ORF con-
structs and found that the expected rank of relative expression
was preserved (
Figure S10
). However, the exact expression
levels varied with different ORF ordering, suggesting that some
iterative tuning is required to achieve specific target values.
Producing gas vesicle ultrasound reporters using 2-ORF
SEMPER
Next, we set out to demonstrate that 2-ORF SEMPER constructs
could be applied to encoding a multimeric protein complex by
expressing gas vesicles (GVs) using mammalian acoustic re-
porter genes (mARGs).
9
Originally evolved in prokaryotes, GVs
were recently introduced as genetically encodable reporters
for ultrasound imaging, enabling the noninvasive imaging of dy-
namic cellular processes in living organisms.
2
,
35
A single GV is
made up of many GvpA structural units that are assembled
together in a helical pattern through the cooperative activity of
six heterologous assembly factors and minor constituents,
referred to collectively as GvpNJKFGW.
36
,
37
Using a two-vector
system, our group has previously expressed GVs in mammalian
cells by co-transfecting one plasmid encoding the structural unit
upstream of IRES-mCherry (pgvpA-IRES-mCherry) along with
another plasmid encoding the assembly factors and a
terminal Emerald GFP (EmGFP), all linked together by P2A ele-
ments (pgvpNJKFGW-EmGFP). Additionally, we have found
that co-transfecting the pgvpA-IRES-mCherry in excess of
pgvpNJKFGW-EmGFP improves acoustic contrast.
9
Likewise,
Anabaena flos-aquae
—the organism from which mARGs used
in this study are derived—contains more copies of gvpA relative
to the other gvps in its GV gene cluster.
38
Using the 2-ORF SEMPER strategy, we cloned single-vector
systems for producing GVs in mammalian cells. We refer to these
constructs as SEMPER mARGs. Because gvpA does not contain
any internal methionines (iMet), we encoded it directly into the
first ORF. We tested our panel of TIS sequences in front of
gvpA while maintaining the strong ACC TIS in front of the
(
p
< 0.0001 for all colors across all bins). Post-hoc Dunnett’s T3 multiple comparisons tests are provided in
Table S4
. (Right) Comparisons of msfBFP[r5M] relative
translation levels between the max control and those TIS combinations with multiple ACC driven ORFs. Error bars indicate mean ± SEM (n = 4 biological re
p-
licates). Post-hoc Dunnet’s T3 comparisons tests demonstrate a significant upregulation in relative translation of msfBFP[r5M] for depicted const
ructs compared
with that observed for ACC/***/***.
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Please cite this article in press as: Duan, Dev, et al., SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact,
ratio-tunable multi-gene expression, Cell Systems (2024), https://doi.org/10.1016/j.cels.2024.06.001