Precise and reliable gene expression via standard transcription and translation initiation elements

Journal name:
Nature Methods
Volume:
10,
Pages:
354–360
Year published:
DOI:
doi:10.1038/nmeth.2404
Received
Accepted
Published online

Abstract

An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ~93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

At a glance

Figures

  1. Rules for regularizing gene expression.
    Figure 1: Rules for regularizing gene expression.

    (a) We defined an expression operating unit (EOU) to set boundaries and junctions of functional genetic elements underlying the expression of heterologous genes (Supplementary Note). The variable regions within each element type (wider icons) and the standard junctions (labeled lines) between elements that best enable reliable reuse of elements in novel combinations are detailed. The bicistronic design (BCD) with its two Shine-Dalgarno motifs (SD1 and SD2) is shown. (b) Rank-ordered library of constitutive promoters that encode an expected common +1 mRNA boundary and 5′ UTR leader sequence. a.u., arbitrary units. (c) Rank-ordered library of SD2 sites that adhere to the BCD and resulting BCD:GOI junction as established here. Error bars, s.d. (n = 3).

  2. Standard translation initiation elements using a bicistronic design are reliably reusable.
    Figure 2: Standard translation initiation elements using a bicistronic design are reliably reusable.

    (a) Gene expression via a regularized medium-strength promoter (Ptrc; asterisk indicates an absent operator sequence) and 22 monocistronic design (MCD) 5′ UTRs of varying expression strength. Eight GOIs coding for a total of 14 chimeric reporter fusions with either gfp or rfp (columns) are shown. The 14 chimeric reporter GOIs are encoded via the first 36 nt of the N-terminal coding sequences of lacI, araC, rfp, gfp, tetR and genes encoding putative cellulase (Cell), phosphomevalonate kinase (PMK) and penicillin acylase (PA) and via the full-length coding sequence of tetR (Online Methods). Variance in mean-centered log2 expression (left) from each MCD across all GOIs sequences (right) and average Spearman rank correlations (bottom) as given (Supplementary Fig. 8). a.u., arbitrary units. (b) The same SD sequences used in a encoded within bicistronic designs (BCDs). Rank orderings for a and b were established via data of b. Variance in mean-centered log2 expression from each BCD across all GOIs (right) and average Spearman rank correlations (bottom) as given (Supplementary Fig. 6). (c,d) Analysis of variance (Online Methods) in total protein synthesis levels realized using the MCDs (c) or BCDs (d). (e) Comparison of absolute GFP synthesis ranges produced using MCDs or BCDs across all tested GOIs. (f) Predicted hybridization free energies between 16S rRNA and SD sequences are better correlated to expression for BCDs than that for MCDs (Supplementary Figs. 11 and 12).

  3. Bicistronic designs (BCDs) retain functional reliability with alternate transcription systems and different leader cistrons.
    Figure 3: Bicistronic designs (BCDs) retain functional reliability with alternate transcription systems and different leader cistrons.

    (a) Correlated gene expression levels from BCDs with an E. coli Ptrc* promoter (x axis) or bacteriophage T7 (y axis) promoter and RNA polymerase. The asterisk indicates that the promoter has no operator sequence and hence is constitutive in expression. a.u., arbitrary units. (b) Correlated gene expression levels from a phage T7 transcription system but with two GOIs. (c) Rank-ordered GFP expression for BCDs (WT-SD1-BCD) compared to expression for those in which SD1 is disrupted (Null-SD1-BCD, schematic). (d) Correlated expression levels from an E. coli promoter but with stop or rare codons inserted in the BCD leader cistron (schematic) across SD2 elements of different expression strengths (x axis, clustered groupings). Error bars, s.d. (n = 3).

  4. Precise and reliable gene expression via standard transcription-control and translation-initiation elements.
    Figure 4: Precise and reliable gene expression via standard transcription-control and translation-initiation elements.

    (a) Standard promoters produce mRNA from a common +1 nucleotide position. Translation initiation is entirely encoded by a separate and independent bicistronic design (BCD). (b,c) Mean-centered log2 expression for green (b) and red (c) fluorescent proteins via a full combinatorial library of standardized promoters (14) and BCDs (22). a.u., arbitrary units. (d) Direct correlation of expression from b and c (red circles) against those generated by use of irregular transcription- and translation-control elements (blue diamonds, data from ref. 15). (e) Factorial analysis of variance for mean-normalized expression from the standard promoter and BCD combinatorial library, with element- and junction-specific contributions to total expression as noted (Online Methods). (f) Correlation of observed versus predicted protein expression for sequence-distinct GOIs, as predicted using expression data from a single GOI (GFP) to estimate activity scores for promoters and BCDs adhering to method for forward-engineering gene expression developed here. Error bars: y axis, s.d. (n = 3); x axis, deviations in predicted values derived from the cross-validated model (Online Methods). Cellulase, putative cellulase; PMK, phosphomevalonate kinase; PA, penicillin acylase.

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Author information

  1. These authors contributed equally to this work.

    • Adam P Arkin &
    • Drew Endy

Affiliations

  1. BIOFAB International Open Facility Advancing Biotechnology, Emeryville, California, USA.

    • Vivek K Mutalik,
    • Joao C Guimaraes,
    • Guillaume Cambray,
    • Colin Lam,
    • Marc Juul Christoffersen,
    • Quynh-Anh Mai,
    • Andrew B Tran,
    • Morgan Paull,
    • Jay D Keasling,
    • Adam P Arkin &
    • Drew Endy
  2. Lawrence Berkeley National Laboratory, Physical Biosciences Division, Berkeley, California, USA.

    • Vivek K Mutalik,
    • Jay D Keasling &
    • Adam P Arkin
  3. Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.

    • Vivek K Mutalik,
    • Joao C Guimaraes,
    • Guillaume Cambray,
    • Colin Lam,
    • Marc Juul Christoffersen,
    • Quynh-Anh Mai,
    • Andrew B Tran,
    • Jay D Keasling &
    • Adam P Arkin
  4. Department of Informatics, Computer Science and Technology Center, University of Minho, Campus de Gualtar, Braga, Portugal.

    • Joao C Guimaraes
  5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA.

    • Jay D Keasling
  6. Joint BioEnergy Institute, Emeryville, California, USA.

    • Jay D Keasling
  7. Department of Bioengineering, Stanford University, Stanford, California, USA.

    • Drew Endy

Contributions

V.K.M., A.P.A. and D.E. conceived the study and designed the experiments. V.K.M., C.L., Q.-A.M., A.B.T. and M.P. performed the experiments. V.K.M., J.C.G., G.C., M.J.C., A.P.A. and D.E. analyzed the data. V.K.M., J.C.G., G.C., J.D.K., A.P.A. and D.E. wrote the manuscript. All authors discussed and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Text and Figures (8 MB)

    Supplementary Figures 1–32, Supplementary Table 1 and Supplementary Note

Excel files

  1. Supplementary Data 1 (475 KB)

    List of parts, plasmids and strains used in the present work. Columns as follows: A, number; B, vector backbone; C, abstract part number for promoter element, indicated as “apFAB#”; D, promoter name; E, abstract part number for 5' UTR element, indicated as “apFAB#”; F, 5' UTR name used in the main text; G, abstract part number for GOI element, indicated as “apFAB#”; H, GOI name; I, plasmid number “pFAB#”; J, antibiotics; K, replication origin; L, strain; M, strain number “sFAB#”; N, project name.

  2. Supplementary Data 2 (135 KB)

    List of primers used in the present work. Columns as follows: A, number; B, oligonucleotide number (“oFAB#”; primers used for sequencing are denoted as “soFAB#”); C, forward and reverse primers are indicated as FW and RV; D, information notes for the primer; E, primer sequence (5' to 3'); F, project name.

Additional data