CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli
Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites.
By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated.
The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.
KeywordsSynthetic biology Chromosomal integration CRIM plasmids MoClo CRIMoClo
Synthetic Biology aims at applying engineering principles to biological systems , yet the complexity of living cells often leads to unpredictable behavior of heterologous genetic circuits. For instance, the expression of synthetic circuits from medium- or high-copy plasmids in E. coli can lead to toxic side effects on the chassis cell, as being the result, e.g., of competition for essential cellular resources, such as metabolites, RNA polymerase or ribosomes . Thus, reducing such undesired cross-reactions with the host are among the key factors for the success of a rational, model-driven design of novel synthetic circuits. While this has led to the development of a number of orthogonal (i.e. context independent) regulators for synthetic circuit construction [3, 4, 5], high plasmid copy numbers can still generate growth defects, as e.g. in the case of the expression of membrane-anchored anti-σ factors regulating the activity of orthogonal extracytoplasmic function (ECF) σ factors .
To circumvent these issues, different methods for integrating DNA from plasmids into the E. coli chromosome have been developed. For instance, recombineering-based strategies [17, 18], such as KIKO vectors , facilitate the integration of large circuits in E. coli . However, these strategies often rely on traditional restriction digestion and ligation, which limit the speed of circuit construction and do not allow for recycling of genetic parts. Recently, an innovation was developed by Schindler et al., providing a series of MoClo-compatible vectors that facilitate lambda Red-based integration . However, recombineering-based strategies tend to suffer from another limitation, which is the lack of well-characterized orthogonal loci. For instance, it was shown that protein expression and metabolite production in E. coli may be influenced by the location of their integrations sites on the genome  and even though five novel open reading frames were identified as suitable integration loci for synthetic circuits in E. coli, the integration efficiency and expression of genetic constructs in these loci varied significantly .
An alternative way to perform chromosomal integrations is based on bacteriophage integrases . A prime example embarking on this strategy is implemented in the “conditional-replication, integration, and modular” (CRIM)-based plasmids, which carry different phage attachment (attP) sites and can be used to insert large DNA fragments at bacterial phage-attachment (attB) sites (Fig. 1b). The site-specific recombination is driven via expression of phage-derived integrase (int) gene encoded on a helper plasmid. The integration procedure is simple, requiring only the co-transformation of the bacterial strain with a CRIM plasmid, together the cognate helper plasmid, and a temperature shift that induces expression of the integrase. Since the helper plasmid is temperature sensitive for replication, the integration of the CRIM and the cure of the helper plasmid occur simultaneously . Moreover, CRIM plasmids possess the γ conditional origin of replication of R6K that depends on the trans-acting π protein (encoded by pir) for replication. Hence, successful integration events can easily be selected by transforming a pir− host with CRIM plasmids under antibiotic selection. These characteristics make CRIM plasmids a fast and reliable strategy for chromosomal integration in E. coli, and even though the system was further improved , so far CRIM-based integration methods lacked standardization, limiting the speed of DNA assembly/integration and not enabling for the reusability of genetic parts.
To fill this gap here we combine the standardization and modularity of the MoClo system, with the high integration efficiency of CRIM plasmids, generating a set of CRIMoClo plasmids (Fig. 1c). We benchmark their suitability for synthetic biology approaches assembling synthetic circuits from reusable Level 0 parts and showing the orthogonality of the four phage attachment sites and four different resistance cassettes. Further, we present a strategy that facilitates the sequential integration of different inducible reporter systems at the four phage attachment sites, showing the modularity and the efficiency of the framework. Finally, we use our fast and reliable assembly/integration strategy to perform a large multi-part assembly (∼10 kb) and integration of synthetic ECF-σ/anti-σ switches, showing that growth defects observed for circuits encoded on medium-copy plasmids are abolished.
Results and discussion
Predicted PCR product sizes for attB sites, using primers P1-P2-P3-P4. Successful integration events at each attB site are revealed by two fragments generated by P1-P2 and P3-P4 (highlighted in green). Recombinants with two (or more) CRIMoClo plasmids at the attB site show in addition a third fragment generated by P2-P3. False positive (non-integrants) are revealed by the PCR product generated by P1 to P4 (highlighted in red)
Based on this design we created a combinatorial set of 32 CRIMoClo plasmids featuring all permutations of four phage attachment sites (attHK022, attP21, attϕ80, attλ), four resistance cassettes (chloramphenicol, kanamycin, spectinomycin, gentamicin) and compatibility with two MoClo Levels (M and P). In theory, this now enables efficient assembly and chromosomal integration of synthetic genetic circuits in only 4 days, starting from Level 1 transcription units (see below). Moreover, the availability of each plasmid with one of four resistance cassettes should allow, in principle, sequential integration in different att sites without the necessity of recombining the resistance cassette after each integration step.
Insulation and robustness of gene expression at attB sites
As a further test to compare gene expression levels from the different loci, we fused the lux operon to two additional inducible promoters, Ptet  and PLlac0–1 , and integrated each of these reporter constructs into the four att sites. The reporter constructs also contain expression cassettes encoding the relative repressors (TetR and LacI, respectively), transcribed in divergent orientation to the Ptet and PLlac0–1 promoters, respectively. For both promoter-lux fusions, quantification of reporter activity showed that the luciferase signals were independent of the integration locus, and varied in a concerted manner with the inducer level, i.e. anhydrotetracycline (ATc) for Ptet and isopropyl-β-D-thiogalactoside (IPTG) for PLlac0–1 (Additional file 1: Figure S2). Concluding, our results show that integration into the four att sites leads to highly reproducible gene expression behavior, suggesting that these sites can be used as interchangeable and orthogonal loci for chromosomal integration of synthetic circuits.
Since CRIMoClo plasmids exist with four different resistance cassettes (Fig. 2a), we wanted to test the potential influence of the resistance cassettes on reporter activity after chromosomal integration. To this end, we integrated and measured the expression level of PBAD-lux integrated into all selected att sites, using CRIMoClo plasmid possessing different selection markers (Fig. 4b and Additional file 1: Figure S3-S5). The results show again virtually no difference in the expression levels, suggesting that also the different resistance cassettes can be used interchangeably.
Finally, we assayed the genomic stability of the integration by using four strains harboring PBAD-lux constructs integrated into the four phage attachment sites (Fig. 3a). Additional file 1: Figure S6A shows that all the colonies of the four strains are luminescent when streaked onto LB plates supplied with 0.2% arabinose, in absence of antibiotic selection. Moreover, Additional file 1: Figure S6B shows that the same strains are still luminescent after being precultured for 7 days in absence of antibiotic selection.
Overall, these results show that the four phage attachment sites are well-insulated from their genomic context and that the different positions and orientations of the transcription units on the chromosome do not influence the dynamics of reporter gene expression. Moreover the four selected att sites guarantee a stable integration of a given genetic construct even in absence of antibiotic selection. Therefore, our experiments demonstrate orthogonality of the four phage attachment sites of the CRIMoClo system and verify the stability of integrated constructs, which makes them ideally suited for synthetic biology applications.
Application of CRIMoClo plasmids to minimize heterologous expression burden
Genetic circuits encoded on plasmids can generate undesired effects on cellular physiology [36, 37]. For instance, plasmid maintenance, as well as high expression of heterologous genetic constructs, can cause a metabolic burden to the cell, and therefore toxicity [38, 39, 40]. Hence, lowering the copy number of the construct using low copy number plasmids or via chromosomal integration is often a solution. Yet, the size of the genetic circuits can often limit the efficiency of chromosomal integration and even though there are strategies that facilitate the integration of large genetic circuits [18, 24], they generally do not support easy, modular circuit generation.
One of the aims of synthetic biology is the design of complex genetic pathways. In such pathways, optimizing the balance in production of different pathway components is often a key step to reach the desired output product. To do so, one of the strategies consists in adjusting the transcription and the translation rate of each gene in the pathway. Using a combinatorial framework like the MoClo system allows for the generation of a library of interchangeable parts (such as promoters, ribosome binding sites, and transcriptional regulators), that can be combined in several permutations in order to find the right balance of gene expression levels that feature functional synthetic circuit activity. With the aim of increasing the spectrum of possible permutations in synthetic circuit generation, the CRIMoClo system designed here represents an additional degree of freedom to the possible configurations of a given synthetic circuit. In particular, the system supports the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites in the genome of E. coli. By combining the properties of the CRIM plasmids with those of the MoClo system, the framework allows for easy generation and rapid integration of large synthetic constructs. In the present work we have assembled and integrated constructs up to ~ 10 kb with high efficiency. Given that under natural conditions the recombination machinery is capable of integrating typical phage genomes of ~ 50 kb, the manipulation and integration of larger synthetic circuits should, in principle, be possible. However, at such sizes a finite plasmid stability and other factors might limit transformation and thus integration efficiency, and further studies are required to probe the upper limits of the approach.
Utilizing four different resistance modules, CRIMoClo system facilitates fast and reliable multiple integrations. Moreover, the modular design of CRIMoClo plasmids allows for an easy, further expansion of the system for compatibility with other Modular Cloning-based frameworks, such as CIDAR MoClo , Start-Stop Assembly , Loop Assembly  and Mobius Assembly . With these features the CRIMoClo system brings the combinatorial assembly to the next step, enabling a seamless transition between plasmid-encoded and chromosomally integrated genetic circuits. Finally, with the CRIMoClo system, it is possible to generate and simultaneously utilize a combination of plasmid-borne and chromosomally integrated genetic modules. Therefore, when used together, the different gene copy numbers implemented in the MoClo and CRIMoClo systems enable adjustment of gene expression levels over wide ranges, thereby facilitating, e.g., the optimization of enzyme expression levels in a biosynthetic pathway in order to maximize downstream product formation.
Materials and methods
Bacterial strains and growth conditions
E. coli strains used in this study are listed in Additional file 2: Table S1. The strains were cultivated in LB (LB Broth Miller, Sigma Aldrich Cat.No. L3522) medium or MOPS minimal medium (TEKNOVA Cat.No. M2106; 0.5% glycerol as carbon source) at 37 °C shaking at 250 rpm. To maintain plasmids, the following antibiotics were used: chloramphenicol at 25 μg/ml, kanamycin at 50 μg/ml, spectinomycin at 100 μg/ml, gentamicin at 10 μg/ml. For the selection of single-copy integrants, antibiotics were added as follows: chloramphenicol at 6 μg/ml, kanamycin at 10 μg/mL, spectinomycin at 35 μg/ml, gentamicin at 5 μg/ml. For the blue-white screening, LB plates containing isopropyl β-D-1-thiogalactopyranoside (IPTG) at 0.1 mM and 5-Bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) at 40 μg/ml were used.
Molecular biology techniques
Oligonucleotides were provided by Sigma-Aldrich. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs) or Taq DNA Polymerase (New England Biolabs). Reaction mixtures were purified using the E.Z.N.A. Cycle-Pure Kit (Omega Bio-Tek). For gel extraction, Zymoclean Gel DNA Recovery Kit (Zymo Research) was used. Type IIs restriction enzymes (BpiI and BsaI) and T4 DNA ligase were purchased from Thermo Scientific. DNA sequence verification was performed by Eurofins Genomics. Transformation of different chemically competent E. coli strains was performed according to the Inoue method  or using the transformation and storage solution (TSS) technique  (see below).
CRIMoClo vector construction
CRIMoClo vectors generated in this study (Additional file 3: Table S2.1) were assembled using Ligase Cycling Reaction (LCR)  and Gibson Assembly . For the construction of the first 8 CRIMoClo vectors (Level M and P with chloramphenicol resistance and 4 att sites), DNA fragments were PCR-amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) with the primers listed in Additional file 4: Table S3. In particular, the tL3 terminator together with one of four different phages attachment sites (attHK022, attP21, attϕ80, attλ) was amplified using the universal forward primer GF0524 in combination with the reverse primers GFC0525, GFC0526 and GFC0530, using CRIM plasmids pAH68, pAH81 and pAH153  as templates, respectively. The amplification of attλ from pAH120  was performed in was performed in two steps, using primers GFC0524/GFC0527 and GFC0528/GFC0529 to remove an undesired BpiI restriction site. The γ conditional origin of replication of R6K was amplified from pAH68  using the primers GFC0531/GFC0532, while the chloramphenicol resistance cassette was amplified from pKD3  using the primers GFC0533/GFC0534. The rgnB terminator was amplified from pAH68  using the primers GFC0535/GFC0536. Finally, the MoClo multicloning region was amplified with the primers GFC0537/GFC0538 using pSVM-mc  and pICH82094  as Level M and Level P templates, respectively. The generated fragments (blunt-end and 5′ phosphorylated) were fused via Ligase Cycling Reaction (LCR) according to de Kok et al. . In particular, a reaction mix of 0.3 U Taq DNA Ligase (New England Biolabs), 3 nM DNA parts, 30 nM bridging oligos (Additional file 4: Table S3), and 8% (v/v) DMSO was used under the following assembly protocol: 2 min at 94 °C, then 50 cycles of 10 s at 94 °C, 30 s at 60 °C, and 60 s at 65 °C, followed by incubation at 4 °C. The newly generated set of plasmids (Additional file 3: Table S2.1) were used as templates for the assembly of the next 8 CRIMoClo plasmids featuring the kanamycin resistance cassette, using Gibson Assembly . In particular, the backbones from pSV004, pSV006, pSV008, pSV077, pSV016, pSV018, pSV080 and pSV079 (Additional file 3: Table S2.1) and the kanamycin cassette from pSVM-mc  were PCR-amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) using the primers GF0807/GF0532 and GF0805/GF0808. The generated fragments were fused via Gibson Assembly , setting a reaction in which 50 ng of backbone DNA (0.03 pmol) were mixed with 0.09 pmol of insert (represented by the resistance cassette) and Gibson Reaction Mix (New England Biolabs) in a final volume of 20 μl. The Gibson Assembly was performed for 1 h at 50 °C. The resulting plasmids were then used as templates to generate eight gentamicin and eight spectinomycin resistant CRIMoClo plasmids, using Gibson Assembly . In particular, the backbones pSV125, pSV126, pSV127, pSV128, pSV219, pSV220, pSV221 and pSV222 were PCR amplified using Q5 High-Fidelity DNA and the primers GF0945/GF0532 in order to maintain the promoter of the kanamycin cassette. These fragments were then fused with the spectinomycin and gentamicin coding sequences (amplified from pMA333  and pABC2  using the primers GF0858/GF0947 and GF0856/GF0949, respectively) via Gibson Assembly, following the protocol described above. The plasmid maps of all 32 CRIMoClo vectors can be found in Additional file 5.
Modular cloning (MoClo) reactions
The integrative CRIMoClo-based plasmids generated in this study (Additional file 3: Table S2.2) were assembled on CRIMoClo vectors, using MoClo-compatible parts listed in Additional file 3: Table S2.2. MoClo reactions were set up using 15 fmol of each DNA part (PCR product or plasmid), 1 μl of the required restriction enzyme (BsaI or BpiI), 1 μl of T4 DNA Ligase (5 U/μl) and 2 μL of T4 DNA Ligase buffer (10x), in a final reaction volume of 20 μL. The reaction was incubated in a thermocycler for 5 h at 37 °C, 10 min at 50 °C and 10 min at 80 °C. Then 2 μl of the reaction mixture was added to 50 μl chemically competent DH5α λpir cells, incubated for 30 min on ice and transformed by heat shock. This was followed by adding 950 μl of liquid LB to the transformation, and cells were recovered for 45 min at 37 °C. Finally, 40 μl of the transformation was plated on selective LB plates and emerging colonies were verified by colony PCR and restriction digestion.
CRIMoClo plasmid integration using competent cells pre-transformed with the helper plasmid
The integration of the CRIMoClo plasmids was performed similarly as described by Haldimann and Wanner . In particular, 2 μl of purified plasmid was added to 50 μl chemically competent E. coli SV01 cells, carrying one of the CRIM helper plasmids (pAH69, pAH121, pINT-ts and pAH123 ). The cells were incubated for 30 min on ice and transformed by heat shock. Then 950 μl of liquid LB was added to the transformation mix, and cells were incubated at 37 °C for 1 h and at 42 °C for 30 min (to induce the phage-derived integrase (int) gene and simultaneously cure the helper plasmid). Then 80 μl of the transformation mixture were spread onto selective agar plates and incubated at 37 °C overnight. Colonies were tested by colony PCR using the primers P1–P2–P3–P4 (Table 1 and Additional file 4: Table S3), pre-cultured once in non-selective medium and then tested for antibiotic resistance for stable integration and loss of the helper plasmid.
CRIMoClo plasmid integration using TSS competent cells
As an alternative way to achieve chromosomal integration (e.g. in the multi-integration experiment) we prepared competent cells using the TSS method . A single clone of E. coli SV01 was picked from LB agar plates and pre-cultured in 3 ml LB media at 37 °C, shaking at 250 rpm. When the OD600 reached 0.5–0.8, cells were chilled in ice for 10 min and then 500 μl of cell culture were mixed with 500 μl of TSS 2x and left in ice for 45 min. Subsequently, 50 μg of purified CRIMoClo-based plasmid and 50 μg of the cognate helper plasmid were added to the cell mixture and left in ice for 45 min, followed by 1 h at 30 °C and 30 min at 42 °C shaking at 250 rpm. Then 200 μl of the cell culture was plated on selective plates and grown overnight at 37 °C. In the case of strains possessing multiple resistance cassettes, we selected only for resistance encoded by the latest integrated construct. The obtained colonies were tested by colony PCR using the primers P1–P2–P3–P4 (Table 1 and Additional file 4: Table S3), pre-cultured once in non-selective medium and then tested for antibiotic resistance for stable integration and loss of the helper plasmid.
Integration stability assay
To assay the stability of the PBAD-lux construct integrated into the four selected att sites, the strains GFC0214, GFC0216, GFC0218, GFC0500 (Additional file 2: Table S1) were streaked onto an LB agar plate without antibiotic selection. The same strains were also streaked onto LB agar plates supplied with 0.2% arabinose to induce luciferase expression. The plates were grown overnight at 37 °C and subsequently screened for light production imaging them using a BioRad Chemidoc MP imaging system, in presence and absence of bright light (Additional file 1: Figure S6A). To assay the stability of the PBAD-lux construct integrated into bacterial genome, a single colony of each strain was grown in liquid LB medium without antibiotic selection for 7 days, re-inoculating the cultures in fresh medium every 24 h. Aliquots of the liquid cultures (5 μl) were then streaked onto LB agar plates and LB agar plates supplied with 0.2% arabinose (both without antibiotic selection) and grown overnight at 37 °C. Subsequently, the plates were screened for luciferase activity as described above (Additional file 1: Figure S6B).
Microplate reader assays
Microplate reader assays were performed as follows. For each E. coli strain, a single bacterial colony was picked from selective plates and grown in liquid LB medium until stationary phase (37 °C shaking at 250 rpm; 7–8 h). These pre-cultures were diluted 1:6000 into MOPS minimal medium and grown overnight (37 °C shaking at 250 rpm) until they reached an OD600 of 0.5–0.6. The cultures were then diluted to an OD600 of 0.05 in fresh MOPS minimal medium (see above) and 100 μl of culture were loaded in the wells of a black 96-well plate (GREINER catalog no.: 655097). Using a Tecan Infinite F200 pro microplate reader the plate was incubated for 10 h (37 °C with shaking) and OD600 as well as luminescence and fluorescence were measured every 5 min (10 min in case of multiple fluorescence/luminescence measurements). For switching the circuits from the OFF to the ON state, after 2 h of incubation cells were induced with the appropriate inducer (arabinose, ATc, IPTG) at the concentrations indicated in the Figures and incubation was resumed.
Luciferase bleed-through correction
The luciferase activity for each replicate in Fig. 3, Fig. 4, Fig. 5 and Fig. 6 was determined as follows. First, the raw luminescence data obtained from microplate reader measurements were background-corrected by subtracting luminescence values obtained from a control well containing the growth medium alone. Then we corrected for luminescence bleedthrough (i.e. light-scattering) from neighboring wells on the microplate, by using a de-convolution algorithm developed in our group . Last, the resulting values were divided by the optical density at each time point during the course of the experiment, which yields the luciferase activity in relative luminescence units per OD600 (RLU/OD).
We would like to thank Anna Staroń and Calin C. Guet (IST Austria) for sharing the CRIM plasmids and Torsten Waldminghaus (Synmikro, Marburg) for many helpful discussions and for providing us with E. coli DH5α λpir cells. We also thank Johannes Döhlemann, Doreen Meyer and Anke Becker (Synmikro, Marburg) for discussing the LCR strategy and sharing the pABC2 plasmid .
SV performed the experiments and analyzed the data. SV and GF conceived the research and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by a grant from the ERA-SynBio program via the Federal Ministry of Education and Research (Germany; grant 031L0010B) and the LOEWE program of the State of Hesse (Germany).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- 32.Bertram R, Hillen W. The application of Tet repressor in prokaryotic gene regulation and expression. Microb Biotechnol. 2008;1:2–16.Google Scholar
- 44.Im H. The Inoue method for preparation and transformation of Competent E. coli: “ultra competent” cells. BIO-PROTOCOL. 2011;1:1–7.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.