Team:TUDelft/DesignTest

Sci-Phi 29

The Sci-Phi 29 Universal Toolkit


Inspired by iGEM’s great contribution in the creation and standardization of parts, our vision is to engineer a universal toolkit which can operate efficiently and predictably in any bacterial chassis (Adams, 2016). We envision that this will encourage iGEM teams and other synthetic biologists to use a wider range of organisms in their projects, saving time and resources in developing species-specific parts, consequently unifying the efforts of Synthetic Biology into host-independent genetic engineering (Kushwaha & Salis, 2015).


In order to create a transferable genetic toolkit whose parts present predictable and standardized behavior across a wide range of bacteria, we have addressed the two main processes in molecular biology: replication of genetic vectors and gene expression. By applying the engineering concepts of orthogonality (Liu, Jewett, Chin, & Voigt, 2018) and systems control (Ma, Trusina, El-Samad, Lim, & Tang, 2009), we aim to construct synthetic devices which decouple replication and gene expression levels from the varying conditions within different bacteria.


We thus have investigated the Φ29 replication system (Blanco & Salas, 1985) for in vivo orthogonal replication to create a universal vector to replicate independently of the host’s replication machinery. We have also engineered an orthogonal incoherent feed-forward loop to achieve stabilization of expression levels across different bacterial species .

  • Why is orthogonality so great? ﹀

    Much like how software developed for one operating system may fail to run in another operating system due to system-based specificities, genetic circuits seldom function effectively when transferred across two host organisms. Orthogonal systems are the “virtual machines” of Synthetic Biology: a network of processes which are insulated and independent from the host’s own native functions . Due to the minimal interactions between the orthogonal circuit and the rest of the cell, orthogonal systems can be highly predictable and transferrable, qualities which make them very promising in universal expression tools. Furthermore, little effect on the host means these systems can be heavily engineered to exhibit new functions (Liu, Jewett, Chin, & Voigt, 2018).


    (image explaining orthogonality)

    Orthogonal DNA replication has been adapted from a natural cytoplasmic selfish plasmid of Kluveromyces lactis and implemented in multiple species of yeast (Ravikumar, Arrieta, & Liu, 2014). However, no orthogonal replication systems have been successfully engineered in bacteria (Ma, Phan, Walsh, & Ye, 2015). We believe that establishing orthogonal replication in prokaryotes would be truly beneficial to Synthetic Biology in order to create a universal genetic vector which is truly transferable across highly divergent species. The further engineering of such a system would benefit Synthetic Biology efforts of every bacterium it works in.


    Orthogonal transcription is ubiquitous in Synthetic Biology, most prominently with the T7 bacteriophage RNA polymerase (T7 RNAP) and its corresponding promoter and terminator. The T7 transcription system has been extensively explored for heterologous expression in a wide range of organisms, as well as applied in logic circuits and cell-free systems (Wang, et al., 2018). They have also been used in universal expression tools, which allowed for effective expression in a diverse set of bacteria without the need for host-specific promoters (Kushwaha & Salis, 2015; Kar & Ellington, 2018; Liang, Li, Wang, & Li, 2018). While two of these systems rely on ribosome binding site (RBS) design for calibrating gene expression (Kushwaha & Salis, 2015; Liang, Li, Wang, & Li, 2018), Kar and Ellington demonstrated the use of different T7 promoter mutants with different strengths to regulate expression in a host-independent manner (Kar & Ellington, 2018). However, these systems are still prone to variations in translation rate which result from transferring the system between bacterial species, requiring constant recalibration for similar expression levels across organisms. To address this issue, we have designed a novel T7-based incoherent feed-foward loop.


    Orthogonal translation can be separated into two processes. The first consists of orthogonal translation initiation, with the engineering of orthogonal 16s rRNA (o-ribosomes) and orthogonal RBS sequences (Darlington, Kim, Jiménez, & Bates, 2018). The second process orthogonal translation elongation, related to codon with the engineering of orthogonal tRNA-aminoacid synthetases and orthogonal tRNAs (Chin, 2014). Unfortunately, even orthogonal translation initiation, which is far simpler to implement, is not appropriate for universal expression tools: o-ribosomes and o-RBSs are designed by computationally predicting Shine-Dalgarno and anti-Shine-Dalgarno sequences which have minimum cross-talk with the host’s translation machinery, making it necessary to re-design these tools for every new organism (Chubiz & Rao, 2008). Fortunately, with our designed incoherent feed-foward loop, it is possible to insulate expression levels from inter-species translational variations without the use of orthogonal translation.



Host Independent Replication and the Phi 29 system


Mobile genetic elements will continue to be essential tools for Synthetic Biology: their flexible interchangeability across strains is crucial to reach a new multi-chassis genetic engineering paradigm (Jain & Srivastava, 2013).


One of the main constraints in genetically engineering novel and unconventional organisms is the need to identify vectors that work in these organisms. Natural broad host range plasmids can be replicated in a wider range of bacteria, but are often shown to be limited to a few specific groups of organisms, and their considerable size and complexity results in large fitness costs. Thus, substantial investigative efforts are put in the determination of host range and reduction of fitness costs of these vectors (Shintani Masaki and Suzuki, 2019).


Our team sees the potential of orthogonal systems in overcoming these issues. We thus have taken a bottom-up approach of engineering an independent DNA replication system to in live bacterial cells. We have turned to the Bacillus subtilis double-stranded DNA bacteriophage known as Φ29 (Blanco & Salas, 1985). A system such as this can be theoretically applied to any bacterium species as well as any other organism, once it only requires the expression of its own replication machinery (Liu, Jewett, Chin, & Voigt, 2018).


The Φ29 replication system has been shown to efficiently amplify DNA sequences in liposome synthetic cells with only 4 relatively small proteins in synthetic liposome cells (Nies, et al. 2018). The mechanisms of the system are explained visually and in text bellow (Blanco, Lazaro, De Vega, Bonnin, & Salast, 1994):


The linear, double-stranded DNA is flanked by two origin of replication sequences, named OriL and OriR. The terminal protein (TP) is linked to the 5’ end of each strand by a aminoacid/nucleotide covalent bond, which protects the DNA from exonucleases.




In initation, multiple double-strand binding proteins (DSB) bind along the origin sequences, changing the conformation change in the DNA and cause opening of the DNA duplex. The TP, forming a heterodimer with the single-subunit DNA Polymerase (DNAP) acts as a primer by recognizing the origins of replication. DNAP then catalyzes a covalent bond between the hydroxyl group of aminoacid Ser-232 of TP with an AMP molecule.


In elongation, DNAP is dissociates from TP and replicates DNA with high processivity, demonstrating helicase activity of strand displacement. Single-strand binding proteins (SSB) binds to the displaced single-strand for stabilization. During DNA synthesis, DNAP displaces both DSB and SSB proteins form double-strands and previously displaced single strands, respectively. When polymerization is completed, the DNAP dissociates from the DNA resulting in termination.


Reaching out to experts in orthogonal replication, we have come to learn that overexpression of the 4 proteins would not be sufficient for establishing the system in vivo, due to possible unintended interaction of the machinery with the host’s DNA. We consequently set out to find optimal concentrations of these proteins in E. coli cells. Furthermore, we explored the cell-free replication of our constructed linear DNA sequences to assess the functionality of replication the system when modified to our particular application.


  • Why do we use the phi 29 replication system? ﹀
    • With only 4 fairly small proteins and 2 replication origin sequences, the Φ29 replication machinery is quite compact and presents less burden to the cells than many replisomes (Nies, et al. 2018).
    • Φ29 is the most studied and understood double-stranded DNA bacteriophage (Blanco, Lazaro, De Vega, Bonnin, & Salast, 1994). The existing literature facilitates further engineering of our proposed plasmid in novel technologies.
    • The Φ29 DNA Polymerase has high processivity and fidelity (Blanco & Salas, 1985).
    • The Φ29 has been demonstrated a powerfull tool for synthetic cell studies (Nies, et al. 2018). In vivo replication of the system can be better engineered based on knowledge obtained with cell-free assays. Furthermore, a system which is easily transferable between in vivo and in vitro would be incredibly useful in many applications.
    • The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences (Gella, Salas, & Mencı, 2016). We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many new engineered protein-DNA complexes.
    • Orthogonal replication not only enables replication independent from the host, but the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome makes in vivo directed evolution a possibility (Ravikumar, Arrieta, & Liu, 2014).
    • The necessity of the 4 proteins for efficient DNA replication, as well as the TP-based DNA priming, make the Φ29 system a viable option of a biologically contained system, contributing to the safety of its biotechnological applications. (Torres, Kr, Csibra, Gianni, & Pinheiro, 2016).

iFFL and control of gene expression across organisms


In order to reliably implement genetic circuits or metabolic pathways in new bacterial chassis organisms, it is necessary to perform extensive characterization of genetic parts. Engineering and screening of new parts are expensive and laborious processes that need to be repeated whenever a new bacterial species is engineered. Furthermore, whenever a new synthetic system is developed in a model organism like Escherichia coli, its implementation in another bacterium requires complete rewiring and retuning with parts that function analogously in the new organism. This incompatibility arises from interspecies variations, such as copy number of plasmids, transcription rates of promoters, translation initiation rates of ribosome binding sites and the codon usage of coding sequences.


Applying again the concept of orthogonality, we approached the issue with the use of the T7 bacteriophage RNA polymerase (T7 RNAP), the most commonly used orthogonal transcription system. This system has been shown to enable host-independent transcription in a wide range of organisms1. Furthermore, multiple research groups have engineered or screened T7 promoter variants with different strengths. A wide range of T7 terminators with different termination efficiencies were also characterized. This copious amount of parts allows for the precise host-independent calibration of the expression of multiple genes. Therefore, we have investigated the implementation of T7 RNAP of a portable system consisting of a Mixed Feedback Loop (MFL), known as Universal Bacterial Expression Resource (UBER)2.


(Explain the UBER system)


  • dropdown 2 ﹀
    • With only 4 fairly small proteins and 2 replication origin sequences, the phi 29 replication machinery is quite compact and presents less burden to the cells.
    • phi29 is the most studied and understood double-stranded DNA bacteriophage. The existing literature facilitates further engineering of our proposed plasmid in novel applications.
    • The phi29 DNA Polymerase has the highest processivity of all known DNA polymerases.
    • The phi29 is widely used in in vitro and artificial cell studies. In vivo replication of the system can be better engineered based on knowledge obtained with cell-free assays. Furthermore, a system which is easily transferable between in vivo and in vitro would be incredibly useful in many applications
    • We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many innovative technologies. For DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences.
    • Orthogonal replication not only enables replication independent of the host, but the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing genomic mutations makes directed evolution a possibility.

Sci-Phi 29 Toolkit and the modular cloning of host-independent systems


We share with other iGEM teams (link to collab) the belief that Golden Gate Cloning is the future of synthetic biology. It enables parallel one-pot assembly of full transcriptional units while not requiring the ordering of primers, as parts are interchangeable and construction is highly modular. This type of assembly fits perfectly with our many other iGEM projects, where a multitude of genetic circuits with single-part changes can be easily constructed, enabling high-throughput characterization.

We have designed our collection of parts to be Type IIS compatible, and all our genetic circuits were assembled by using the MoClo Toolkit. Although being an older method of Golden Gate cloning, it was the best choice for our project. We also have expanded Golden Gate assembly in two ways: the modular assembly of 5’ UTR parts by combining different ribozyme transcriptional insulators and...

This enables independent characterization of each part, which can then be used to predict its behavior when assembled into other circuits, this being the foundation of engineering biological systems. Unfortunately, real genetic circuits often behave unpredictably when implemented in different genetic contexts, which limits Synthetic Biology as an engineering discipline. For example, engineered promoter parts may contain operator sequences downstream of the transcription start site. Although one would expect promoters...


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