Host Independent Replication and the Phi 29 system

One of the main constraints in genetically engineering novel and unconventional organisms is the need to identify vectors that work in these organisms. Broad host range plasmids can be replicated in a wider range of bacteria, but are often limited to a specific group of organisms and, due to their size, present a great resource burden to the host. Furthermore, it is quite unpredictable how or whether these plasmids will function for every new species they are transformed into. Genome integration is a commonly used alternative, but it is strain-specific and not as robust. Based on the concept of orthogonal replication, we have turned to the Phi 29 bacteriophage replication system to develop a plasmid that can replicate independently of the host’s machinery.

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...