The ability to manipulate and transfer the high molecular weight DNA molecules between different chassis constitutes one of the main bottlenecks of the rational genome engineering. Bacterium Escherichia coli and plant Marchantia polymorpha are both well-characterized model organisms that are frequently used as chassis for biotechnology and synthetic biology applications. Although the vast majority of the good DNA assembly and editing tools are in E. coli, M. polymorpha is better host for certain applications. Novel tools combining the benefits of M. polymorpha chassis and the reliable DNA recombineering approaches developed for E. coli are critical for developing a robust synthetic biology toolkit. Bacterial artificial chromosomes (BACs) based on the E. coli fertility factor (F-factor) are frequently used for engineering high molecular weight DNA fragments in E. coli. We have recently engineered integrative bacterial artificial chromosomes (iBACs) that can accept virtually any high molecular weight DNA fragment for integration into B. subtilis chromosome and allow rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. This project aims to develop an IP-free system for transfer of the high molecular weight DNA from E. coli to M. polymorpha. This includes development of the plant-specific iBACs for the reliable transfer and integration of the high molecular weight DNA into the M. polymorpha genome. Alternative methods for interchassis DNA transfer, such as integrative and conjugative elements (ICEs) will be also explored.
Who we are
The project will benefit from the collaboration with the Jim Haseloff's lab at the Department of Plant Sciences of the University of Cambridge with expertise in the M. polymorpha genome modification and analysis
Collaborator from the Faculty of Law might join the project later (for IP related issues)
The main goal, to develop a reliable, IP-free and safe system for inter-chassis transfer of the high molecular weight DNA fits the remit of the SynBio Fund that encompasses development of open technologies for synthetic biology. The project fits with the existing work in our laboratory. At the Department of Pathology (University of Cambridge) we have recently developed methods (such as PFGE) for constructing and analyzing iBACs for the high molecular weight DNA fragment integration into B. subtilis chromosome. These methods will be used to engineer a reliable and IP-free iBAC- based system for the high molecular weight DNA transfer and integration into M. polymorpha genome. This project will benefit from the M. polymorpha genome modification and analysis expertise of Jim Haseloff's laboratory at the Department of Plant Biology (University of Cambridge). It will lead to tangible, publicly documented and open outcomes, including educational resource (protocol for interchassis high molecular weight DNA transfer), synthesis and sharing of useful DNA parts (engineered iBAC for transfer and integration into M. polymorpha genome will be IP-free and available to synthetic biology community) and publication in a peer-reviewed journal.
Benefits and outcomes
The main goal is to develop a reliable and IP-free system for the inter-chassis transfer of the high molecular weight DNA. This is critical for the synthetic biology community as the ability to manipulate and transfer large DNA fragments between different chassis constitutes one of the main bottlenecks of the rational genome engineering.
This encompasses three key aims:
1. Engineering IBACs (from IP-free parts) for the reliable transfer and integration of high molecular weight DNA into the M. polymorpha genome.
2. Development of the alternative (such as integrative and conjugative elements (ICEs)-based) methods for interchassis DNA transfer
3. Dissemination of the achieved outcomes (protocols and materials, such as iBACs will be open and publicly available, information will be stored on the website provided by the SynBio Fund and in a peer-reviewed journal)
The key methods used here will be:
Lambda red recombineering (for iBAC engineering)
Pulsed field gel electrophoresis (PFGE) (this method will be adapted for iBAC modifications analysis)
Phi29 polymerase-based amplification of M. polymorpha genome sequences out of crude extracts
Reagents and materials for Lambda red recombineering (£1000)
Reagents and materials for PFGE, PFGE agarose, standards, buffers, gel and gel plug forms and combs (£1000)
Reagents and materials for Phi29 polymerase based method (£250)
Reagents and materials for M. polymorpha DNA extraction (£250)
Publication costs (£1500)
Additional funding is available from the EPSRC grant to meet the aims of the project