Project Proposal: Developing teaching resources for rapid, open and combinatorial genetic circuit fabrication in cell-free systems.
The Idea
We propose:
- To develop an efficient system for the fabrication of elementary genetic function and the assembly of higher order circuits to be tested in cell-free systems.
- To adopt and promote the OpenPlant syntax to facilitate community-based expansion of these resources (ie distributed development)
- To provide instructions for simple mathematical model building to fit the data obtained with these resources
- To adapt these resources to be used with open source hardware developed to track the dynamics of genetic reactions. We will use fluorescent proteins that are excited by the same emission source (low cost 470 nm LEDs and Raspberry Pi Camera).
- To develop a NCBE-style manual and slides accompanying the resources to facilitate their use in classrooms
Synthetic Biology is changing the perception of biology as a source of materials and food to a vision of biology as a programmable substrate for controlling matter, energy and information processing. This transition has been fuelled by the advent of new enabling technologies such as large scale DNA fabrication and the use of mathematical modelling, part characterisation and design specifications. These efforts have led to a significant abstraction of the complexity involved in engineering biological systems trough genetic programming, which has simplified operational procedures and protocols. This abstraction, supported by the open access to elemental components and techniques, has not only led to rapid advances of the field (Nielsen et al., 2016) but it has also fuelled the emergence of new bioengineering communities such as iGEM, bio-make-spaces and DIY-bio labs around the globe. This democratisation of biological technology resembles the expansion of DIY movements based on open source hardware and off-the-shelf electronics. Together, these developments hold a great potential for transforming secondary and tertiary education by providing easy-to-operate and low cost tools and resources for fabricating genetic components in classrooms. We seek to adopt a DNA fabrication method, called LOOP, recently developed for low cost and efficient building of genetic instructions for plant systems (Pollak et al., unpublished). This system will permit the exponential assembly of transcriptional units, facilitating the construction of higher order circuits from modular pieces. In other words, this system would allow greater complexity of gene circuits at each assembly step and maintain the complexity of assembly constant.
Programmable biology on a test tube
Recent progress on the use of transcription-translation cell-free extracts has opened a new horizon for the democratisation of biological engineering techniques and hands-on bioengineering activities in schools. Given the non-GMO nature of these procedures (since no living systems are involved), genetic programming can be performed in schools without concerns of biological containment and GMO disposal. Cell-free systems also offer more control over biological variables and parameters by fine-tuning the composition of the extracts. We seek to develop protocols for teaching basic gene regulation and simple dynamic behaviour such as auto-regulation and FFLs that can be conceived from level 0 parts and LOOP plasmids.
Distributed user-developer ecosystems for collaborative projects
The rapid growth of online libraries and web-based platforms where users and developers share design files (e.g. GitHub) has led to the birth of new collaborative networks and distributed technological developments. Tools are developed collaboratively, with members across the world contributing and modifying designs that are curated publicly online. We seek to adopt these structures and set a platform for distributed development of bioengineering teaching resources. For this, we propose the generation of a web-page in which developers across the globe meet virtually to share parts and protocols that are compatible with the DNA assembly standards here proposed. Given that this aim escapes the reach of this proposal, we propose two strategies: i) using existing networks such as protocols.io and Google groups; and congregate links on a webpage to track developments, ii) propose to all the applicants the creation of an online platform where users and developers meet and share resources, ideas and protocols, similar to the Raspberry PI Teaching Resources section. We envision an Arduino-like platform for molecular programming that follows the OP syntax to be compatible to our LOOP system. The use of standardised modules and syntax makes assemblies by different users identical if their composition is maintained. We would provide all of these resources in English and Spanish.
Who We Are
Fernán Federici Noe ffederici@bio.puc.cl http://federicilab.org
Pontificia Universidad Católica de Chile and OpenPlant Synthetic Biology Centre, University of Cambridge, Cambridge, UK
Fernan is a synthetic biologist and microscopist interested in the development of open source tools for bioengineering and the translation of cutting-edge technology into teaching resources. He has been involved in the development of open source teaching resources in Chile and the UK. He has also been part of the organization of educational initiatives such as TECNOx (http://tecnox.exp.dc.uba.ar) and advising high school teams such as the BioBuilderChile team (http://biobuilderchile.weebly.com).
Dean Madden D.R.Madden@reading.ac.uk www.ncbe.reading.ac.uk
Director, National Centre for Biotechnology Education (NCBE), University of Reading, UK.
Dean has worked at the NCBE for 26 years and has been responsible for developing most of the Centre's educational materials, which are aimed predominantly at post-16 biology students in UK schools. This includes trehalose-stabilised DNA, restriction enzymes and DNA ligase, and protocols for their use in gel electrophoresis, DNA assembly and bacterial transformation.
Nicola Patron Nicola.Patron@earlham.ac.uk
Earlham Institute, UK
Nicola has long led the development of DNA assembly methods for plant systems. She is now working on the optimisation of small scale DNA fab protocols. Nicola is collaborating with Fernán and Bernardo on the development of the LOOP system. She has been involved in a number of educational initiatives such as being a judge for the iGEM competition.
Bernardo Pollak bp358@cam.ac.uk
PhD student at Jim Haseloff’s lab. University of Cambridge
Bernardo is a biochemist who is leading with Fernán the work on the LOOP DNA Fab system. Bernardo has been involved in several outreach and educational initiatives such as being an instructor of iGEM teams from Chile.
Implementation
Bioware:
We will develop DNA fab tools for the fabrication of elementary genetic function and the assembly of higher order circuits for cell-free reactions. This tool will be based on the LOOP system, which consists of Golden Gate one pot reactions with BsaI and SapI type IIs enzymes and T4 Ligase. Elementary parts (level 0) such as promoters and repressors (Nielsen et al., 2016) will be generated following the OpenPlant syntax (ie sites ABCEF of CIDAR). We seek to facilitate community-based expansion of these resources (ie distributed development) by inviting developers to create level 0 parts that follow the same syntax.
We will test and optimise our system to be expressed in cell-free systems. For instance, we will construct level 0 modules of fluorescent proteins that have been selected to work with single excitation (ie 470 nm blue LEDs; Figures 1A, B and C). We will provide instructions for simple mathematical model building to fit the data obtained with these resources.We will adapt these resources to be used with opensource hardware developed to track the dynamics of genetic reactions.
We will develop:
● auto-regulation feedback by using a series of repressors and their promoters from the Voigt lab (Nielsen et al., 2016)
● incoherent feed forward loop with CRISPRi and protein repressors
● simple logic gate (e-g NAND, NOR)
Hardware design
We will adapt low-cost 470 nm LEDs and Raspberry Pi cameras to be used with cell free reactions (Figure 1A). This work would probably involve the redesign of the imaging station to be suitable for paper-based or 96-well plate imaging.
Manuals and teaching materials:
To facilitate the use of these resources in classrooms, we will develop manual and slides following design and graphics already used by NCBE (Figure 2). These resources will be provided in both Spanish and English.
Outreach and promotion
We will promote the use of these resources through existing networks such as TECNOx. We will also organise two biohackthons to use these resources in classrooms, one in a high school in Chile and another in an undergraduate SynBio course in Chile and Buenos Aires. We are currently working in collaboration with a Chilean High School (Colegio Blest Gana).
Figure 1: A) top, Raspberry Pi imaging station; Bottom, fluorescent images taken with the camera B) Analysis of different settings, shutter speed (-ss in raspian shell) = 50000 and 100000. C) Example of cell free reactions run on the imaging station.
Figure 2: Example of graphics developed by NCBE for teaching transformation.
Benefits and outcomes
This collaboration will result in the invention, publication, and distribution of open source resources for teaching genetic programming. We envision the development of a community that will further contribute with resources to be used with this system. We will also deliver opensource and adaptable hardware for imaging and performing time-lapse experiments. The data obtained could be analysed with simple models that will be also provided with these resources.
We will work closely to teachers in both high schools and universities to be able to use these resources for curriculum development in Latin America. We are currently working in collaboration with a Chilean High School (Colegio Blest Gana).
Budget
First £4000
We would outsource the TxTL extracts from MyTXTL, US, via Cambridge.
● MyTxTL cell free extracts and shipping (http://www.mycroarray.com) 4 x 96 reactions plus shipping: £2500
● 96 well plates (V bottom) and lids: £200
● DNA synthesis and primers: £1300
● Subtotal: £4000
Final £1000
We plan to run two biohackathons; one in Santiago, Chile and the other in Buenos Aires, Argentina with these resources.
To complete two imaging stations for the schools, we need:
● 2x Raspberry Pi + Camera (from waveshare)= £100
● 2x Micro SD card (8gb + Noobs) = £50
● 2 USB-integrated Mouse + Keyboard systems = £50
● Subtotal: £200
Materials:
● printing of manuals: £150
● tips, dry ice and bed heating (for 30 °C reactions): £150
● Subtotal: £300
Trips:
● airfare to Argentina and accommodation for 2 people: £500
● Subtotal: £500