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In vitro synthetic biology: surface modified, 3D printed molecule machines

Title of project: In vitro synthetic biology: surface modified, 3D printed molecule machines
This PhD proposal focuses on the development of 3D printer compatible chemical linkers to enable functional biomolecules (or any type of macromolecule) to be printed onto the surfaces of 3D printed objects during their production. The potential impacts of this basic technology are diverse and game‐changing, since it enables bespoke objects, with a large range of biological and chemical properties to be designed and printed. This research is broadly in the area of in vitro synthetic biology 1, whilst there are many potential applications. Two key applications are in natural product synthesis, where the proteins that synthesise a biomolecule are printed into a device for in situ deployment in biomedical devices or small scale syntheses, and semi‐biotic devices, biological devices with the transcription/translation modules that facilitate the self‐repair of their biological parts.
The use of 3D printers in chemistry is growing but marginal. In contrast, bioengineering disciplines have been exploring the use of 3D printers in R&D for a number of years. Typically, bioengineering uses of 3D printing are restricted to microfabrication of existing technologies in microfluidic devices. The increasing availability of 3D printers and the modular nature of some models make an attractive research area for applied and fundamental chemistry research as it overlaps with biotechnology. Biotechnology is tipped to be one of the largest growth areas in science in the coming decades, evidenced by the recently announced Horizon 2020 industry led funding streams and the commitment of RCUK to synthetic biology research. One of the uncertainties that surrounds the expansion of biotechnology is its public acceptance, the impact of which can be seen in the analogous GM food debate, thus in vivo synthetic biology and biotechnology research is frequently the subject of ethical concern and debate. Last year a number of large organisations (Friends of the Earth, Greenpeace, WWF) called for a voluntary ban in synthetic biology research in vivo. This project is in the general area of in vitro synthetic biology, thus is highly topical research without the ethical baggage of in vivo synthetic biology, making it an excellent focus for public engagement.

Introduction –

rationale for research: Focus will be on methods that demonstrate the functional capacity of DNA molecules within 3D printed devices. This will be achieved with a commercially available, adaptable 3D printer with interchangeable extruders. One application of these DNA printed objects is in continuous transcription/translation devices (these can be used to make functional proteins e.g. insulin and peptides for medical devices and wound healing applications).

A literature review


Discussion of method/methodologies


To develop methods that demonstrate the functional capacity of DNA molecules within 3D printed devices (by the use of commercially available adaptable 3D printer with interchangeable extruders)
The DNA printed objects is in continuous transcription/translation devices that can be used to make functional protein such as insulin and peptide for medical devices and wound healing applications.

There are two key challenges in the project: the first is developing a method of making the printed polymer reactive to a tether, the second is making a reactive tether initiated by the heat of the 3D printer extruder to enable molecular printing. The first challenge will be approached by mixing the thermosetting plastics used by the 3D printer with colloidal gold particles. A small layer of this material will be printed in areas where tethered molecules are required. This approach builds on the well established molecular tethering technique where thiol linkers covalently attach to gold particles.
 Alternatively, a range of functionalised polymer methodologies used to link proteins to surfaces in biomedical assays and the food technology industry offer potential, such as the commonly used streptavidin‐avidin interaction. Both of these approaches have been used to prepare functional DNA on surfaces, terminal modification of the DNA is achieved using modified PCR primers ‐ a cost‐effective way to make ‘printable’ DNA molecules. It is also possible to use these methods to terminally label proteins such that their functionality is maintained when surface tethered, however it may be that protein‐protein interactions are better suited to enable printing.
In tackling the second challenge it is an advantage to have a range of supramolecular strategies available to us, since it will be critical to balance tethering yields printing speed and molecular stability. For example, double‐stranded DNA molecules ‘melt’ into single‐stranded DNA at circa 65°C and many proteins denature at a similar temperature. This may compromise the printing of DNA/protein molecules necessitating a customised molecular linker, in turn linked to gold particles via thiols to the printed surface, but containing a reactive biocompatible functional group. A modified biomolecule (e.g. lipid or synthetic ribosome) with a modified reactive terminus would then be required. Recent work in the field of ‘click’ chemistry” has given rise to an extensive number of biocompatible chemical linkers that offer a definite research direction to tackle this challenge.
Background info for help with wright up
This is how I like the literature review to follow
Synthetic biology; 3D printing
Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.
Surface and DNA modification techniques for attaching DNA to gold
I have attached my PowerPoint presentation and some papers for writing about this.
The chosen DNA modification attachment to attach DNA to gold is SH ( thiol) as many paper estate they managed to do so with high yield of result. also labeling kit are available which shows that this is a fast process.
I have chosen gold for attachment because?

Gold nanoparticles were chosen because it can provide a catalytically active metallic surface; if coated with a biomolecules,
They can obtain an alternative source of functionality much like an enzyme.

These residues can act as a nucleophile, general acid, and or general base, exploiting cooperatively between different groups of adjacent peptides at catalytic site imparted by nanoparticle surface and creation of a localized water free environment similar to an enzyme.

Gold nanoparticles can be reconfigured to sense many different analytes by simply changing the peptide or DNA sequence with which they are functionalized.
Gold nanoparticles have more functionality in term of nanodevices ……..
Can be detected easily by colorimetric assay


“Click Chemistry” and use of ligation of DNA functionalized gold nanoparticles.

Explain what is click chemistry and for your info I have chosen this technique because It is
Fast, Stable, Simple process, High yielded, Achievable, Cost effective
Ways to make printable DNA molecules.
4) Then finish it by anything you like to link all of this together and a root for making natural product within 3D printer.
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