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Biology Team's Pilot Project: A DNA Synthesizer for Space.

Biology Team's Pilot Project: A DNA Synthesizer for Space.

==SSI Bio 2016 Pilot Project==

==SSI Biology Pilot Project==

SSI Bio is launching 2016 by sending a DNA synthesizer into space. For information on how to join the project, join the SSI Slack and go to the #biology channel, and read information on how to get [[Wet Lab Access]] on this wiki.

The DNA synthesizer project was started in 2016 as SSI Biology Team's first project. For information on how to join the project, join the SSI Slack and go to the #biology channel. Read information on how to get [[Wet Lab Access]] on this wiki.

==Components of the Synthesis Project==

==Components of the Synthesis Project==

SSI Bio will be breaking up this DNA synthesizer project into several smaller subteams to tackle each critical component of the synthesizer.

SSI Bio will be breaking up this DNA synthesizer project into several smaller and more defined subcomponents.

===Enzymatic Synthesis Chemistry===

===Enzymatic Synthesis Chemistry===

DNA synthesis is currently done using the [https://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Synthetic_cycle phosphoramidite method]. However, phosphoramidite chemistry produced large volumes of hazardous waste, requires precise chemical conditions, and is limited to producing short strands of DNA (oligonucleotides) on the order of ~100 basepairs. Most genes of interest are in the ~5,000 basepair range, so current synthesis methods cannot create a gene-length piece of DNA without further processing. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846215/ TdT is one possible enzyme to consider,] and has previously been [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735642/pdf/11693_2009_Article_9023.pdf the subject of some prior theoretical work.]

DNA synthesis is currently done using the [https://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Synthetic_cycle phosphoramidite method], which involves hazardous chemicals and solvents like acetonitrile. Phosphoramidite chemistry is also restricted to centralized synthesis facilities that are hard for scientists in remote locations (like space) to access reliably. Using [[enzymatic synthesis methods]] rather than phosphoramidites would enable small-scale distributed synthesis of short single strands of DNA (oligonucleotides) on the order of 100 base pairs. We are currently working with the enzyme [[Terminal Deoxynucleotidyl Transferase]] (TdT), which has previously been the subject of some prior [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735642/pdf/11693_2009_Article_9023.pdf theoretical work.]

Enzymatic DNA synthesis would be a great alternative way to synthesize DNA in space, for the following reasons:

Enzymatic DNA synthesis would be a great alternative way to synthesize DNA in space, for the following reasons:

====Improved Speed and Efficiency====

====Improved Speed and Efficiency====

It may be possible that an enzymatic method could improve the speed and efficiency of synthesizing DNA in space. This would be split into two effects. First, being able to make longer strands of DNA (oligonucleotides) would mean that the final product could be composed of fewer parts, which makes the creation of algorithms and strategies for reassembling this DNA to become much easier. Second, being able to make longer strands of DNA faster would cut down substantially on the complexity and time involved with synthesizing DNA.

It may be possible that an enzymatic method could improve the speed and efficiency of synthesizing DNA in space. This would be split into two effects. First, being able to make longer strands of DNA would mean that the final product could be composed of fewer parts, which makes the creation of algorithms and strategies for reassembling this DNA to become much easier. Second, being able to make longer strands of DNA faster would cut down substantially on the complexity and time involved with synthesizing DNA.

===Safe Phosphoramidite Chemistry===

===Microfluidic Synthesizer Design===

A more conventional alternative to aiming for enzymatic synthesis would be to try to adjust conventional phosphoramidite chemistry to be safer for use in space. This might involve creating a system that can synthesize DNA using safer organic solvents and less toxic reagents.

To run a DNA synthesis reaction in a small autonomous payload, we need to precisely move tiny nanoliter-scale amounts of liquid. Microfluidics is a good way to do this. Similar oligonucleotide synthesizers that don't quite fit our needs [http://scholarbank.nus.edu.sg/bitstream/handle/10635/20904/WangC.pdf?sequence=1 have been made in the past]. Good things to know about when designing a device like this: [https://en.wikipedia.org/wiki/Diaphragm_pump diaphragm pumps], [https://en.wikipedia.org/wiki/Solenoid_valve solenoid valves], and of course [https://en.wikipedia.org/wiki/Microfluidics microfluidics in general].

We are currently designing and prototyping an autonomous fluid handling system based on [[Electrowetting on Dielectric]] (EWOD) technology.

To fit all of these fluidic parts into a small enough payload, we have to make everything quite small. Microfluidics is a good way to do this. Similar oligonucleotide synthesizers that don't quite fit our needs [[http://scholarbank.nus.edu.sg/bitstream/handle/10635/20904/WangC.pdf?sequence=1 have been made in the past,] although our final design will most likely look different. Good things to know about when designing a device like this: [https://en.wikipedia.org/wiki/Diaphragm_pump diaphragm pumps], [https://en.wikipedia.org/wiki/Solenoid_valve solenoid valves], and of course [https://en.wikipedia.org/wiki/Microfluidics microfluidics in general].

===Reassembly Chemistry and Algorithm===

===Reassembly Chemistry and Algorithm===

Our chemistry may look something like [https://en.wikipedia.org/wiki/Polymerase_cycling_assembly Polymerase Chain Assembly, also called Assembly PCR].

Our oligonucleotides will likely be used directly in downstream applications (as PCR primers, for example), but they could also be put together into biologically functional genes. Most genes of interest are in the ~5,000 basepair range, so current synthesis methods cannot create a gene-length piece of DNA without further processing and assembly. Our assembly chemistry may look something like [https://en.wikipedia.org/wiki/Polymerase_cycling_assembly Polymerase Chain Assembly, also called Assembly PCR].

===DNA Product Verification===

===DNA Product Verification===

Once a strand of DNA is made, we will need to check to make sure that it is the correct desired sequence. [http://scholarbank.nus.edu.sg/bitstream/handle/10635/20904/WangC.pdf?sequence=1 One possible strategy comes from this paper] - essentially tagging oligo and looking for fluoresence as a sign that homology was sufficiently similar to produce a result.

Once a strand of DNA is made, we will need to purify it, check to make sure that it is the correct desired sequence, and purify it for use in downstream applications. This verification can be done a variety of different ways, including [[Polyacrylamide Gel Electrophoresis]] (PAGE), [[Pyrosequencing]], [[Ligation and Sequencing]], or [https://en.wikipedia.org/wiki/Matrix-assisted_laser_desorption/ionization MALDI-TOF Mass Spectrometry].

 

==Effects of Space on Synthesizer==

==Effects of Space on Synthesizer==

===Physical Stress of Launch===

===Physical Stress of Launch===

Similar to any other payload, our DNA synthesizer will have to be durable enough to withstand the stresses and forces associated with launch.

Similar to any other payload, our DNA synthesizer will have to be durable enough to withstand the stresses and forces associated with launch.

===Payload Size and Power Constraints===

===Payload Size and Power Constraints===

We'd like to fit our synthesizer into a 10 centimeter cube, so that it could be launched on a [https://en.wikipedia.org/wiki/CubeSat CubeSat] or another standardized research payload.

We'd like to fit our synthesizer into a 10 centimeter cube, so that it could be launched on and powered by a [https://en.wikipedia.org/wiki/CubeSat CubeSat] as a standardized research payload. To do this, we'll need to design and optimize our fluid handling system, microprocessor, power supply, temperature regulator, and verification technology with size and other constraints in mind.

===Shielding Requirements===

===Shielding Requirements===

===History of DNA Synthesizer Idea in SSI===

===History of DNA Synthesizer Idea in SSI===

The idea of a DNA synthesizer for space has been floating around SSI for some time. [[John Cumbers - Synthetic Biology | One of the earliest recordings stretches back to 2013, in a talk given John Cumbers.]] John Cumbers was also consulted during the initial conception and planning of the project in the summer of 2015.

The idea of a DNA synthesizer for space has been floating around SSI for some time. One of the earliest records stretches back to 2013, in a [[John Cumbers - Synthetic Biology |talk given by John Cumbers.]] John Cumbers was also consulted during the initial conception and planning of the project in the summer of 2015.