Ciro Santilli @cirosantilli 35

Using de novo DNA synthesis to synthesize a genes to later insert somewhere.

Note that this is a specific application of de novo DNA synthesis, e.g. polymerase chain reaction primers is another major application that does not imply creating genes.

As of 2018, Ciro Santilli believes that this could be the next big thing in biology technology.

"De novo" means "starting from scratch", that is: you type the desired sequence into a computer, and the synthesize it.

The "de novo" part is important, because it distinguishes this from the already well solved problem of duplicating DNA from an existing DNA template, which is what all our cells do daily, and which can already be done very efficiently in vitro with polymerase chain reaction.

Many startup companies are attempting to create more efficient de novo synthesis methods:

Notably, the dream of most of those companies is to have a machine that sits on a lab bench, which synthesises whatever you want.

TODO current de novo synthesis costs/time to delivery after ordering a custom sequence.

The initial main applications are likely going to be:but the real pipe dream is building and bootstraping entire artificial chromosomes

News coverage:

Isothermal means "at fixed temperature".

This is to contrast with the more well established polymerase chain reaction, which requires heating and cooling the sample several times.

The obvious advantage of isothermal methods is that their machinery can be simpler and cheaper, and the process can happen faster, since you don't have to do through heating and cooling cycles.

More generic PCR information at: Section "Polymerase chain reaction".

Because it is considered the less interesting step, and because it takes quite some time, this step was done by the event organizers between the two event days, so participants did not get to take many photos.

PCR protocols are very standard it seems, all that biologists need to know to reproduce is the time and temperature of each step.

We did 35 cycles of:

This process used a Marshal Scientific MJ Research PTC-200 Thermal Cycler:

We added PCR primers for regions that surround the 16S DNA. The primers are just bought from a vendor, and we used well known regions are called 27F and 1492R. Here is a paper that analyzes other choices: academic.oup.com/femsle/article/221/2/299/630719 (archive) "Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment" by Yuichi Hongoh, Hiroe Yuzawa, Moriya Ohkuma, Toshiaki Kudo (2003)

One cool thing about the PCR is that we can also add a known barcode at the end of each primer as shown at Code 1. "PCR diagram".

This means that we bought a few different versions of our 27F/1492R primers, each with a different small DNA tag attached directly to them in addition to the matching sequence.

This way, we were able to:

Input: Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...

|||
|||
vvv

Output: PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492R

Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.