This was a long project that involved:

• [1] The robotic automatization of biotechnological protocols.

  All the custom hardware design can be found at OT_Robot/harware_design.

• [2] The usage of Artifical Intelligence.

• [3] A search engine.

• [4] A tool to migrate WordPress websites to the iGem servers.

• [5] And many, many custom hardware.

You can find information on installation and usage at the end [5] and our website.

Folder OT_Robot contains biotechnological protocols for obtaining trained libraries of DNA aptamer molecules automated with the Opentrons OT-2 robot. This protocol is part of our contribution to the 2019 iGEM international biotechnology competition. For the thermocycler we built the Ninja-PCR(original from hisashin). More information about the entire project can be found in our website.

Aptamers are a cutting-edge technology that is revolutionizing biotechnology, from biosensing to synthetic biology. Aptamers are single-strand DNA molecules that hold nature’s most important information: our genetic code. But instead of using DNA for carrying information, our aptamers depend on DNA’s 3-dimensional shape.

We genetically engineer this shape to take hold of and mesh with our target molecule. DNA’s unique role in nature gives aptamers amazing characteristics, making them robust, stable, and cheap to produce.

The small-sized thermal cycler is controlled via motherboard Ninja-PCR Arduino-alike, as shown above, manually welded, whose code is in NinjaPCR folder. You can update a new binary since ESP8266 SystemOnChip includes the TCP/IP stack. We also recommend the usage of an external Raspberry Pi to uninterruptedly provide the robot with Wi-Fi. You can find more information about usage at [5].

We also did an Artificial Intelligence algorithm to predict the optimal DNA molecule shapes. It is a Generative adversarial network, divided in two neuronal networks: the Generative and the Discriminative. CNN nets are used.

In addition, you can use our Search Engine for words in previous or this year's team's websites. This is a python search engine with GUI for searching words in each competition year and team's website.

We also developed a tool to make life easier to web page developers at the iGem competition. You can use WordPress templates to develop your site and then migrate it easily with this multi-threaded script located at Tool_Migrate folder.

To run the script you can do:

$ bash treatment.sh [url_1] -nm - - [url_2]

Some additional options are available, feel free to navigate throw our code!

To assemble the potentiostat, first you should send to manufacture the PCB, in our instance we sent it to JLPCB (https://jlcpcb.com/) and buy the components listed on the Bill of Materials file. After that, you should solder those materials on the PCB. You can also request them to solder the components, if they have them.

Any team could reproduce this potentiostat sending the gerber and the NC Drill files to the manufacturer.

Regarding to the case, we designed it with a 3D modelling software, and we cut the flat faces in methacrylate with a laser cutter machine and otherwise we printed it with a 3D printer.

The PCB was made with the Altium Designer software. Altium can export PCB and schematic files as ACCEL-ASCII files. With these files you can import them to Eagle.

To assemble the fluorimeter, you should send to manufacture the PCB, for instance we sent it to JLPCB (https://jlcpcb.com/) and buy the components listed on the Bill of Materials file. After that, you should solder those materials on the PCB. You can also request them to solder the components, if they have them.

Any team could reproduce this fluorimeter sending the gerber and the NC Drill files to the manufacturer. The PCB was made with the Altium Designer software. Altium can export PCB and schematic files as ACCEL-ASCII files. With these files you can import them to Eagle.

In relation to the case, we designed it with a 3D modelling software, and we cut the faces in methacrylate with a laser cutter machine.

Wax Module To assemble the Wax Module, you should send to manufacture the PCB, in our instance we sent it to JLPCB (https://jlcpcb.com/) and buy the components listed on the Bill of Materials file. After that, you should solder those materials on the PCB. You can also request them to solder the components, if they have them.

Any team could reproduce this module sending the gerber and the NC Drill files to the manufacturer.

The PCB was made with the Eagle software. To generate the necessary files, you can follow the JLPCB guide (https://support.jlcpcb.com/article/43-how-to-export-eagle-pcb-to-gerber-files).

In relation to the case, we designed it with a 3D modelling software, and we cut the faces with a laser cutter and the part where the resistances are located, was printed with an aluminium printer. Also, the part where the stamps are placed can be printed with a 3D printer.

To assemble the NinjaPCR, you can make it yourself following the instructions written on their webpage or buy it (https://ninjapcr.tori.st/en/index.html).

To assemble the Thermal Module, you can send to manufacture the PCB, in our instance we sent it to JLPCB (https://jlcpcb.com/) and buy the components listed on the Bill of Materials file. After that, you should solder those materials on the PCB. You can also request them to solder the components, if they have them. The other option you could buy it from their webpage.

In relation to the case, we redesigned the NinjaPCR case with a 3D modelling software, and we cut the faces with a laser cutter in methacrylate and in wood.

We redesigned it because we used another fan.

To assemble the Shaker Module, you should send to manufacture the PCB, in our instance we sent it to JLPCB (https://jlcpcb.com/) and buy the components listed on the Bill of Materials file. After that, you should solder those materials on the PCB. You can also request them to solder the components, if they have them.

Any team could reproduce this module sending the gerber and the NC Drill files to the manufacturer.

The PCB was made with the Eagle software. To generate the necessary files, you can follow the JLPCB guide (https://support.jlcpcb.com/article/43-how-to-export-eagle-pcb-to-gerber-files).

In relation to the case, we designed it with a 3D modelling software, and we printed the module with a 3D printer.

We also designed some PCBs from ourselves to make things easier and make them fit in the robot. We used a 3D-printer for some of our designs to store Eppendorf and Falcon tipracks. We also deisgned a PCB for a potenciostat microcontroller Teensy 3.0 and an auxiliar thermic module to cool to 4 degrees Celsius, and so on.

The 3D-designs will are available at the folder OT_Robot/hardware_design

Our project has a lot of software involved, including:

1. A graphical Search Engine for words in websites of previous years of iGem:

You can run it from your terminal using:

$ python search.py

You might have some dependencies not installed, you just need to do:

$ sudo pip install <dependencies>	

2. The Aptamer Folding:

In the folder DB_Creation, execute (if you use Mac or Linux), for terminal:

$ python DB_Creation_Terminal.py

You can also speed things up with threads with bash run_several.sh, but be aware that you would need to modify the absolute path to the Rosetta software.

In the folder GAN_Network, from terminal:

$ python Main.py

Other files that could be used:

3D_structure_creation_in_pdb.py Creates only one PDB for the DDBB (testing puposes)

GAN_Network.py (Same thing as GAN_Network folder but in one file)

3. OT_Robot protocols

It contains tons of biotechnological protocols automated for the Opentrons OT2 robot in Python. The most important one is the SELEX process. To run these protocols you just need to drug and drop the protocols into the OT2 app.

You can also find in here the hardware design.

You can clone this repo or download the zip. Make sure all the dependencies are covered. First line is for installation of pip at most Linux distributions.

$ sudo apt install python-pip || pacman -S python-pip
$ sudo pip install opentrons subprocess time os

The robot uses the audio robot.mp3 and gets it from Robot Raspberry's /mnt/usbdrive/ (put it there or edit the location). You can load the protocol in the Opentrons application, available here. We also recommend the usage of an external Raspberry Pi to uninterruptedly provide the robot with Wi-Fi. Once you have stablished the Wi-Fi network at the Raspberry, you can connect the Robot too:

# nmcli dev wifi connect <userSSID> password <passwdSSID>

After the last update of the Robot -by the time this is written- the SSH connection has changed and now you need to exchange with it a public key for a more secure connection. Then when connected the robot will request the passphrase, at least the first time. Here is how you create the public key (you probably already have one):

$ ssh-keygen
Generating public/private rsa key pair.
Enter file in which to save the key (/home/.ssh/id_rsa):
/home/pepe/.ssh/id_rsa already exists.
Overwrite (y/n)?
Enter passphrase (empty for no passphrase):
Your identification has been saved in /home/.ssh/id_rsa.
Your public key has been saved in /home/.ssh/id_rsa.pub.
The key fingerprint is:
a9:49:2e:2a:5e:33:3e:a9:de:4e:77:11:58:b6:90:26 pepe@remote_host
The key's randomart image is:
+--[ RSA 2048]----+
|     ..o         |
|   E o= .        |
|    o. o         |
|        ..       |
|      ..S        |
|     o o.        |
|   =o.+.         |
|. =++..          |
|o=++.            |
+-----------------+

• Pablo Villalobos
• Carlos Bilbao
• Ana Matesanz
• Irene Guardiola
• Francisco Javier Quero

This project's source code is licensed under the GNU-GPL License - see the LICENSE.md file for details. The other parts of the project have the Creative Commons license, as stated in the competition guidelines.