Puppeteer Overview

Description

Lab protocols often require careful and precise execution of many individual steps to achieve the desired result. Slight inaccuracies and miscalculations can lead to invalid conclusions. We understand this crucial need to develop a less error prone workflow that will allow us to execute complicated and repetitive lab protocols in a more systematic and infallible matter. Our solution to this problem is Puppeteer, a high-level protocol specifying language that will allow users to construct protocols using our library of Common Human Robot Instruction Set (CHRIS). Using this language, users will be able to create protocols and save them into a Protocol Repository that we have created. This will allow synthetic biologists to share their work among themselves, therefore promoting collaboration within the synthetic biology community.

To aid the creation of new protocols using Puppeteer, we have also created a GUI front-end for our language called PuppetShow. Within PuppetShow, users can create protocols in the editor panel and execute the protocol they just wrote with the click of a button. Given that the user’s computer is hooked up to a compatible robot, resource allocation of liquids and plates will happen automatically and a report will be generated containing a resource report, an output report, instructions for the deck setup, and a pipette verification report. For now, we have the software bridge written for the Evoware 150 API and support all major robot commands through this bridge. So far, we have created Restriction Digest and Ligation protocol using PuppetShow and successfully verified their execution on our robot.

Using this tool we have created, we aim to improve accuracy and save time. A protocol only needs to be written once and can be run repeatedly with the click of a button. Using PuppetShow, synthetic biologists can create and run protocols without worrying about any low level details or error.

Results:

We have finished implementation of an initial version of the Puppeteer stack; it is fully integrated with the Clotho platform. We have implemented basic BioBricks assembly protocols and validated them in the wet lab by assembling basic devices.

Collaboration with other iGEM teams:

In order to ensure reproducibility and promote collaboration, we visited the Weiss lab at MIT to set up the Puppeteer stack on their robot. We met a few of MIT’s iGem team members and coordinated with them to test the Puppeteer flow on the MIT robot. During this process, we noticed some differences between our robot deck setups and the way this setup was handled in the Puppeteer flow. We separated such settings into an easily edited settings file to ensure that Puppeteer was easily configured for different deck setups. We successfully tested the Puppeteer flow on the MIT deck and everything ran as expected. This collaborative effort with MIT’s iGem team is definitely fruitful because it made Puppeteer more robust and ensured functionality across multiple robots.

Future Work:

Integration between Puppeteer/Puppetshow and the eLabNotebook is planned for implementation.

Ethical User Study practices:

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Demo Video

Constructing a combinatorial library of devices is tedious using manual laboratory techniques and would require hundreds of hours of careful work. To remedy this, we are implementing the Puppeteer Biological Protocol Automation Suite. This suite includes a high level programming language for specifying biological protocols commonly used in the laboratory, which are then executed by a liquid-handling robot with minimal user intervention.

Demo Video

Wetlab Verification

We implemented two protocols central to BioBricks assembly---Restriction Digestion and Ligation---in Puppeteer. We validated the Puppeteer implementation by executing multiple trials of both protocols and verifying the result by running a gel.

Restriction Digest:

For restriction digest we digested the plasmid containing the BioBrick BBa_J52028 in order to isolate the GFP. After cutting the plasmid with EcoRI and SpeI we ran it on a one percent gel along with a ladder in order to determine the amount of base pairs for each dna part. As GFP is about 1221 bp the location in which it is located on the gel is correct. Also the backbone was about 3189 bp which is also located in the correct location on the gel.

Results:

The images show the verification results.

Safety practices:

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