Tool Overview

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.

Purpose and Design

Laboratory protocols often require careful and precise execution of numerous procedures to achieve desired results. However, slight inaccuracies and miscalculations attributable to human-error continually arise and often lead to invalid conclusions. For this reason, we have developed an accurate workflow that executes biological protocols in a more systematic manner. The solution we propose is the Puppeteer Biological Protocol Automation Suite. Our suite centers on the execution of Puppeteer, a high-level protocol specification language used to programmatically describe biological protocols. Puppeteer allows users to create protocols locally, but will eventually incorporate a “Protocol Repository” that will retain version-controlled instances of uploaded protocols. This feature will promote all forms of collaboration within the synthetic biology community, with a predominant focus on inter-laboratory protocol sharing.

Puppeteer Assembly Flow

Our assembly flow incorporates a five-layer stack as illustrated in the adjacent figure. The Assembly Planner produces an assembly plan for synthetic biological devices, with each assembly step annotated with the name of a biological protocol. Each protocol may be fully specified using PuppetShow.

The protocols are written in the high-level language called Puppeteer. The Language layer includes a Puppeteer interpreter and linker. A protocol specified in Puppeteer may contain Puppeteer instructions as well as references to previously created Puppeteer programs available in the protocol repository. The Language layer expands and translates Puppeteer protocols to a sequence of low-level commands expressed in a Common Human Robot Instruction Set (CHRIS). Any high-level language may produce CHRIS programs and any robot vendor may support a superset of CHRIS. The Hardware Layer---the external control and I/O interface of a robot---is wrapped under a Hardware Abstraction Layer (HAL). Vendor-provided software for programming the robot may be proprietary and is used to control the robot. An interface to it is provided by a software bridge, which maps protocols expressed in CHRIS to sequences of native robot instructions.

The Resource Management layer maintains resource state information and provides a standardizable high-level interface for initializing, requesting, naming, aggregating, and accessing resources to the Language layer, analogous to a ``system call'' suite. This interface supports our goal of removing the minutiae of resource management from the protocol specification language.

Demo and Download

Downloads:

Demo Video

Results

In the wet lab, 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. 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.

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.

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.

Ligation:

The images show the verification results.

Safety practices: Aliquam in felis sit amet eros pharetra volutpat.

Future Work

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