For my final project, I plan to utilize automation tools, particularly the Opentrons OT-2, to optimize the transformation process of Sporosarcina pasteurii for enhanced microbial-induced calcium carbonate precipitation (MICP). Transformation in S. pasteurii presents several challenges, including low efficiency and difficulty in integrating foreign DNA. Automating this process will not only improve reproducibility but also streamline key steps such as plasmid assembly, transformation, and screening for successful integrations. The Opentrons OT-2 will handle liquid handling tasks for plasmid assembly, transformation preparation, and colony screening, significantly reducing manual labor and error.

The first step in automation will involve plasmid assembly using Golden Gate or Gibson Assembly protocols. Opentrons OT-2 will be programmed to handle the precise pipetting of DNA fragments, ligase, and reaction buffers to construct transformation vectors. To ensure successful assembly, an automated PCR verification step will be incorporated, along with an Opentrons-assisted gel electrophoresis loading process. This automation will allow for the rapid construction and validation of various genetic constructs designed to enhance MICP efficiency in S. pasteurii.

Once the plasmid is successfully assembled, the next stage will focus on automating the transformation workflow. This will include the preparation of competent S. pasteurii cells through heat shock or electroporation protocols. The Opentrons OT-2 will be used to aliquot transformation mixtures, perform recovery steps, and plate transformed cells onto selective media. Automating these steps systematically improves transformation efficiency while minimizing contamination and variability.

Screening and characterization of transformed colonies will also be automated. I will perform colony PCR using the Opentrons OT-2. This crucial step confirms successful integration of the desired genetic elements. Fluorescent protein reporters or antibiotic resistance markers will be used to quickly identify successful transformants, eliminating the need for time-consuming manual screening. Additionally, a high-throughput colony counter could be integrated to quantify transformation efficiency and evaluate construct performance.

A relevant study that demonstrates the power of automation in synthetic biology is titled "AssemblyTron: Flexible Automation of DNA Assembly with Opentrons OT-2 Lab Robots" by Bryant et al. (2023). This paper introduces AssemblyTron, an open-source Python tool designed to integrate j5 DNA assembly software with Opentrons OT-2 for automated plasmid assembly. The researchers demonstrated the ability to perform polymerase chain reactions (PCR), Golden Gate assembly, and in vivo homology-dependent assembly (IVA) with minimal human intervention. Their results showed that AssemblyTron significantly reduced human error while maintaining high transformation efficiency and construct fidelity.

The homology-based assembly strategies described in the study could be adapted for constructing optimized plasmids for S. pasteurii. The study shows how automation can facilitate high-throughput screening, which could be useful for evaluating different genetic modifications aimed at improving biocementation.

I plan to automate DNA assembly, transformation, and screening, thereby reducing manual errors and accelerating the development of genetic tools for sustainable construction applications. I hope to contribute to the growing field of engineered living materials by leveraging automation for precise and scalable microbial transformations.

John A Bryant, Mason Kellinger, Cameron Longmire, Ryan Miller, R Clay Wright, AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots, Synthetic Biology, Volume 8, Issue 1, 2023, ysac032, https://doi.org/10.1093/synbio/ysac032

from opentrons import protocol_api

metadata = {
    'protocolName': 'Automated Golden Gate Assembly for S. pasteurii',
    'author': 'Your Name',
    'apiLevel': '2.11'
}

def run(protocol: protocol_api.ProtocolContext):
    p300 = protocol.load_instrument('p300_single', 'right', tip_racks=[protocol.load_labware('opentrons_96_tiprack_300ul', '1')])
    plate = protocol.load_labware('corning_96_wellplate_360ul_flat', '2')
    
    # Transfer DNA fragments for assembly
    p300.transfer(5, plate.wells_by_name()['A1'], plate.wells_by_name()['B1'], mix_after=(3, 10))
    
    # Add ligase & buffer
    p300.transfer(2, plate.wells_by_name()['C1'], plate.wells_by_name()['B1'], mix_after=(2, 5))
    
    # Incubation step (to be performed externally)
    protocol.comment('Move the plate to a thermocycler for ligation')