合成生物学自动化：改进您的分子克隆过程的五个技巧

全球最受关注的问题之一是过度使用资源及其对环境造成不可否认的影响。In particular, manufacturing processes require enormous amounts of energy derived from fossil fuels such as oil and gas, which are decreasing in availability and increasing in price, making them unsustainable. By re-engineering the functions of microorganisms, researchers have successfully started to generate food, textile, and biopharmaceutical products that consume much fewer resources during their manufacturing process. If you have ever eaten the Impossible Burger sold at Burger King, you have taken a bite of an engineered food product. That distinct meat taste is a product of molecular cloning, an integral part of synthetic biology. To achieve the right set of features (e.g., taste and texture), manufacturers isolate and introduce DNA fragments into microbial strains for mass protein production.

That distinct meat taste [in an Impossible Burger for example] is a product of molecular cloning, an integral part of synthetic biology.

Today, we see the benefits of synthetic biology methods gaining momentum in various industries. With an increased demand for synthetic products derived from re-engineered biology, quick and accurate methods for high-throughput microbial strain engineering and molecular cloning have become necessary. Unfortunately, manual synthetic biology workflows are still labor-intensive, time-consuming, and prone to human error.

This article summarizes the common bottlenecks in synthetic biology workflows and how automation can overcome these challenges to increase throughput and walkaway time.

From manual picking to automated picking using a high-throughput colony picker
Integrate lab devices for a fully automated molecular cloning workflow
Reducing cross-contamination
Versatility for colony screening
Data management in synthetic biology workflows

1. From manual picking to automated picking using a high-throughput colony picker

After genes of interest are amplified, assembled into vectors, and transformed into microbes, you need to screen colonies for several features from size to fluorescence intensity. Doing this manually with pipette tips, toothpicks, or inoculation loops can cause uneven streaking, especially when high throughput is the goal. This hinders the further analysis of your colonies.

An automated colony picker could save you from the trouble of manual picking. For example, the QPix Microbial Colony Picker is a useful tool for rapidly picking and inoculating colonies into deep-well blocks with growth media. If it is your first attempt at automating colony picking, you might opt for an entry-level colony picker, such as QPix 420 system, which has a quick setup ability for picking up to 12, 96 well plates automatically. This translates to up to 3000 colonies screened per hour. As it requires minimum intervention, you reduce the risk of human errors and cross contamination while providing your team extra walkaway time to multitask.

Throughput and Labor Ditribution Graph

2。Integrate lab devices for a fully automated molecular cloning workflow

While manual pipetting can be practical in transferring liquid in small-scale applications, it is impractical for processing hundreds of samples. Instead, you can integrate automated pipetting into your molecular cloning workflow for DNA prep and then perform heat shock transformation of microbes with DNA plasmids using on-deck thermocyclers.

Robotic arm integration is the most common method for automated integration of multiple instruments that accept the standard micro-titer plate formats such as standard and deep-well SBS footprint plates without moving a muscle. More importantly, robotic automation can be adjusted to screen and pick colonies with favorable traits, accelerating hit colony isolation which can be validated using our multi-assay plate readers.

Before integrating a robotic arm into your experimental design, you need to make sure that it is ISO 15066 compliant.

3。Reducing cross-contamination

Cross-contamination is a significant risk factor that can occur on several occasions, such as an unsterile loop, dropping the lid, or the plate being unlid for too long. This can invalidate your results and negatively affect their reproducibility, meaning you cannot compare them with results from other research laboratories or previous rounds of cloning.

Avoiding cross-contamination during molecular cloning processes requires meticulous hygiene practices. You can flame the loop or use disposable loops, but neither is a practical solution for high throughput streaking.

Sterilization is yet another aspect of molecular cloning that can benefit from automation. With automated colony pickers, you can find built-in sterilization tools requiring minimum human intervention. For example, QPix Microbial Colony Pickers contains wash baths and halogen heat sterilization to eliminate cross-contamination among pins.

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4。Versatility for colony screening

Another challenge in colony picking workflows is efficiently screening for the best performing colonies. The user typically chooses their best colonies based on custom parameters and experience in manual picking, which is often highly subjective. The problem arises if a large number of colonies have to be screened. Even if sample preparation and plating is automated, the colony picker still needs to learn what characteristics to look for in a colony before colony selection.

Automated colony pickers such as the QPix 420 system are advantageous because the software is designed to quickly gate colonies based on parameters such as shape, size, proximity, and fluorescence intensity. Furthermore, you can screen the colonies in several selection modalities, including fluorescence intensity, blue/white screening, and zone of inhibition.

The QPix 420 system boasts the versatility required for precise colony selection. For example, you can adjust settings to correct for hollowness to pick colonies that appear hollow in the center despite exhibiting desired expression levels and characteristics.

自动化微生物克隆筛选系统

The QPix 420 system is capable of imaging in four different fluorescence channels, which allows you to potentially monitor four different fluorescently tagged protein expressions in the same organism.

The advantage of using automated colony picking is the ability to replicate your master plate. You can analyze your clones on newly generated sub-plates while retaining the master plate for further colony picking.

5。Data management in synthetic biology workflows

Manual data collection can be cumbersome in synthetic biology workflows, especially when data is collected across multiple instruments. This creates extra FTE hours, fatigue, and a higher risk of data loss.

The QPix 420 system can assist with data collection and storage. The actuator head contains a built-in high-resolution camera and a barcode reader for reliable data traceability. Image data gets recorded into the built-in database with an extensive audit trail and sample tracking options. Based on the barcode reading, the QPix software can be used to track information about a colony, such as its location in the source and destination plates and the date and time of picking. You also have the option to customize tags for important samples and group your colonies based on their morphological traits.
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Qpix Library management

The data collected from our automated colony picker, as well as the data collected from multiple instruments on a synthetic biology workcell, is easily exported into widely accepted formats into your favorite laboratory information management system.

Learn more about automated synthetic biology processes

Automation of synthetic biology, especially molecular cloning, not only saves you a tremendous amount of time and cost but also increases the accuracy of your results. If you want to know more about the foundations of automated synthetic biology workflows, you can register for our recent webinar by Dwayne E. Carter, PHD, BioPharma Field Application Scientist. In this webinar, you can find illustrations and diagrams of every step involved in automation with concrete examples.