Automation for High-Throughput Identification and Picking of GFP Expressing Colonies

Stephens, Sarah; Figg, Aaron; Mann, Christopher; Atkinson, R. J. A.; Woodrough, Robert

doi:10.1016/s1535-5535-04-00193-5

Cited by 4 publications

(5 citation statements)

References 3 publications

(3 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this regard, the current robotic platforms reduce the culture volume by replacing shaker flasks with microplates [ 21 , 22 ]. Similarly, our screening protocol was optimized at microscale [ 4 ]. The correlation analysis of fluorescent signals and enzyme activity revealed that there is a positive relationship between the expression of eGFP and model protein.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, these platforms require high-resolution imaging systems to monitor the colonies growing on the agar surface and discriminate them based on user-selectable criteria, such as diameter and roundness. Therefore, these platforms are not cost-effective and in the practice, the screening of expression is carried out semi-quantitatively [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

“…The development of enhanced green fluorescent protein (eGFP) and other fluorescence-based proteins has opened new opportunities for screening purposes. These protein reporters do not need an external substrate or cofactor, work in a wide range of conditions (pH, temperature, salt concentration, detergents, and protease), are non-toxic, and are detectable quantitatively by fluorimetry [ 4 ]. However, removal of the reporter is usually necessary following expression since it may affect the structure and function of the target protein [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

et al. 2021

View full text Add to dashboard Cite

Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

et al. 2021

View full text Add to dashboard Cite

show abstract

“…These tasks are well performed by molecular biological workstations, which are capable to accommodate special equipment such as vacuum manifolds, magnetic separators, shakers, incubators or indexed centrifuges on, beneath or under the work deck. Examples for genomics specific tasks reported in the literature include identification and picking of colonies, 15 purification of DNA from large samples, 16 automated sample-preparation technologies in genome sequencing projects, 17 high throughput probe production for DNA microarrays, 18 plasmid purification, 19 high throughput DNA extraction methods for PCR, 20 automated cycle sequencing, 21 purification of BigDye Ter-minator fluorescent DNA sequencing reaction, 22 purification of PCR products, 23 automated agarose gel electrophoresis, 24 high throughput RNA purification, 25,26 and printing of microarrays using liquid-handling robots. 27 However, some tasks are difficult to automate at all, such as initial sample (e.g.…”

Section: Considerations For Choosing Automationsmentioning

confidence: 99%

Liquid-Handling Robotic Workstations for Functional Genomics

Lorenz

2004

JALA: Journal of the Association for Laboratory Automation

View full text Add to dashboard Cite

More and more functional genomics laboratories are willing to invest in robotic workstations due to the higher throughput liquid-handling intensive nature of the work. In this report, the features of robotic workstations important for functional genomics are discussed. Workstations for functional genomics are useful for replication of clone sets, PCR and sequencing set-up and clean-up, hit picking, gel loading, and nucleic acid purification procedures. Workstations not only increase throughput, but also ensure that assay steps are performed consistently, human error- and learning curve-free from run to run. Workstations fit on a laboratory bench and may have several robotic arms in different configurations. Available configurations include grippers and single-, oligo-, or multi-probe heads, with uniform or independent spanning, and liquid-level detection or tracking options. Most hardware and software packages can be designed for integration with other automated devices, building towards a fully automated system.

show abstract

“…Prior to picking, a specialized software processes the image to detect, locate and characterize the colonies in the plate. This enables the discrimination and selection of the best colonies according to flexible criteria: colony size, geometry, color, luminescence or fluorescence ( Stephens et al, 2002 ; Fabritius et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Technical upgrade of an open-source liquid handler to support bacterial colony screening

Lianes

Yubero

Gómez-Luengo

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The optimization of genetically engineered biological constructs is a key step to deliver high-impact biotechnological applications. The use of high-throughput DNA assembly methods allows the construction of enough genotypic variants to successfully cover the target design space. This, however, entails extra workload for researchers during the screening stage of candidate variants. Despite the existence of commercial colony pickers, their high price excludes small research laboratories and budget-adjusted institutions from accessing such extensive screening capability. In this work we present COPICK, a technical solution to automatize colony picking in an open-source liquid handler Opentrons OT-2. COPICK relies on a mounted camera to capture images of regular Petri dishes and detect microbial colonies for automated screening. COPICK’s software can then automatically select the best colonies according to different criteria (size, color and fluorescence) and execute a protocol to pick them for further analysis. Benchmark tests performed for E. coli and P. putida colonies delivers a raw picking performance over pickable colonies of 82% with an accuracy of 73.4% at an estimated rate of 240 colonies/h. These results validate the utility of COPICK, and highlight the importance of ongoing technical improvements in open-source laboratory equipment to support smaller research teams.

show abstract

Automation for High-Throughput Identification and Picking of GFP Expressing Colonies

Cited by 4 publications

References 3 publications

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

Liquid-Handling Robotic Workstations for Functional Genomics

Technical upgrade of an open-source liquid handler to support bacterial colony screening

Contact Info

Product

Resources

About