High-throughput Cell Plating Using the ROTOR HDA

Jay Yang1, Michaela Spitzer1, Adison Wong2, Wen Shan Yew3, and Matthew Chang3

1Singer Instruments, Roadwater, TA23 0RE, UK2Singapore Institute of Technology, 10 Dover Drive, Singapore 138682, Singapore3Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore

INTRODUCTIONAdvances in robotic automation help standardise repetitive procedures in labs. This in return increases throughput and reliability. The plating and isolation of single colonies is indispensable in the course of any micro- and molecular biology study. Despite the clear benefits of automation, plating cells on robotic platforms is not widely adopted. This is largely due to the inconsistency and the low throughput of current robotic solution – plating one plate at any one time. This is not practical if one is working with hundreds of thousands of samples at a time. At Singer Instruments, we have developed a solution for this problem: the high-throughput plating protocol using the ROTOR HDA. This protocol provides researchers the capacity to plate 96 samples in under two minutes on a single plate, a truly high-throughput approach saving valuable time and resources!

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ROTOR PLATES CELLS USING A CUSTOM PINNING MATRIXTo take advantage of the ROTOR’s liquid to agar transfer functionality, we have developed a plating protocol that uses 96-density long pin RePads to transfer cells from a 96-well microplate to an agar plate. For this protocol the ROTOR pins a 7x7 matrix for each of the 96 samples on a rectangular agar plate. Once the ROTOR has picked up samples from the 96-well plate, it repeatedly spots the pintip of the RePad onto the agar plate without revisiting the source plate. The repeated spotting of the pintips dilutes the cells on the pintips as the ROTOR progresses through the matrix, resulting in pinning of single colony forming units (Figure 1).

THE ROTOR PLATING PROTOCOL IS CAPABLE OF PLATING YEAST AND BACTERIAL CELLSTo assess the effectiveness of this protocol, we grew an overnight culture of S. cerevisiae, performed a 2-fold dilution series of the overnight culture, and pinned the cells onto PlusPlates (Figure 2a). After 48 hours incubation at 30°C we observed single colonies across a range of dilutions. We repeated the same experiment with P. pastoris (Figure 2b) and E. coli (Figure 2c). The results were consistent and we observed growth of single colonies at various dilutions. This demonstrates that the 7x7 matrix plating protocol is applicable to a range of microorganisms, including yeast and bacterial cells.

Figure 1: Schematic diagram illustrating the 7x7 matrix protocolThe ROTOR pins samples from a 96-well source plate in the 7x7 matrix onto an agar plate. After incubation single colonies can be observed for all 96 samples.

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2c

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2-fold Serial Dilutions

2-fold Serial Dilutions

2-fold Serial Dilutions

Figure 2: Testing the 7x7 matrix plating protocol with different model organisms.Overnight cultures for different model organisms were 2-fold dilution series and plated onto an agar. A) Plated S. cerevisiae was incubated at 30°C for 48 hours. B) Plated P. pastoris was incubated at 30°C for 36 hours. C) Plated E. coli was incubated at 37°C for 18 hours.

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THE ROTOR PREPARES A PLATE OF 96 SAMPLES OF YEAST CELLSThe 2-fold dilution series suggests that 1:16 to 1:128 dilutions result in single colonies for many samples. Therefore we repeated the experiment using a 1:32 dilution of a yeast overnight culture to confirm our conclusion. The resulting image (Figure 3) shows that every sample was clearly defined in its matrix with single colonies and without cross-contamination.

THE PROTOCOL IS ABLE TO PLATE CELLS FROM E. COLI TRANSFORMATIONE. coli transformation is the most common protocol where large numbers of transformants need to be plated to pick single colonies. To determine whether the ROTOR’s 7x7 pinning protocol is able to plate E. coli cells after transformation, we transformed E. coli with either a) a plasmid encoding GFP, b) an empty vector, or c) dH2O without any plasmid. Our data indicates that we were able to obtain single colony forming units on LB + Kan plates when cells were transformed with plasmids, while the cells transformed without plasmids were not viable and did not grow on the selective media (Figure 4a). To test the specificity of the transformation, we imaged the plate using the PhenoBooth, which is able to detect GFP signals. As expected, the cells that were transformed with GFP plasmid showed strong GFP signals, while the cells that were transformed with empty vector did not (Figure 4b).

Figure 3: Samples are plated without cross-contamination.A S. cerevisiae overnight culture was diluted 1:32 and plated onto agar. The plate was incubated at 30C° for 36 hours.

3 1:32 Dilution

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COLONY PICKING AND SAMPLE TRACKING WITH THE PHENOBOOTH AND THE STINGERThe high-throughput cell plating and colony picking process can be fully automated with the PhenoBooth and The Stinger. The single colonies on the SBS-format agar plate can be imaged with the PhenoBooth. Colonies for all 96 samples are detected by the PhenoBooth in the corresponding sectors and the user can specify how many colonies to pick per sample. The positions of the selected colonies are then exported from the PhenoBooth and used by the ROTOR to pick the colonies with The Stinger. Sample tracking is easy since colonies from sample A1 on the source agar plate get picked into position A1 of the target agar or 96-well plate (Figure 5).

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dH2O Control

GFP Plasmid

Empty Vector

White light4aFigure 4: 7x7 matrix protocol is able to plate E. coli transformants.E. coli cells were transformed with either GFP-vector, empty vector, or water. After transformation, the 7x7 matrix protocol was used to plate these cells for single colonies. A) Cells transformed with either GFP-vector or empty vector formed colonies on the plate, while cells transformed with water failed to form colonies. B) The plate was imaged with the PhenoBooth using UV light to detect GFP signal. Cells transformed with GFP-vector formed colonies with a GFP signal, while cells transformed with empty vector failed to fluoresce.

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CONCLUSIONThe ROTOR 7x7 matrix protocol automates high throughput plating of microbial organisms for single colonies. This protocol allows researchers to plate 96 samples onto one target plate in less than two minutes, using a single 96-long pin RePad. Altogether, the ROTOR provides a truly high-throughput and low cost solution that merely takes 1.25 seconds and costs 4.2 cents per sample. Our cutting edge protocol will save time, resources, and money.

This new matrix plugin for the ROTOR provides a protocol that adds significant value and flexibility to an already robust system. The default 7x7 matrix is fully customisable as is the location where the matrix is spotted. If you are working with a different model organism, and would like to be the first to test this pioneering technology, please contact us at contact@singerinstruments.com

5Figure 5: Sample tracking using The Stinger.