Bacteria such as E. coli are popular model systems for engineering and production of modified proteins. Yet the idea of putting bacteria in a cell sorter conjures unwelcome images. Take heart, the Sony SH800 cell sorter, simplifies decontamination by quickly and easily letting researchers replace key components that come in contact with the sample. Here are some examples, and publication citations.

Here are some examples of publications and applications where transfected bacteria are detected and sorted on the basis of either permeant fluorescent dyes or by the fluorescent proteins that they express using  the SH800 has been used to sort bacteria:

CRISPR/Cas9 optimization

The CRISPR/Cas9 system has opened new possibilities in the treatment of disease. However, for the CRISPR system to reach its full clinical utility better understanding of targeting and improved function are critical. Scientists at the University of California, Berkley have transformed bacteria with constructs containing GFP, and sorted cells to identify insertion hot spots to engineer Cas9 for enhanced function.

References

Oakes, Benjamin L., et al. "Protein engineering of Cas9 for enhanced function." Methods Enzymol. 546 (2014): 491. PubMed
This paper details methods for screening engineered Cas9 protein. The strategy described may be useful for the screening of other engineered proteins.

Oakes, Benjamin L., et al. "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch." Nat Biotechnol. 6 (2016): 646-651. PubMed

Engineering of proteins for enhanced production

E. coli are routinely used for protein production but they can also be used for the production of other molecules such as polyester. Many bacteria, including E. coli produce polymer inclusions, which serve as stockpiled carbon storage material during periods of nutrient imbalance. These polymer inclusions can be leveraged to make polyesters that are an alternative to petroleum based-plastics. To increase the yield of these polyesters efforts have been made to engineer the bacteria. The SH800 was used in these studies to study the cells density of the bacteria.

References

Kadoya, R., et al. "MtgA deletion-triggered cell enlargement of Escherichia coli for enhanced intracellular polyester accumulation." PLoS One. 6 (2015): e0125163. PubMed

Kadoya, R., et al. "Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters." J Biosci Bioeng. (2017). PubMed

Development of new expression systems

Inducible expression systems can turn protein production on or off to varying degrees by treating the culture with a drug or other molecule. They are particularly useful for the expression proteins that may be toxic to the cell when expressed at high levels. Most available inducible expression systems are known to be “leaky” meaning that there may still be some expression even when expression is “off”. Researchers at the University of Manchester have created a systems that contains both an inducible promoter and an orthogonal riboswitch to control gene expression in several strains of E. coli. The constructs contained both the inducible elements and eGFP (green fluorescent protein). The SH800 was used to identify subpopulations of bacteria with differing levels of GFP expression.

References

Morra, R., et al. "Dual transcriptional-translational cascade permits cellular level tuneable expression control."Nucleic acids research (2015): gkv912.( PubMed
This paper also contains a nice introduction on the principles of inducible expression systems.

Identification and enumeration of hard to culture bacteria. While many bacteria such as E. coli are relatively easy to grow in culture, some such as those found in the gut microbiome are not. Cell sorting is a useful way to enumerate bacteria from natural sources that contain mixtures of cells. To better understand the role of the bacteria Candidatus Erwinia dacicola in the life cycle of the olive fly, it was isolated by sorting cells from the gut of the fly and sequenced.

References

Blow, Frances, et al. "Draft Genome Sequence of the Bactrocera oleae Symbiont “Candidatus Erwinia dacicola”. Genome Announcements 5 (2016): e00896-16. PubMed

Sorting of bacteria is a useful tool for protein engineering and the study of bacteria. The studies cited above are practical examples of how you can incorporate the sorting of bacteria into your standard laboratory practices. But don’t worry, post bacteria sort your Sony SH800 will be easy to clean and set up for your next mammalian cell experiment.