The Danelon lab proposes new methods to help construct synthetic cells through evolution

Imaging Flow Cytometry for High-Throughput Phenotyping. Cover of ACS Synthetic Biology, Volume 12, Issue 7. Image credit: Ana Restrepo Sierra.

The team led by Christophe Danelon has published two methods designed to accelerate the construction of synthetic cells. The methods, which could be used more widely for in vitro evolutionary research, were published in April and May 2023 in the journal ACS Synthetic Biology.

Life adapts and evolves in response to environmental changes. To understand how natural organisms evolve, and to exploit evolution as an engineering tool, we need to explore DNA, where the encoded program of life lies. DNA contains all the instructions necessary for the functioning of living organisms and can undergo modifications. These modifications may include mutations that can have a direct effect on certain functions or characteristics of the organism, such as the appearance of the colour blue in human eyes and the ability to digest lactose in adulthood. Interestingly, DNA mutations can sometimes confer evolutionary advantages on individuals that can be passed on to future generations.

The research group led by Christophe Danelon, from Delft University of Technology (Netherlands) and Toulouse Biotechnology Institute (France), uses evolution as nature does, as an engineering tool to build synthetic cells. The group recently published two papers in ACS Synthetic Biology that provide easy-to-follow methods for facilitating the screening and engineering of synthetic cells: CADGE and Imaging Flow Cytometry.

“Both techniques are easy to use and reproducible for other researchers using in vitro strategies to build synthetic cells. These methods may also benefit a wider community of researchers working on cell-like compartments or in vitro evolutionary systems,” said Ana Restrepo Sierra, co-first author of the two papers.

1# The CADGE method for increasing protein production and enhancing in vitro evolutionary processes

Cells use the information encoded in DNA to produce proteins that perform almost all the tasks required for organisms to function properly. Protein functions translate into observable features that can be labelled as phenotypic outputs. When performing evolution, researchers often want to optimize a specific protein function or phenotypic output. In the Danelon Lab, in particular, the team wants to utilize in vitro evolution to accelerate the integration of the biological elements and modules needed to build a synthetic cell. To do so, they screen and select the best phenotypes from a set of DNA variants encapsulated in vesicles.

Ana Restrepo

In in vivo evolution, i.e. in the context of living cells, each phenotype is intrinsically connected to the genotype, or DNA, from which it is derived. In In vitro, however, researchers need to provide the means to establish a strong genotype-phenotype link. Therefore, only one DNA variant from a large library needs to be present per synthetic vesicle. DNA dilution is often the method of choice, but such low DNA concentrations inside synthetic vesicles usually compromise gene expression, i.e. the process by which the information encoded in a gene is used to make proteins: protein production is low and phenotypic outputs can then be hindered.

How to efficiently increase protein production per synthetic vesicle while maintaining a genotype-phenotype link? The answer is not so simple. “Increasing the amount of the same DNA in a closed compartment to obtain higher protein expression can be difficult. DNA replication or polymerase chain reaction [PCR] are processes for constructing new DNA copies that are not very compatible with gene expression. There are temperature conflicts, for example between PCR and gene expression, but also inhibitory effects between some of the reaction components that have been tried so far” explained Ana.

The Danelon lab has overcome this challenge by developing a strategy called Clonal Amplification-Enhanced Gene Expression (CADGE). This strategy successfully couples isothermal clonal DNA amplification with gene expression inside liposomes, resulting in increased protein yield and phenotypic output.

“We see many advantages to this method. We can increase protein production while maintaining a link between genotype and phenotype. We also believe that CADGE will be instrumental in paving the way for the integration of modules and their subsequent evolution in a synthetic cell. For example, we are now working on the integration of DNA replication with more essential synthetic cell modules, such as membrane synthesis in liposomes and division,” remarked Ana.

2# The team goes beyond the limits of flow cytometry technology to rapidly screen the complex phenotypic traits of large populations of synthetic cells

Microscopy and flow cytometry are powerful technologies for analyzing biological processes in natural cells or cell-like structures such as liposomes. However, when using each method separately, researchers have to compromise between robust statistical population analysis by flow cytometry and information-rich images provided by microscopy.

To overcome this limiting compromise and enable the imaging of millions of individual synthetic cells, the Danelon lab has developed a user-friendly pipeline for screening gene-expressing liposomes using Imaging Flow Cytometry technology. As Ana explained, “With our method, we can extract robust statistical information on complex phenotypes from large liposome populations. This is something that goes beyond the capabilities of each instrument when used separately.”

For more information on the Danelon lab

Video: Christophe Danelon on constructing minimal cells that can evolve