HyperXpress: High-throughput, one-pot DNA assembly and protein production in low microliter volumes using cell-free extracts

For academic and industrial life science research, efficient DNA assembly methods and gene expression systems are an essential tool for realizing numerous applications and gaining far-reaching knowledge. In this context, high-throughput methods have a prominent position by allowing a large number of experiments in a short time.^[1] Moreover, one-pot processes are favored for the actual experiments because they are quick, inexpensive and easy to use.^[2]

HyperXpress is intended to provide a high-throughput, one-pot process for a successive sequence of DNA assembly and in vitro gene expression. As part of HyperXpress, an optimized ligase cycling reaction (LCR)^[3], multiply-primed rolling circle amplification (RCA)^[4] and cell extract-based in vitro cell-free protein synthesis (CFPS)^[5] are carried out step by step in a single well of a 384-well plate.

We present some of the optimization of LCR, RCA and CFPS performed to enable protein production in 3.6 µl in 10 hours starting from DNA fragments as well as a software addition to the DiVA-Software^[6] which aids in bridging oligo design and liquid handling. As a proof-of-concept a family shuffling of GFP-ß-strands was performed. The resulting 51 variants consisted of ß-strand deletions, ß-strand-subtstitutions as well as of heterologous ß-strand additions.

2. References

[1]: „Direct assembling methodologies for high-throughput bioscreening” (2012) by Jorge I. Rodríguez-Dévora, Zhi-dong Shi and Tao Xu;
Biotechnol J. 2011 Dec; 6(12): 1454–1465. DOI: 10.1002/biot.201100100
[2]: „Pot economy and one-pot synthesis” (2016) by Yujiro Hayashi,
Chem Sci. 2016 Feb 1; 7(2): 866–880. DOI: 10.1039/c5sc02913a
[3]: „Optimization of the experimental parameters of the ligase cycling reaction” (2019)
by Niels Schlichting, Felix Reinhardt, Sven Jager, Michael Schmidt
and Johannes Kabisch;
Synthetic Biology, Volume 4, Issue 1, 2019, ysz020, DOI: 10.1093/synbio/ysz020
[4]: „Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and
Multiply-Primed Rolling Circle Amplification” (2001) by Frank B. Dean, John R.
Nelson, Theresa L. Giesler and Roger S. Lasken;
Genome Res. 2001 Jun; 11(6): 1095–1099; DOI: 10.1101/gr.180501
[5]: „Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression
System for Synthetic Biology” (2013) by Zachary Z. Sun, Clarmyra A. Hayes,
Jonghyeon Shin, Filippo Caschera, Richard M. Murray and Vincent Noireaux;
J Vis Exp. 2013; (79): 50762 ; DOI: 10.3791/50762
[6]: DiVA - Design, Implementation, and Verification Automation Platform, https://public-diva.jbei.org/