View source for SURF 2023: Lysate Optimization to Extend Cell-Free Reaction Lifetime

'''[[SURF 2023|2023 SURF]] project description'''
__NOTOC__

* Mentor: Richard Murray
* Co-mentor: Yan Zhang
 
=== Introduction: ===
 
Cell-free expression systems harness the transcriptional and translational machinery of living cells to enable in vitro functions that are usually only effective in vivo [1]. Cell-free reactions can be created from purified recombinant elements comprising the minimum set of necessary transcriptional and translational machinery (PURE systems) [2] or via crude cell lysate from cells [3]. Of which, the crude cell lysate-based system, particularly those harvested from E. coli cells, is more widely used and cost-effective.
 
However, a significant gap exists in understanding the background metabolism in lysate-based cell-free systems. Recent works have shown that endogenous metabolism is active cell-free lysates, regardless of whether the reaction produces proteins [4, 5]. Thus, being able to identify major “waste” pathways and reroute energy consumption to focus only on protein production can potentially extend the lifetime of cell-free reactions.
 
=== Project Overview: ===
 
E. coli BL21 will be the primary strain for lysate-based cell-free protein expression. The SURF student will first identify potential gene candidates for “waste” metabolic branches responsible for diverting energy away from protein synthesis. Following branch point identification, the student will determine the optimal method for “waste” pathway removal. For example, the relevant enzyme involved in a pathway’s entry point can be removed via gene knockout [6]. However, these gene knockouts can also make cells non-viable for culturing during the lysate preparation. To circumvent this problem, alternative strategies, such as inserting affinity tags and removing proteins via affinity columns, will be explored [7]. Once gene candidates and their optimal method of removal are determined, the student will be trained by the co-mentor to carry out gene removal, cell-free lysate preparation, cell-free reaction assembly, and metabolite profile assessments.
 
=== SURF Student Qualifications: ===

Prerequisite coursework: Bi1x (or a similar introductory biology course that provides a basic foundation in molecular biology and biochemistry)

Preferred coursework: BE 150, BE/APh 161, and at least one biology course with a lab component

Other: basic familiarity with coding preferred to aid in experiment design and data analysis (Python, etc.)

=== References: ===

[1] Gregorio, N.E.; Levine, M.Z.; Oza, J.P. A User’s Guide to Cell-Free Protein Synthesis. Methods Protoc. 2019, 2, 24. https://doi.org/10.3390/mps2010024
 
[2] Lavickova, B. and Maerkl, S.J. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell-Free System. ACS Synthetic Biology 2019 8 (2), 455-462. https://doi.org/10.1021/acssynbio.8b00427
 
[3] Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x
 
[4] Miguez, A.M., McNerney, M.P., Styczynski, M.P., Metabolic Profiling of Escherichia coli-Based Cell-Free Expression Systems for Process Optimization. Industrial & Engineering Chemistry Research 2019 58 (50), 22472-22482. https://doi.org/10.1021/acs.iecr.9b03565
 
[5] Miguez, A.M., Zhang, Y., Piorino, F., Styczynski, M.P., Metabolic Dynamics in Escherichia coli-Based Cell-Free Systems. ACS Synthetic Biology 2021 10 (9), 2252-2265. https://doi.org/10.1021/acssynbio.1c00167
 
[6] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. https://doi.org/10.1073/pnas.120163297
 
[7] Garcia, D.C., Dinglasan, J.L.N, Shrestha, H., Abraham, P.E., Hettich, R.L, Doktycz, M.J., Lysate proteome engineering strategy for enhancing cell-free metabolite production, Metabolic Engineering Communications, 12, 2021, e00162,
https://doi.org/10.1016/j.mec.2021.e00162