Week 3 HW: Cell Free

A. COMPARE CELL-FREE REACTION COMPOSITIONS (20 minutes)

1. Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?

Magnesium Glutamate is more sensitive when compared to Potassium Glutamate, small deviations from the optimal concentration can lead to significant drops in protein production [20] where as research has shown that protein production is relatively independent of potassium glutamate concentration over a wide range [20]. Magnesium (Mg²⁺) binds to ATP and other nucleotides, and phosphate can pull it out of solution. So a recipe with more nucleotides or phosphate needs more Mg²⁺ to keep enough free for the ribosomes and T7 RNA polymerase. Potassium (K⁺) mostly sets the overall ionic strength and helps ribosomes and translation factors work, so it can vary over a wide range, often roughly 80-200 mM.

2. Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.

PEP acts as a high-energy phosphate donor, allowing for the regeneration of ATP from ADP and GTP from GDP via endogenous pyruvate kinase [7] while Ribose+NMP system utilizes NMPs instead of expensive NTPs which are significantly more cost-effective. The cell extract’s endogenous metabolic pathways (such as the pentose phosphate pathway or glycolysis) are harnessed to phosphorylate NMPs into NTPs using energy derived from the catabolism of ribose, glucose, or maltodextrin [9][15][16]

3. Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.

Composition A: Li et al. (2022) The reaction mixture used by Li et al. is optimized for supramolecular protein assembly[2]. Glutamate Salts: 175 mM potassium glutamate, 10 mM magnesium glutamate, and 10 mM ammonium glutamate. Energy Source: 3 mM PEP, 1.2 mM ATP, and 0.86 mM each of CTP, GTP, and UTP. Other Reagents: Includes 2.7 mM potassium oxalate monohydrate (to inhibit PEP consumption by competing pathways), 1.5 mM spermidine, 1 mM putrescine, 0.33 mM NAD, 0.27 mM coenzyme A, and folinic acid. Special Components: T7 RNA polymerase and a high concentration of amino acids (specified as 50 mM each of 19 amino acids without glutamic acid)[2].

Composition B: Vezeau & Salis (2021) This baseline composition was designed to control the dynamics and time-delay of gene expression[5]. Glutamate Salts: 100 mM total potassium glutamate and 8.67 mM total magnesium glutamate. Energy Source: 30 mM PEP, 1.5 mM each of ATP and GTP, and 0.9 mM each of CTP and UTP. Other Reagents: 50 mM HEPES, 1 mM putrescine, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, and 0.068 mM folinic acid. Amino Acids: 1.5 mM each (with leucine at 1.25 mM)[5].

Contrast and Discussion

The two compositions represent different optimization goals. Li et al. use a significantly higher potassium glutamate concentration (175 mM vs 100 mM) and a much lower PEP concentration (3 mM vs 30 mM). The inclusion of potassium oxalate in the Li et al. formulation is a notable difference, intended to stabilize the PEP energy source [2]. Conversely, the Vezeau & Salis composition includes cyclic AMP (cAMP), which is often used to stimulate transcription in systems using endogenous RNA polymerase, whereas Li et al. utilize exogenous T7 RNA polymerase [2], [5]. The extreme difference in reported amino acid concentrations (50 mM vs 1.5 mM) suggests that the Li et al. system is geared toward high-intensity synthesis, though such high concentrations are atypical for standard TX-TL reactions [2],[5],[11].

B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS (40 minutes)

Reteplese

  1. Potassium Glutamate 209.84 mM
  2. HEPES-KOH pH 7.5 75.00 mM
  3. Ribose 49.96 mM
  4. Magnesium Glutamate 10.10 mM
  5. 17 Amino Acid Mix 5.00 mM
  6. Nicotinamide 4.00 mM
  7. AMP 3.00 mM
  8. Oxidized Glutathione (GSSG) 2.55 mM
  9. CMP 2.13 mM
  10. GMP 2.13 mM
  11. UMP 2.13 mM
  12. ATP 1.25 mM
  13. Reduced Glutathione (GSH) 1.00 mM
  14. DsbC (E. coli, 25.5 kDa) 750.0 nM
  15. Water 2.900 uL

sfGFP

  1. Potassium Glutamate 209.84 mM
  2. HEPES-KOH pH 7.5 67.50 mM
  3. Ribose 40.80 mM
  4. Magnesium Glutamate 8.22 mM
  5. 17 Amino Acid Mix 5.00 mM
  6. Nicotinamide 4.00 mM
  7. AMP 3.00 mM
  8. CMP 2.13 mM
  9. GMP 2.13 mM
  10. UMP 2.13 mM
  11. Maltose 1.10 mM
  12. Water 4.150 uL

PETase

  1. Potassium Glutamate 205.47 mM
  2. HEPES-KOH pH 7.5 67.50 mM
  3. Ribose 40.80 mM
  4. Magnesium Glutamate 8.22 mM
  5. 17 Amino Acid Mix 5.00 mM
  6. Nicotinamide 4.00 mM
  7. AMP 3.00 mM
  8. CMP 2.13 mM
  9. GMP 2.13 mM
  10. UMP 2.13 mM
  11. Maltose 1.10 mM
  12. DsbC (E. coli, 25.5 kDa) 2.63 uM
  13. Water 3.725 uL

C. ASIMOV KERNAL (OPTIONAL)

  1. Build T7 compatible plasmids for cell-free expression using the amino acid coding sequences below: sfGFP None MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQER TISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGP VLLPDNHYLSTQTVLSKDPNEKGTRDHMVLHEYVNAAGITWSHPQFEK PETase None MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGT EASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNK NWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEY RSTCPFGGSGVSGWRLFKKIS Reteplase None MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDA KPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTV ILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEA HVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP GGSGVSGWRLFKKIS
  2. Bonus question: design a T7 helper plasmid that you could add to the cell-free reaction to improve the yield of functional reteplase (general chaperones, disulfide-bond machinery, etc).

References: *[1] F. Guzmán-Chávez et al., “Constructing Cell-Free Expression Systems for Low-Cost Access,” ACS Synthetic Biology, vol. 11, no. 3, pp. 1114–1128, Mar. 2022, doi: 10.1021/acssynbio.1c00342.

[2] Z. Li et al., “Supramolecular protein assembly in cell-free protein synthesis system,” Bioresources and Bioprocessing, vol. 9, no. 1, pp. 1–14, Mar. 2022, doi: 10.1186/s40643-022-00520-8.

[3] “Cell-Free Synthetic Biology,” Jan. 2022, doi: 10.3389/978-2-88974-080-2.

[4] A. C. Hunt et al., “A rapid cell-free expression and screening platform for antibody discovery,” Nature Communications, vol. 14, no. 1, July 2023, doi: 10.1038/s41467-023-38965-w.

[5] G. E. Vezeau and H. M. Salis, “Tuning Cell-free Composition Controls the Time-delay, Dynamics, and Productivity of TX-TL Expression,” bioRxiv, Apr. 2021, doi: 10.1101/2021.04.02.438196.

[6] F. Ba, Y. Zhang, X. Ji, W.-Q. Liu, S. B. Ling, and J. Li, “Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology,” bioRxiv, June 2023, doi: 10.1101/2023.06.05.543671.

[7] Y. Liu, B. R. Fritz, M. J. Anderson, J. A. Schoborg, and M. C. Jewett, “Characterizing and alleviating substrate limitations for improved in vitro ribosome construction.,” ACS Synthetic Biology, vol. 4, no. 4, pp. 454–462, Apr. 2015, doi: 10.1021/SB5002467.

[8] Y. Zhang et al., “Protocell Arrays for Simultaneous Detection of Diverse Analytes,” bioRxiv, Feb. 2021, doi: 10.1101/2021.02.13.431022.

[9] K. Calhoun and J. R. Swartz, “An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates.,” Biotechnology Progress, vol. 21, no. 4, pp. 1146–1153, Sept. 2008, doi: 10.1021/BP050052Y.

[10] J. Failmezger, R. Nitschel, A. Sánchez-Kopper, M. Kraml, and M. Siemann-Herzberg, “Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems.,” PLOS ONE, vol. 11, no. 12, Dec. 2016, doi: 10.1371/JOURNAL.PONE.0168764.

[11] R. B. Ferguson, “Probing metabolism in anE. coli-based cell-free system reveals a trade-off between transcription and translation,” Mar. 2023, doi: 10.1101/2023.03.22.533877.

[12] J. Failmezger, M. Rauter, R. Nitschel, M. Kraml, and M. Siemann-Herzberg, “Cell-free protein synthesis from non-growing, stressed Escherichia coli,” Scientific Reports, vol. 7, no. 1, pp. 16524–16524, Nov. 2017, doi: 10.1038/S41598-017-16767-7.

[13] C. J. Whitfield et al., “Cell-free genetic devices confer autonomic and adaptive properties to hydrogels,” bioRxiv, Dec. 2019, doi: 10.1101/2019.12.12.872622.

[14] J. Shin and V. Noireaux, “Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system,” Journal of Biological Engineering, vol. 4, no. 1, pp. 9–9, July 2010, doi: 10.1186/1754-1611-4-9.

[15] C. Albayrak and J. R. Swartz, “Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair,” Biochemical and Biophysical Research Communications, Feb. 2013, doi: 10.1016/J.BBRC.2012.12.108.

[16] “Development of a cell-free strategy for the directed evolution of enzymes for high-value natural products,” Dec. 2022, doi: 10.22024/unikent/01.02.99209.

[17] K. Calhoun, “Analysis and control of central metabolic pathways during cell-free protein synthesis reactions”, [Online]. Available: https://search.proquest.com/openview/03583edd376ff2cf7ec651b6a894f872/1?pq-origsite=gscholar&cbl=18750&diss=y

[18] C. A. Elena-Real et al., “The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity,” bioRxiv, vol. 30, no. 3, pp. 309–320, Oct. 2022, doi: 10.1038/s41594-023-00920-0.

[19] M. Neerathilingam, S. Bairy, and S. Mysore, “Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase),” PLOS ONE, vol. 11, no. 2, Feb. 2016, doi: 10.1371/JOURNAL.PONE.0150153.

[20] J. Shin and V. Noireaux, “Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70.,” Journal of Biological Engineering, vol. 4, no. 1, pp. 8–8, June 2010, doi: 10.1186/1754-1611-4-8.*