Week 3 HW: CFPS for sfGFP, PETase, Reteplase

WEEK 3 ASSIGNMENT: CFPS REACTION DESIGN FOR SFGFP, PETASE, RETEPLASE

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

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

Referance concentration: K-glutamate (279.84 mM), Mg-glutamate (8.22 mM) are to mimic and maintain osmotic parameters of lysate in CFPS, assure ribosome, energy-replenishing enzymes, polymerase function, stabilise DNA/RNA structure etc.; both glutamic acid salts are usually found in high concentrations.

• Mg²⁺ is needed by ribosomes, T7 polymerase, and ADP phosphorylating enzymes; nucleotides, phosphate groups and PEP all bind it => optimum Mg level shifts with the rest of the recipe.

• K⁺ is a broader background that tolerates tens of mM shifts (Mg is the more sensitive one).

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

• PEP+NTP: PEP regenerates ATP directly (+the four triphosphates) - quick, expensive, risky (PEP degradation releases phosphate that can tie up Mg);

• Ribose+NMP: cheap sugar + monophosphates that will be metabolised by the lysate and supposedly turned into ATP (i.e. GMP can be partly replaced by guanine + ribose, like one paper specifies) - cheaper, harder to nail (reactions dependent on phosphate availablity, cofactors, pH etc)

“Surprisingly, we found that GMP could be replaced with a combination of guanine and ribose without sacrificing protein yield and that UMP could be replaced with its corresponding base and ribose, albeit with reduced yields.”

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

Note: A recommended starting point would be to review a least one published formulation, such as PANOx-SP (Jewett & Swartz 2004), a cost-optimized formulation from Olsen et al. (2026), and/or an AI-designed formulation from Smith et al. (2026).

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

Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for each of the following (don’t worry, you can resubmit later): sfGFP PETase Reteplase

Note: if you’re unable to login with your HTGAA keycloak account, please post in Zulip so that you can participate in the coopetition!

For each composition, specify the reagents you would adjust from the relevant categories, including salts, energy sources, building blocks, cofactors, and redox components.

C. ASIMOV KERNAL

Build T7 compatible plasmids for cell-free expression using the amino acid coding sequences below:

sfGFP MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRDHMVLHEYVNAAGITWSHPQFEK

PETase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFGGSGVSGWRLFKKIS

Reteplase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRPGGSGVSGWRLFKKIS

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).