Week 13 HW: Principles and Practices

fire fire

1. Prove lead-sensing logic

Aim: I am designing a modular genetic cassette in which a heavy-metal sensing element, likely pbrR/pbr, controls either a standard reporter or a geosmin output module, with HSR considered as an auxiliary stress-sensitive element rather than the primary detector

pb pb

Design intent: Detect bioavailable Pb(II) using a PbrR/pbr lead-responsive sensing module. Initial output will be a fluorescent reporter to validate sensing behavior. A later version may replace the reporter with a geosmin-associated output module for sensory repair signaling.

Cassette v1 should respond to bioavailable Pb(II) PbrR + pbr promoter/operator → reporter

think think

Building aa Benching Circuit Template Promoter → pbrR → Terminator → pbr promoter → sfGFP → Terminator benchling benchling benchling

Assuming E. coli as initial chassis for circuit implementation

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2. Make this biologically plausible + lab-ready

I updated the annotiations

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Design uses J23100 to constitutively express pbrR (from C. metallidurans). Lead-bound PbrR activates the pbr promoter controlling sfGFP.

Terminators used to isolate transcriptional units. Designed for E. coli chassis. ##

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Questions for Elliot

  • I designed a lead-specific PbrR system. I’m wondering if you’d like me to instead—or additionally—explore an HSR-based stress-responsive promoter
  • Is PbrR/pbr a good system in your lab’s E. coli setup?
  • Would you recommend validating with GFP before attempting a geosmin output?
  • What are my next steps?

  • Anwers from Eliot

    To validate put a small amoont of Lead nitrate ((\text{Pb(NO}{3}\text{)}{2})) or Lead acetate in an agar plate and grow ecoli in the presence of lead. Go to addgene. search PbrR and get the plasmid pjl74y. Regarding petrachor/ geosmin you need to add a that oroduces a similar smell. Use Goldergate to design DNA that emits geosmin smell.List all the supplies i need to conduct the basic experiment and email Elliot.