Results

Overview

Our main goal was to build a dual-plasmid biosensor system that could detect nitric oxide (NO) and hydrogen peroxide (H₂O₂) and produce GFP as a reporter to temporarily replace IAA. We went through several rounds of cloning to get there, adjusting our process each time based on what went wrong before. Although we weren’t able to fully confirm the constructs by the deadline, we learned a lot from the build process and used modeling and literature to support the logic behind our design.

Experimental Results

We completed multiple Build cycles, each one focused on troubleshooting issues from the last:

Step	Observations	Notes
Round 1 – Digestion & Ligation	Confirmed through gel electrophoresis	DNA yield was low after gel extraction, it is uncertain whether vectors are properly digested and ligated or not based on band size
Round 1 – Separate Transformation	Colonies grew, but sequencing results showed empty backbones and recombination	Insert:vector ratio was likely too low due to lost DNA yield in the gel extraction step
Round 2 – Digestion & Ligation	Switched to PCR clean up protocol after insert PCR to increase yield of insert Incubated the digestion reaction overnight for increased effectivity, ran gel, and ligated Gel results: digestion of pUC19 and pJUMP worked. For pUC19, the restriction enzymes weren’t cutting out a section of the plasmid, which is why the digested and undigested are similar in length. However, the undigested is still slightly further down because it is in circular form while the digested is linearized. Linear will move slightly slower in the gel than the circular DNA. For the pJUMP vector, the undigested form is heavier and intact while the digested moved farther down, which means it has been successfully digested by the restriction enzymes.	DNA recovery improved slightly through PCR cleanup as opposed to gel extraction
Round 2 – Transformation	Standard transformation protocol Colonies did not grow on the antibiotic plates	Possible reasons: incomplete ligation despite longer incubation time, ineffective PCR

The high background vectors and possibly incomplete digestion were our main issues, but we also realized that our strain choice (TOP10) may have been causing the recombination with the E. Coli genome.

Plasmid Maps

Plasmid Sequencing results from Plasmidsaurus during Round 1 of the Transformation

Puc19-PnorV refers to the plasmid containing the insert responsible for detecting nitric oxide and producing LuxR

pJUMP-PoxyS refers to the plasmid containing the insert responsible for detecting hydrogen peroxide and producing AHL, and then finally GFP.

puc19-PnorV from colony 5

Puc19-PnorV from colony 7

Both Ampicillin 5 and 7 vectors were empty, indicating that they consisted of an undigested pUC19 backbone without any DNA fragments from the insert. The ligation process was unsuccessful for ampicillin vectors.

Kanamycin vector 1 has recombined with some chromosomal DNA from E. Coli, resulting in an incorporation and expression of the GFP gene, which was not part of what we had intended to include. Kanamycin vector 2, similar to the ampicillin plasmids, was also an undigested vector.

For more confirmation steps, please visit our lab notebook, which includes a detailed list of all procedures we performed in this process, along with analysis of any validation steps.

pJUMP-PoxyS from colony 1

pJUMP-PoxyS from colony 7

Analysis

While we couldn’t confirm the plasmids experimentally, our modeling results and research back up the design:

• Using Hill’s Equation, we modeled promoter activation for NO and H₂O₂ and found the expected response curves.
• Literature supports that IAA reduces inflammation by blocking p65 nuclear translocation, lowering NF-κB activity, and neutralizing ROS.

Together, that gives us confidence that our system design should work once the build is complete.

Troubleshooting Takeaways

• Low insert DNA concentration from PCR → redo with longer elongation step and more cycles
• Low DNA concentration after gel extraction → switch to PCR clean up
• No colonies for initial choice of chloramphenicol resistance vector → switch to pUC19 instead, it has a more compatible origin of replication too with pJUMP-1A and decrease antibiotic concentration
• Low vector concentration after vector-only transformation and miniprep → inoculate E. Coli again with the vector and grow for a longer period of time in a liquid culture before miniprepping
• Low vector concentration post-restriction → inoculate E. Coli again with the vector, grow in SOC media and improve DNA yield
• No colonies after post-ligation vectors were transformed → Re-do transformation, allow the bacteria incubate on ice for 30 minutes before the heat shock and let the transformed cells recover in SOC media for one hour before plating on selective media.
• Sequencing showed incomplete digestion and ligation → optimize PCR for longer elongation time and more cycles, let the restriction digest incubate overnight at room temperature, ligate with higher insert:vector concentrations.

Next Steps

For our next engineering cycle, we plan to:

• Repeat digestion with fresh restriction enzymes and extended incubation.
• Research maximal optimized protocols for each step in the cloning protocol.
• The PCR protocol will be improved through a longer elongation step and more cycles to improve insert concentration after performing gel extraction.
• Use PCR cleanup exclusively for inserts.
• Increase insert DNA concentration and ligation ratio.
• Transform into NEB Stable cells and test with GFP with fluorescence assays to validate the predicted AND gate logic.
• Measure different concentrations of inducers to create a dose-response curve and compare with our Hill’s equation model
• Conduct research into safe and optimal concentrations of IAA to use in our plasmid system such that the final product will be both safe to apply on the skin and effective in reducing inflammation.
• Replace GFP with indole-3-acetic acid complex (iaaM and iaaH genes) and test inducer and output concentrations both in-vitro and in-vivo.