This page documents the outline of expected experiments to be conducted during the wet lab phase.
To build and test a synthetic AND logic gate system in E. Coli that detects two signs of skin inflammation, which are nitric oxide (NO) and hydrogen peroxide (H₂O₂), and activates downstream gene expression only when both signals are present.
| Part | Name |
|---|---|
| BBa_K1153000 | NorV promoter. NOx detector. |
| BBa_K1216007 | wild-type pLuxR promoter |
| BBa_C0062 | luxR repressor/activator |
| BBa_C0062 | LuxI |
| BBa_E0040 | GFP |
| BBa_K4216007 | pOxyS |
| BBa_B0015 | double terminator |
| BBa_B0034 | RBS (Elowitz) |
(More detailed versions in our lab notebook)
Order custom inserts from IDT:
Digest Plasmid Backbones [puc19 & pJUMP-1A(sfGFP)] - NEB protocol (Total reaction volume 50 uL):
Mix reaction by pipetting, centrifuge briefly. Incubate at 37°C for 1 hour. Heat-inactivate at 65°C for 20 minutes.
Analysis of Digest and Gel Purification - Run digested products on 1% agarose gel. Use gel extraction kit to purify plasmids and backbones.
Ligation - Use a 1:3 vector to insert ratio. Make ligation mixture (20 uL Total Volume):
Mix components, incubate at 16°C overnight or 10 minutes at room temp. Include a vector-only negative control.
Use E. coli TOP10. Thaw 50 uL of competent cells on ice, add 2-5 uL of DNA. Incubate on ice for 30 minutes. Heat shock at 42°C for 45 seconds, then return to ice for 2 minutes. Recover in LB (no antibiotics) with shaking at 37°C for 1 hour. Plate on LB + Ampicillin (30 µg/mL) for puc19 construct or Kanamycin (50 µg/mL) for pJUMP construct. Incubate at 37°C overnight.
Plasmid miniprep + Digest - Grow colonies in 5 mL LB + respective antibiotic overnight. Perform plasmid miniprep. Digest minipreps with EcoRI + PstI and run on a 1% agarose gel to confirm insert size.
Co-transform both plasmids into fresh E. coli TOP10. Plate on LB + Ampicillin + Kanamycin. Pick colonies and grow in 5 mL LB with both antibiotics overnight. Subculture 1:100 into fresh media. At mid-log phase (OD600 ~0.4–0.6), split into tubes for induction:
Incubate 4–6 hrs at 37°C with shaking. Measure GFP fluorescence (Ex: ~488 nm, Em: ~510 nm), normalized to OD600.
Replace GFP with therapeutic gene (iaaM+iaaH complex for indole-3-acetic acid synthesis). Re-characterize using the same protocol.
To verify the function of our dual-sensor AND gate system, we designed both positive and negative controls. Negative control: E. coli transformed with only one plasmid (either the NO or H₂O₂ plasmid) and grown without inducers. This ensures that GFP expression does not occur in the absence of one or both signals. Additionally, E. coli cotransformed with both plasmids but with only one inducer added should not produce GFP either. We will test this as well.
Positive control: E. coli transformed with both plasmids and treated with both NO (DETA-NONOate) and H₂O₂ to activate the AND gate. This condition should produce the highest GFP fluorescence.
Replicates: Each condition will be performed in triples to ensure reproducibility and allow calculation of standard deviation.
This design confirms whether both signals are required for gene expression and helps validate the function of the regulatory elements (PnorV, PoxyS, Plux).
Our team followed the engineering design cycle—design, build, test, learn—in order to conduct our wet lab protocols. This page further documents our steps in the lab and how we iterated on the protocols for optimized results.
We conducted a series of validation steps to ensure that we were on the right track when performing the lab protocols. This page documents all results that were obtained from our experimentation.
During the cloning process, potential challenges included incomplete digestion, low PCR efficiency (likely due to machine errors), or ligation inefficiency. To address this, we planned to run all digests on a 1% agarose gel to confirm fragment sizes before ligation and use a 1:3 vector-to-insert ratio based on NEB’s ligation calculator to maximize success.
Improved steps:
If transformation yields few colonies, we will check antibiotic concentrations, repeat with freshly prepared competent cells, and include a vector-only negative control to confirm background levels. To prevent recombination, we will grow in NEB Stable Competent cells. For fluorescence readings, if signal strength is low, we will verify that the cultures reached mid-log phase (OD₆₀₀ 0.4–0.6) before induction, as promoter activity depends on growth phase.
The expected outcome is that E. coli cotransformed with both plasmids will only express GFP when exposed to both nitric oxide and hydrogen peroxide, confirming proper AND gate behavior. This would demonstrate that the system can detect inflammatory signals and produce a measurable output.
Successful GFP expression will validate our design and cloning process, allowing us to later replace GFP with the therapeutic genes for indole-3-acetic acid synthesis. These results will inform the next engineering cycle by confirming promoter functionality, regulatory response, and compatibility between plasmids.
Note: Complete documentation of all lab protocols we used along with exact volumes and concentrations that we changed can be found in our lab notebook.
For our next engineering cycle, we plan to: