Design
We began by identifying the challenge of developing acne treatments that could prevent inflammation before it occurs. Our goal was to engineer a biosensor system that detects inflammatory biomarkers and produces an anti-inflammatory output.
Based on literature review and mentor guidance, we designed a dual-plasmid AND gate system consisting of:
Biosensor modules:
- Nitric oxide (NO) detection via the PnorV, controlling LuxR expression.
- Hydrogen peroxide (H₂O₂) detection via the PoxyS, controlling LuxI.
Responder module:
GFP as a fluorescent reporter temporarily replacing the therapeutic iaaM/iaaH genes (for IAA synthesis) for system validation.
Modeling tools:
The Hill's Equation for predicting promoter dose-response levels and validating system sensitivity.
These tools helped us visualize circuit logic, select compatible plasmids (pUC19/pSB1C3 and pJUMP-1A), and determine the optimal concentration range of nitric oxide donor and hydrogen peroxide for future characterization.
Build
After finalizing the design, we synthesized custom inserts through Twist Bioscience containing EcoRI and PstI restriction sites.
We began by amplifying the gene inserts via PCR. The PCR conditions were carefully chosen based on the length of the inserts and primer design, with initial annealing temperatures set according to Tm calculations. Once amplified, the inserts were purified and analyzed using gel electrophoresis to confirm the size and yield.
PCR Protocol:
- Make master mix
Component Volume 2X Buffer 50 μL Molecular grade H₂O 32 μL Forward Primers 1.5 μL Reverse Primers 1.5 μL - Add 17 μL of master mix into a tube
- Final concentrations of PCR Buffer at 1X and primers at 0.15 μM each
- Transfer into PCR tubes and add 3 μL of DNA (PnorV insert and PoxyS insert) into each tube
- Put the tubes into a thermal cycle for 72 minutes (95°C for denaturing, 59°C for annealing, 72°C for elongation). The cycle was repeated 40 times.
In parallel, plasmids from the iGEM distribution kit were purified using miniprep protocols. The Kanamycin-resistant plasmid (pJUMP-1A) grew successfully in the TOP10 cells, while the Chloramphenicol-resistant plasmid did not yield colonies, indicating either transformation failure or issues with plasmid concentration. Instead, we chose the pUC19 vector, which was already available in our lab, and contained the correct restriction sites and a compatible ORI site with pJUMP-1A.
Transformation Protocol for Vectors:
- Used the iGEM distribution kit plates for this
- Each well has 1-2 ng of DNA, add 10 μL of distilled water to each well needed by punching a hole through the foil—pipette up and down, let sit for five minutes. The resulting mixture should be red due to cresol red dye (it was decidedly NOT red, rather a very bright yellow)
- Used competent TOP10 E. coli cells from -80°C
- Used 3 μL of each vector (chloramphenicol resistant and kanamycin resistant) and added it to 25 μL of TOP10 E. coli
- Get 4 different tubes: chloramphenicol resistant vector + TOP10 E. coli, kanamycin resistant vector + TOP10 E. coli, negative control with TOP10 only and no vector
- Let it sit for 5 minutes on ice
- Heat shock it for 45 seconds at 42°C
- Return to ice immediately for recovery for 1 minute
- Add 100 μL SOC media into each tube (over open flame for sterilization)
- Plate 100 μL from each tube onto LB + chloramphenicol and LB + kanamycin plates. 100 μL of chloramphenicol resistant vector + TOP10 E. coli (positive control) onto LB + chloramphenicol plate and 100 μL of kanamycin resistant vector + TOP10 E. coli (positive control) onto LB + kanamycin plate. 100 μL of TOP10 E. coli onto each LB + chloramphenicol and LB + kanamycin plate (negative control)
- Let them dry by the flame and incubate at 37°C
Results from transformation:
Miniprep Protocol:
Inoculated a liquid culture with the Kan resistant vector.
- Grew overnight in 37 degree Celsius shaker
- Centrifuge the liquid culture at 12,500 g for 5 minutes and discard all the medium
- Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and pipette up and down to ensure mixing (RNase was used to remove all the RNA within the bacteria)
- Add 250 μL Lysis Buffer (L7) to open up the bacteria. Mix gently by inverting the capped tube until the mixture is homogeneous. Incubate at room temperature for 5 minutes
- Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the capped tube
- Centrifuge at 12,500 g for 10 minutes. After centrifuging, all the bacteria debris should be found at the bottom of the tube
- Load the supernatant onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,500 g for 1 minute
- Discard all the flowthrough
- Add 700 μL Wash Buffer (W9) with ethanol to the column
- Centrifuge the column at 12,500 g for 1 minute
- Discard all the flowthrough
- Repeat steps 9 and 10 to ensure everything except DNA is removed
- Place the spin column in a clean 1.5-mL elution tube
- Add 75 μL of preheated TE buffer to the column (this buffer helps detach DNA from the column)
- Incubate the column for 1 minute at room temperature
- Centrifuge the column at 12,500 g for 2 minutes
- Discard the column
- Store the elution tube (contains purified plasmid DNA) at -20°C
The next step was the restriction digestion of pUC19 and pJUMP-1A backbones with EcoRI and PstI. Insert DNA was also digested with the same enzymes to generate compatible sticky ends for ligation. Ligation reactions were performed with around 1:2.5 vector:insert molar ratio, which follows the recommendations for complex constructs.
Restriction Digest Steps:
| DNA (PnorV, PoxyS, pJUMP-1A) | DNA (pUC19) | ||
|---|---|---|---|
| Component | Volume | Component | Volume |
| DNA | 12 μL | DNA | 3 μL |
| 10x Buffer | 3 μL | 10x Buffer | 3 μL |
| EcoR1 restriction enzyme | 1 μL | EcoR1 restriction enzyme | 1 μL |
| Pst1 Restriction enzyme | 1 μL | Pst1 Restriction enzyme | 1 μL |
| Molecular H₂O | 13 μL | Molecular H₂O | 22 μL |
| Total | 30 μL | Total | 30 μL |
- Add all components in a tube
- Incubate for 1 hour at 37°C or at room temperature overnight
- Gel electrophoresis and gel extraction to confirm the lengths of the inserts and vectors post digestion
Ligation Protocol:
Plasmid sizes:
- pJUMP-1A vector: 3163 base pairs
- pUC19: 2686 base pairs
- PoxyS: 1.9 kb
- PnorV: 1.2 kb
PoxyS + Kan vector Ligation:
| 10X T4 Ligase Buffer | 2 μL |
| Kan vector | 3 μL |
| PoxyS | 8 μL |
| Ligase | 1 μL |
| Nuclease-free water | 6 μL |
pUC19 + PnorV Ligation:
| 10X T4 Ligase Buffer | 2 μL |
| pUC19 | 2.5 μL |
| PnorV | 5.5 μL |
| Ligase | 1 μL |
| Nuclease-free water | 9 μL |
Combine and incubate overnight at 16 degrees Celsius in thermocycler
Initial transformations of ligation products into TOP10 cells failed to produce colonies. To improve efficiency, we allowed the bacteria to 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. This adjustment resulted in successful colonies, which we picked 10 of each plasmid type to grow in LB overnight to increase plasmid yield. Minipreps were performed to isolate the plasmids, which then went through PCR with the insert primers. This step was performed so that if the insert had been successfully ligated into the plasmid, the primers would anneal to the flanking regions and amplify the insert. Gel electrophoresis of these PCR products confirmed the presence or absence of bands corresponding to the expected insert size.
We selected plasmids from colonies 5 and 7 from the pUC19-PnorV plasmids and plasmids from colonies 1 and 7 from the pJUMP-PoxyS plasmids.
Test
To validate the assembly, we:
- Performed transformations into E. coli TOP10 using heat shock and antibiotic selection
- Sent plasmids for sequencing to confirm correct assembly
Sequencing results revealed that most plasmids were empty backbones without inserts. One Kanamycin vector showed recombination with E. coli genomic DNA, resulting in unintended GFP expression. This indicated that while some ligations had occurred, the majority of colonies did not carry the intended constructs.
Results:
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
pJUMP-PoxyS from Colony 1
pJUMP-PoxyS from Colony 7
Build issues:
- Sequencing results showed high levels of undigested vector
- We also had unwanted plasmid recombination with the E. coli genome
These outcomes may be due to digestion failure and low insert:vector ratios despite continued PCR.
Learn
From our troubleshooting and results, we gained several key insights:
We learned several key lessons from Build 1:
- Complete digestion and higher insert:vector ratios are critical for ligation success
- TOP10 cells may undergo higher recombination rates, making them suboptimal for preventing recombination in our constructs
- PCR efficiency can be limited by errors in machine cycling or elongation times, affecting insert concentration
- Gel extraction led to significant DNA loss; alternative purification methods like PCR cleanup may preserve more material for downstream applications
Design
Based on the lessons learned from our first iteration, we optimized our approach for the second build cycle.
Build
In Build 2, we implemented lessons from the first round to improve our workflow. We performed an optimized PCR to maximize insert concentration, extending elongation times and adjusting cycle numbers. PCR cleanup was used instead of gel extraction to prevent DNA loss through the extraction process.
Restriction digests were repeated and incubated overnight to ensure complete digestion. Gel electrophoresis was used to confirm the digest success.
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.
Loading order (R → L): ladder, undigested pUC19, digested pUC19, undigested pJUMP, digested pJUMP, digested PnorV, digested PoxyS.
Insert bands were faint which may be due to low PCR yield, so we reused digested inserts from Build 1 for ligation. Ligation reactions were performed using the optimized 3:1 insert:vector ratios. We cut out the bands for the digested vectors and performed gel extraction to only use the digested vectors. We then transformed the ligated product.
Transformed products were plated on selective media. TOP10 cells were used as NEB Stable competent cells had not yet arrived. Unfortunately, no colonies were obtained, and only the negative control showed bacterial growth. This suggested possible errors in PCR amplification during the elongation cycles, resulting in insufficient concentration of insert DNA despite using a maximally optimized protocol, which may have affected the ligation reaction.
Ligation and Transformation Protocol (Build 2):
Ligation set up (aiming for 3:1 or 4:1 ratio). Used 2μL 10× Ligase Buffer and 1μL Ligase per reaction. Total volume 20μL.
- pUC19 + PnorV: 2μL pUC19 (2686 bp), 15μL PnorV (1.2 kb).
- Kan + PoxyS: 3.5μL Kan (3163 bp), 13.5μL PoxyS (1.9 kb).
Ligation mixes incubated at RT for 2 hours.
Transformation into Top10 E. coli cells from −80°C. Positive and negative controls included.
Protocol:
- 25μL Top10 cells added to all tubes.
- 5μL ligation mix added to positive control.
- 5μL digested backbone (no ligase) added to negative control.
- Incubated on ice for 30 min.
- Heat shock in 42°C water bath for 45 sec.
- Incubated on ice for 5 min.
- Added 200μL SOC media (near flame) → vortexed.
- Incubated in 37°C shaker for 1 hr.
- Plating: 100μL plated onto corresponding antibiotic plates. Plates dried, inverted, and put into incubator O/N.
Learn
Lessons learned:
- Maintaining high insert concentration is essential; even small decreases in DNA quantity can prevent colony formation
- Digestion efficiency must be monitored closely; faint insert bands indicate lower yield that can compromise ligation
- Using recombination-resistant strains such as NEB Stable cells may be necessary for our constructs to avoid unintended genome integration into our plasmids
- Iterative troubleshooting, including PCR optimization, digestion confirmation, and ligation efficiency adjustments, is critical for successful assembly
These steps provided extensive hands-on experience with PCR optimization, plasmid miniprep, gel extraction, restriction digestion, NanoDrop spectrophotometry, ligation, transformation, miniprep, troubleshooting, and insert confirmation.
All Troubleshooting Insights
- PCR step didn't yield high enough insert DNA concentration → re-do with longer elongation step and more cycles
- Low insert DNA concentration after gel extraction → switch to PCR cleanup
- 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 concentration of pJUMP 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 concentration of pJUMP 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 to 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 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 concentrations in vitro
- Follow-up with potential stakeholders regarding introducing this system into research studies and trials