iGEM 2025 — Team bostonu

[![Back to top](https://static.igem.wiki/teams/5875/menu/vibi-arrows-totop.webp)](#top)

* [Plasmid Design](#plasmid)
* [Backbone Preparation](#backbone)
* [HiFi Assembly and Transformation](#hifi)
* [Growth Curve](#growth)
* [Dose Responses](#dose)
* [Validation Tests](#valid)

![Hardware header](https://static.igem.wiki/teams/5875/notebook/notebook.webp)

As members of the iGEM Community, we strongly believe that sharing our process along with our results will contribute to the integrity of science. As such, we have provided the following drop down menus of our experimentation. We have done our best to provide details such that another team could replicate our results. If anyone is unable to do this, or has questions about our procedural methods, please do not hesitate to reach out to our email. Science is collaborative and we are willing to help anyone who asks.

[ ]
Week of 6/2/25: Plasmid Design

Purpose:

Design a putrescine-responsive circuit in  *E. coli*

Methods:

We designed the genetic circuit shown below. This circuit constitutively expresses PuuR, a protein that binds to the pHub(1B) inducible promoter. When putrescine is present, the repressor protein PuuR is stopped and eGFP is produced. mCherry is included downstream of PuuR to ensure expression of the PuuR protein.

![Putrescine Circuit](https://static.igem.wiki/teams/5875/notebook/notebook1.webp)

Figure 1. Putrescine Repressor Circuit

We obtained sequences for the PuuR repressor protein and Phyb1B promoter from the literature (Selim et al., “A Synthetic Biosensor for Detecting Putrescine in Beef Samples.”). We chose to use a plasmid with mCherry (Addgene #68142) as our backbone, to shorten the length of the gBlock we would order. The sequences were all imported into SnapGene for plasmid design.
The assembly was simulated using the SnapGene HiFi tool. The backbone was cut with HindIII and XbaI and the following insert was cloned. Homology regions were designed for each end of the insert, with the following specifications: melting temperature of 60 degrees C and 20-30 base pairs long. Cut sites on the backbone and exposed ends on the insert were checked for hairpins and altered such that no hairpins formed above 50 degrees C.

![Inducible Insert](https://static.igem.wiki/teams/5875/notebook/notebook2.webp)

Figure 2. Inducible Insert Design

We also designed a similar insert, with the only change being swapping a T7 constitutive promoter instead of the pHyb(1B) inducible promoter. The purpose of this design was to observe GFP expression, and to have a measure to quantify the inducible promoter expression against.

![Constitutive Insert](https://static.igem.wiki/teams/5875/notebook/notebook3.webp)

Figure 3. Constitutive Insert Design

Results:

Finalized simulated assembly of the gBlock into the mCherry backbone.

![Plasmid Assembled](https://static.igem.wiki/teams/5875/notebook/notebook4.webp)

Figure 4. Plasmid Assembled in SnapGene

Notes:

We initially ran into challenges choosing cut sites on the backbone, as hairpins were predicted to form above 50 degrees C. This caused us to pivot to using different enzymes than we initially planned.

[ ]
Week of 6/9/25: Plasmid Design

Purpose:

Order gBlocks

Methods:

We finalized the insert designs and ensured the complexity was appropriate for IDT to synthesize.

Results:

Ordered 2 gblocks from IDT, the mCherry backbone from Addgene, and restriction enzymes from NEB

[ ]
Week of 6/16/25: Backbone Preparation

Purpose:

Prepare for plasmid assembly and cloning.

Methods:

Streaked out plates with mCherry.

Notes:

We noticed that the agar stab delivered by Addgene had both green and pink bacteria growing. The plate that was streaked out also had pink and green colonies. See images below.

![Agar Stab](https://static.igem.wiki/teams/5875/notebook/notebook5.webp)

Figure 5. Agar Stab

![Streaked Plate](https://static.igem.wiki/teams/5875/notebook/notebook6.webp)

Figure 6. Streaked Plate

[ ]
Week of 6/23/25: Backbone Preparation

Purpose:

Prepare for plasmid assembly and cloning.

Methods:

We made overnights of the mCherry backbone, miniprepped, stocked, and sent for sequencing. We miniprepped using the “low-copy” protocol for better results.

Results:

Initially the concentration of DNA after miniprepping was less than 100 ng/uL, which was lower than we expected for a high-copy plasmid. The 260/280 ratio was within the normal range of 180-2, so we didn’t think contamination was an issue.
After switching to the low-copy miniprep protocol we obtained eluted DNA with a concentration above 120 ng/uL.

Notes:

The sequencing results were perfect except for a 1 bp mutation in the origin of replication.

[ ]
Week of 6/30/25: HiFi Assembly & Transformation

Purpose:

Clone the inserts into the backbone.

Methods:

We performed a restriction digest on the isolated mCherry backbone DNA. After rehydrating the gblocks with our DNA inserts, we performed HiFi assembly and transformed the pHyb(1B)-plasmid into NEB Dh5-α.

Results:

Correct sequencing results for the pHyb(1B)-plasmid in NEB Dh5-α.

![Restriction Digest](https://static.igem.wiki/teams/5875/notebook/notebook7.webp)

Figure 7. Restriction Digest of mCherry Backbone

[ ]
Week of 7/7/25: HiFi Assembly & Transformation

#### HiFi Assembly

Purpose:

Clone the constitutive-insert into the mCherry backbone.

Methods:

We performed HiFi assembly and transformed the constitutive-plasmid into NEB Dh5-α.

Results:

Correct sequencing results for the constitutive-plasmid in NEB Dh5-α.

#### Transformation into BL21(DE3)

Purpose:

It was necessary to transform the plasmids into BL21 (DE3) because the design includes T7 components, which require T7 RNA polymerase (only in BL21).

Methods:

We followed the protocol for transformation into ThermoFisher BL21 (DE3) competent cells.

![Thermo Transformation Protocol](https://static.igem.wiki/teams/5875/notebook/notebook8.webp)

Figure 8. ThermoFisher's Transformation Protocol

Results:

Transformation was unsuccessful with the ThermoFisher competent cells, and the puc19 positive control showed no growth.

[ ]
Week of 7/14/25: Transformation into BL21(DE3)

Purpose:

Alter the transformation protocol to obtain successful transformants.

Methods:

We switched to NEB BL21 (DE3) competent cells and followed the protocol below.

![NEB Transformation Protocol](https://static.igem.wiki/teams/5875/notebook/notebook9.webp)

Figure 9. NEB Transformation Protocol

The initial transformation showed one constitutive-plasmid colony, which we cultured, miniprepped, and sequenced.
In order to transform the inducible-plasmid, we altered the NEB transformation protocol by heat-shocking for 30 seconds at 40 degrees C.

Results:

The sequencing of both plasmids came back with perfect results.

![Success Constitutive-plasmid](https://static.igem.wiki/teams/5875/notebook/notebook10.webp)

Figure 10. Successful Transformant of the Constitutive-plasmid into BL21

![Success inducible plasmid](https://static.igem.wiki/teams/5875/notebook/notebook11.webp)

Figure 11. Successful transformant of the inducible-plasmid into BL21

[ ]
Week of 7/28/25: Growth Curve

Purpose:

Gain insight into the growth mechanics of our plasmid.

Methods:

We performed a growth assay on both the plasmids, as detailed below

1. Grow overnights in culture tubes with 5mL LB and 5uL of ampicillin. Incubate at 37 C for 15-18 hours.
2. Measure OD600.
3. Spin down culture such that OD = 1 in a volume of 1mL. Resuspend the pelleted cells in 1mL of LB + ampicillin.
4. Dilute the resuspended cells at 1:100 in LB + amp, for a starting OD of 0.01
5. In a 96 well plate, add 200uL of diluted resuspended cells per well in triplicate.
6. Measure OD600 every hour, with the plate reader at 37C, shaking 800rpm, for 22 hours.

Results:

There were differences in growth between the three plasmids. This could be due to differing metabolic burdens on the cells.

![growth curve](https://static.igem.wiki/teams/5875/notebook/exp7-growthcurve.webp)

Figure 12. Growth Curve

[ ]
Week of 8/4/25: Dose Responses

#### Dose Response: IPTG

Purpose:

Determine the optimal concentration of IPTG for induction.

Methods:

1. Grow overnights in culture tubes with 5mL LB and 5uL of ampicillin. Incubate at 37 C for 15-18 hours.
2. Measure OD600.
3. Spin down culture such that OD = 1 in a volume of 13mL. Resuspend the pelleted cells in 1mL of LB + ampicillin.
4. Incubate at 37C until it reaches an OD of ~1 (~4hours). Measure OD each hour.
5. In a 96-well black-wall clear-bottom plate, induce cultures with varying concentrations of IPTG {0, 0.1, 0.3, 0.5, 0.7, 1} mM.
6. Incubate for 18 hours shaking at 37C in a plate reader. Measure OD600, GFP, and RFP fluorescence every 20 min.

Results:

We determined 0.5mM to be the optimal concentration of IPTG to use for future testing. See the <experiments> page for more details.

![Dose Response GFP](https://static.igem.wiki/teams/5875/notebook/exp8-2-voo4-gfp.webp)

Figure 13. GFP values divided by corrected OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

![Dose Response RFP](https://static.igem.wiki/teams/5875/notebook/mean-baseline-corrected-of-rfp-od-v004.webp)

Figure 14. mCherry values divided by corrected OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

Figures show fluorescent signal after 14 hours incubation with IPTG

#### Dose Response: Putrescine

Purpose:

Determine the response of the inducible promoter to exogenous putrescine.

Methods:

1. Grow overnights in culture tubes with 5mL LB and 5uL of ampicillin. Incubate at 37 C for 15-18 hours.
2. Measure OD600.
3. Spin down culture such that OD = 1 in a volume of 13mL. Resuspend the pelleted cells in 1mL of LB + ampicillin.
4. Incubate at 37C until it reaches an OD of ~1 (~4hours). Measure OD each hour.
5. In a 96-well black-wall clear-bottom plate, induce cultures with 0.5mM IPTG and varying concentrations of putrescine {0, 10, 100, 500} mM.
6. Incubate for 18 hours shaking at 37C in a plate reader. Measure OD600, GFP, and RFP fluorescence every 20 min.

Results:

![GFP](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-15.webp)

Figure 15. GFP divided by OD600 (corrected) for V004 induced with putrescine and IPTG. Wells with clumped cells not plotted

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-16.webp)

Figure 16. Raw mCherry fluorescent values for V004 induced with putrescine and IPTG. Wells with clumped cells not plotted

Notes:

There was significant clumping of cells observed with high concentrations of putrescine.

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-17.webp)

Figure 17. Settling and clumping of cells during the kinetic run.

#### Putrescine Dose Response 2.0

Purpose:

Avoid clumping of cells during the dose response assay.

Methods:

Same protocol as above, but use new varying concentrations of putrescine {0, 1, 10, 50} mM.

Results:

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-18.webp)

Figure 18. GFP divided by OD600 (corrected) for V004 induced with putrescine and IPTG

Notes:

There was much less, but still some visible clumping of cells observed with higher concentrations of putrescine.

![clumping](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-19.webp)

Figure 19. Settling and clumping of cells

[ ]
Week of 9/22/25: Validation Test

Validation Test 1.0

We performed the Validation Test, first version, as described on the Experiments page.

Validation Test 2.0

We performed the Validation Test, second version, as described on the Experiments page.

Genetic Design

We designed an alternative gblock that replaces the T7 promoter with a constitutive, non-inducible promoter (pVeg1). Based on our experimental results, we hypothesized that IPTG was having an unwanted influence on our biosensor. By removing the T7 promoter, we eliminate the need to induce the cells with IPTG, thus simplifying the genetic system.
Additionally, this new design will always repress GFP, ensuring that GFP is only turned on when putrescine is present. This will allow for tighter control of GFP, and thus stronger and more reliable quantification of putrescine.

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-20.webp)

Figure 20. New genetic design, with T7 promoter swapped for pVeg1

[ ]
Week of 9/29/25: Validation Test & Dose Response

Validation Test 3.0

We performed the Validation Test, third version, as described on the Experiments page.

IPTG Dose Response in M9 Media

We performed an IPTG dose response in M9 media, as described on the Experiments page.

[ ]
Week of 10/6/25: Eliminating Promoters

Group Meeting:

During our group meeting this week, we presented confusing experimental data we collected to Professor Jimenez. He noticed that our plasmid included two additional promoters that could be influencing our system unintentionally: a LacUV5 promoter upstream of the PuuO synthetic promoter, and a reverse T7 promoter downstream of the mCherry CDS.

We developed a plan to cut our plasmid to eliminate these additional promoters.
![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-21.webp)

Figure 21. Whiteboard brainstorming of our plan to remove the undesired sections on the biosensor

1. Fix V002 (mCherry backbone with V004 design in DH5a).

1. To do this, we first need to cut out the lacUV5 promoter with NspI enzyme. This will result in the plasmid shown below.
   ![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-22.webp)

   Figure 22. V004 with the LavUV5 promoter removed.
2. Second, we need to remove the reserve T7 promoter. To do this, we need a few things: NotI enzyme, DraIII enzyme, and Quick Blunt kit.
   ![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-23.webp)

   Figure 23. V004 with the LavUV5 promoter and reverse T7 promoter removed.

2. Clone the new gblock into the backbone, but without the bad promoters.

1. We will cut out the LavUV5 promoter and reverse T7 promoter with the same method as above. This will result in a “clean” backbone to clone into.
   ![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-24.webp)

   Figure 24. “Clean” backbone with the LavUV5 promoter and reverse T7 promoter removed.
2. Use HiFi assembly to clone the new gblock (with pVeg1) into the “clean” backbone.
   ![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-25.webp)

   Figure 25. “Clean” backbone with the LavUV5 promoter and reverse T7 promoter removed, and new gblock inserted.

After these steps are complete, we plan to perform more experimental testing to determine if changing the promoters will improve the response of our biosensor.

Plates:

We made plates of our backbone (mCherry), V002 (PuuR/PuuO system in Dh5a), and V004 (PuuR/PuuO system in BL21).

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-26-1.webp)

Figure 26. Images of plates

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-26-2.webp)

Figure 26. Images of plates

From these plates, we made overnights to begin the process of cutting out the promoters.

Miniprep:

Colonies we miniprepped:

* V004.1
* V004.2
* V002.1
* V002.3
* V002.. → from a good colony
* V002 big → from a big colony
* V001.1
* V001.2

We obtained high yield from V002 and V004 colonies. V001 did not produce high enough yield to continue through the process of digestion.

Digest with NspI:

We followed the lab protocol for restriction digestion.

Notes:

* Digest V001.2 and V002.big and V004.2.
* Run at 37 C for 30 minutes.

After the digestion, we added loading dye and ran it on a gel to separate the linearized backbone DNA from the insert that was cut out.

Preparative Gel

After the digestion, we added loading dye and ran it on a gel to separate the linearized backbone DNA from the insert that was excised.

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-27.webp)

Figure 27. Predicted gel simulated on snapgene

DNA Cleanup

We performed a cleanup on the DNA to extract the DNA from the gel. We selected the highest-yield sample to continue with for a ligation. We performed the ligation twice in parallel, one with Hi-T4 quick ligase, and the other with T4 ligase, which requires overnight incubation. We hope to compare them and select the best result.

1. Quick

1. Ligate with-Hi T4 Quick Ligase using the NEB protocol for Hi-T4™ DNA Ligase (NEB #M2622)
2. Add 50ng of DNA for a final concentration of 2.5ng/uL
   1. Concentration of DNA from cleanup: 137.87 ng/uL
   2. 2.5 ng/uL \* 20 uL = 137ng/uL \* x → 0.36uL
3. Put on thermocycler

1. 10 min 25 C
2. 10 min at 65C
3. Hold at 4

4. Transform

Components to combine:

1. 2uL buffer
2. 0.36uL DNA
3. 16.64 NFW
4. 1 uL Hi-Ligase

2. Slow

1. Ligate with T4 Ligase using the NEB protocol for Ligation Protocol with T4 DNA Ligase (M0202)

Components to combine:

1. 2uL buffer
2. 0.36uL DNA
3. 16.64 NFW
4. 1 uL (normal) Ligase

Transformation of Quick Ligase

1. Only transform Hi-T4 Quick Ligated DNA
2. Need 4 tubes of comp cells (DH5a): C2987

* One for pUC19
* One for V002\_qx outgrowth
* One for V002 outgrowth
* One for V002\_minusLigase outgrowth

3. Samples to plate: total of 13 plates

* Puc19 : transformation control
* V002\_qx (1x) 100uL plated
* V002\_qx (3x) 100uL plated
* V002\_qx (3x) 100uL plated
* V002\_qx (3x) 100uL plated
* V002 (1x) 100uL plated
* V002 (3x) 100uL plated
* V002 (3x) 100uL plated
* V002 (3x) 100uL plated
* V002\_minusLigase (1x) 100uL plated
* V002\_minusLigase (3x) 100uL plated
* V002\_minusLigase (3x) 100uL plated
* V002\_minusLigase (3x) 100uL plated

Steps for making 3X DNA

1. Plate 100uL of 1X (normal DNA)
2. Spin down for 1 min the remaining 900uL of DNA in SOC media
3. Discard 600uL of media
4. Resuspend
5. Plate 100uL on 3 plates

Quick Ligase Transformation Results

![](https://static.igem.wiki/teams/5875/notebook/notebook/notebook-28.webp)

Figure 28. Images of transformation plates for V002\_2 after the quick ligation.

We transformed V002\_2 (which is V004 in Dh5alpha, missing the LacUV5 promoter). We then picked 8 samples to send for sequencing, and 6 out of 8 came back with correct sequences.

[ ]
Future Directions

In the upcoming weeks, we will continue with the plan listed above to alter our genetic design with the aim of increasing the desired response of our biosensor.

We plan to push two genetic design plans forward:

1. Edit our current biosensor. This will include the following steps:
   1. Digest with NotI and DraIII
   2. Use blunting kit
   3. Ligation
   4. Transformation into DH5α
   5. Miniprep
   6. Transformation into BL21

1. Insert the new gBlock into the original backbone. This will include the following steps:
   1. Miniprep successfully
   2. Digest with NotI
   3. Gel
   4. Cleanup
   5. Sticky end ligation
   6. Transformation into DH5α
   7. Miniprep
   8. Digest with NotI and DraIII
   9. Blunting kit
   10. Ligation
   11. Transformation into DH5α
   12. Miniprep
   13. Transformation into BL21

Once these plans have been accomplished, we will perform testing on our new plasmid designs and compare to our previous results.

##

[![Back to top](https://static.igem.wiki/teams/5875/menu/vibi-arrows-totop.webp)](#top)

* [Putrescine Biosensor](#putrescine)
* [Optical System](#optical)
* [Hydrogel Tray and Mechanics](#hydrogel)
* [Graphical User Interface](#graphical)

![Engineering header](https://static.igem.wiki/teams/5875/engineering/engineering.webp)

# Overview

As engineers, it is important that we follow a cycle based on learning and improvement to creat the best, most effective product we can. Therefore the following page describes our iteration through the design, build, test, learn cycle and how it applies to all aspects of our project.

![DBTL Cycle Diagram](https://static.igem.wiki/teams/5875/engineering/dbtl-final.webp)

Figure 1. DBTL Cycle

# Putrescine Biosensor

---

#### Reason

Objective:

* Engineer a genetic circuit that allows *E. coli* biosensors to detect putrescine concentration and convert it into an optical fluorescence output.

Need:

* A sensing mechanism that specifically responds to putrescine, produces a measurable fluorescence signal, and includes internal controls to ensure circuit reliability.

#### Research

Promoter Selection:

* Adopted a custom promoter design from published work that is regulated by the putrescine-sensitive protein PuuR.

Regulatory Mechanism:

* Studied how PuuR acts as a transcriptional repressor, binding to the promoter in the absence of putrescine and releasing upon ligand binding.

#### Design

Primary Circuit:

* Placed GFP downstream of the putrescine-sensitive promoter. In the absence of putrescine, GFP is repressed; in its presence, PuuR binds putrescine instead of the promoter, enabling GFP expression.

Secondary Circuit:

* Added a constitutive promoter upstream of the puuR gene to ensure continuous production of the PuuR protein. Coupled this with an mCherry reporter downstream of puuR to verify repressor production and provide a baseline fluorescence reference.

Dual-Color Output:

* Established a two-signal system: GFP as the dynamic putrescine-responsive signal, mCherry as a constitutive control for normalization.

#### Build

Plasmid Construction:

* Cloned the custom promoter, GFP, constitutive promoter, puuR gene, and mCherry reporter into a single plasmid, ensuring compatibility of expression elements.

Host Transformation:

* Introduced the plasmid into *E. coli* strains to generate stable putrescine biosensors.

#### Test

Functional Validation:

* Exposed biosensors to increasing concentrations of putrescine and confirmed that GFP fluorescence increased in proportion to putrescine levels.

Growth Assessment:

* Monitored growth kinetics of engineered plasmids to evaluate metabolic burden, revealing an extended lag phase in V004 likely due to uninduced GFP expression.

Induction Testing:

* Performed IPTG dose–response experiments to identify optimal induction levels without negatively impacting cell growth.

Biosensor Characterization:

* Exposed biosensors to varying putrescine concentrations, refined conditions to reduce clumping, and validated consistent fluorescence trends across replicates and culture formats.

Characterization results can be found on our registry part page [here](https://registry.igem.org/parts/bba-25087kbx%20) under the experimentation section. Part Number: BBa\_25087KBX

#### Learn

Improvements:

* Identified unintended regulatory elements: the LacUV5 and reverse T7 promoters. These elements potentially affect system behavior and could contribute to unexpected expression patterns.

Optimization:

* Planned plasmid modifications to remove these extraneous promoters, refining construct design to ensure signal specificity and accurate characterization of the Putrescine-driven response.

# Optical System

---

#### Reason

Objective:

* Develop a compact fluorescence optical system to accurately quantify GFP expression from putrescine-responsive bacteria.

Need:

* A system capable of exciting GFP at its optimal wavelength and capturing its fluorescence emission while minimizing ambient light interference and maximizing signal-to-noise ratio.

#### Research

Component Selection:

* Selected a high-power 465 nm monochromatic LED (OSRAM OSCONIQ P 3030) for GFP excitation, a 525 nm-specific photodiode (Marktech MTD5052D3) for emission detection, an aspheric lens for light focusing, and a HOYA Y52 longpass filter to reduce background interference.

Circuit Testing:

* Investigated photodiode sensitivity by building a simple op amp circuit (LMP7721, non-inverting configuration, gain = 2) with two 1 kΩ resistors to characterize response under controlled dark-box conditions.

#### Design

System Layout:

* Designed a compact layout positioning the excitation LED, aspheric lens, longpass filter, and photodiode to optimize GFP fluorescence detection while reducing direct LED leakage.

Thermal Management:

* Integrated a copper heatsink into the PCB-mounted LED design to manage thermal resistance during prolonged operation.

#### Build

Assembly:

* Assembled the optical components within a shielded enclosure to block stray light and preserve signal integrity.

Positioning:

* Mounted the 520 nm green LED directly above the sample chamber and aligned the 525 nm photodiode with focusing optics and filter to maximize emission capture efficiency.

#### Test

Calibration:

* Tested the system in a dark box, using a 520 nm green LED as a controlled excitation source to verify photodiode response and sensitivity.

Sensitivity Testing:

* Recorded photodiode voltage output while decreasing LED drive voltage in 0.1 V steps, producing a calibration curve of sensor response versus input light intensity.

#### Learn

Improvements:

* Identified the need for signal amplification between the photodiode and data acquisition system, addressed with an op amp gain stage.

PCB:

* Designed custom PCBs for stable LED mounting and wiring, improving reliability and ease of integration into the optical housing.

# Hydrogel Tray and Mechanics

---

#### Reason

Objective:

* Develop a reliable housing method for putrescine-responsive bacteria that enables consistent daily sensing while preserving bacterial viability.

Need:

* A system that maintains living biosensors over multiple days, allows for simple handling by the user, and ensures correct alignment with the optical detection system.

#### Research

Hydrogel Formulation:

* Investigated alginate-based hydrogels for their ability to support bacterial viability while permitting diffusion of analytes needed for biosensing.

Cassette Design:

* Explored modular cassette approaches to allow multiple tests from a single preparation while minimizing contamination risk.

#### Design

Cassette Layout:

* Designed circular cassettes partitioned into seven compartments—one per day of the week—to provide daily testing without requiring hydrogel replacement.

Tray Mechanism:

* Created a sliding tray with an alignment notch to ensure cassettes are consistently and correctly positioned within the device.

#### Build

Fabrication:

* Manufactured hydrogel cassettes and trays using biocompatible materials that support bacterial function and withstand repeated handling.

Integration:

* Incorporated the cassette and tray system into the device housing, ensuring compatibility with the optical detection system and user workflow.

#### Test

Viability Testing:

* Confirmed that bacteria embedded in the alginate hydrogel remain viable and responsive across seven days of storage within the cassette.

Usability Testing:

* Verified that the sliding tray and notch system reliably aligns cassettes and simplifies hydrogel replacement by non-expert users.

#### Learn

Improvements:

* Identified the potential for expanding cassette designs to accommodate additional tests per week given long biosensor viability lifetime.

User Experience:

* Found that the sliding tray design significantly reduces handling errors, improving test consistency across users.

# Graphical User Interface

---

#### Reason

Objective:

* Create an intuitive graphical interface that allows users to easily interpret biosensor outputs and track BV progression from FloraDX.

Need:

* A user-friendly display that can present complex fluorescence and pH data in a simple, actionable format, both on-device and via a companion app.

#### Research

User Needs:

* Studied how non-expert users interact with diagnostic devices, emphasizing clarity, minimal steps, and immediate interpretation of results.

Display Options:

* Compared different display technologies and identified the LILYGO T-RGB 2.1-inch LCD touchscreen as optimal for compact size, high visibility, and multi-color output.

#### Design

Interface Layout:

* Designed a touchscreen interface that displays numeric readouts for fluorescence intensity and pH alongside color-coded indicators (e.g., green = safe, red = alert).

Dual Interaction Modes:

* Developed both on-device GUI and a mobile app interface, ensuring consistency across platforms and giving users flexibility in how they access results.

Feedback Features:

* Incorporated simple prompts and confirmation messages on the LCD to guide users through testing steps and confirm cassette alignment or test completion.

#### Build

Integration:

* Embedded the LILYGO LCD touchscreen into the front panel of the device for direct access, wired to the microcontroller for real-time data transfer.

App Development:

* Built a companion app that syncs with the device via Bluetooth/Wi-Fi, allowing users to log and review historical test results.

#### Test

Usability Testing:

* Conducted trials with users to ensure the interface was clear, intuitive, and required minimal training to operate.

Performance Validation:

* Verified that the GUI correctly translated raw fluorescence and pH sensor data into accurate readouts and alerts.

#### Learn

Improvements:

* Determined that multi-color alerts alone were insufficient for some users, so numeric readouts and trend graphs were added for clarity.

User Experience:

* Found that combining simple color indicators with numeric precision boosted user confidence and improved repeatability in interpreting results.

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}
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[![Back to top](https://static.igem.wiki/teams/5875/menu/vibi-arrows-totop.webp)](#top)

* [Biosensor Validation](#biosensor)
  + [Important info](#important)
  + [Growth Curve](#growth)
  + [IPTG Dose Response](#iptg)
  + [Putrescine Dose Response 1.0](#put1)
  + [Putrescine Dose Response 2.0](#put2)
  + [Validation Test 1.0](#valid1)
  + [Validation Test 2.0](#valid2)
  + [Validation Test 3.0](#valid3)
  + [IPTG Dose Response in M9](#m9)
  + [Future Directions](#fut)
* [Hydrogel Experiments](#hydrogel)
* [Hardware Experiments](#hardware)
* [Lab Protocols](#lab)

![Description header](https://static.igem.wiki/teams/5875/experiments/experiments.webp)

# Biosensor Validation

#### Important info

GENETIC DESIGN:
In the experiments below, “V004” refers to the genetic circuit with a synthetic promoter, PuuO, that is responsive to putrescine.
![](https://static.igem.wiki/teams/5875/experiments/experiments-1.webp)

Figure 1. Genetic circuit of V004

“V005” refers to a similar circuit, with the only change being a T7 constitutive promoter in the place of the synthetic PuuO promoter.
![](https://static.igem.wiki/teams/5875/experiments/experiments-2.webp)

Figure 2. Genetic circuit of V005

OPTICAL DENSITY CORRECTION:
To process our experimental data, we corrected the raw optical density (OD) values from the plate reader to more accurately reflect the true OD. The reason for this is because plate reader measurements can be affected by the geometry of the plate and the path length of the light. Applying this correction formula ensures our data is accurate. The correction formula is:

![](https://static.igem.wiki/teams/5875/experiments/experiments-formula.webp)

#### Growth Curve

DESIGN:

Goal:

Goal: The goal of this experiment was to understand the growth patterns of our engineered plasmids, V004 and V005. To accomplish this, we set up a kinetic experiment in a plate reader.

Notes:

Growth curves can be used to inform timing in future experiments. Also, cell growth is a measure of metabolic burden of the cells. Metabolic burden in cells can be affected by many factors, including the production of proteins.

We measured the growth of our 2 engineered plasmids (V004-synthetic promoter, V005-T7 promoters). Under no induction, no proteins should be produced, with the exception of GFP in V004. OD measurements were corrected to account for the specifics of the plate reader used, as described above.

BUILD:

Procedure:

Make overnight cultures from a single colony off a fresh streaked-out plate in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking at 275 rpm. Spin down and resuspend cells in fresh LB and ampicillin such that OD = 0.01. Plate in a 96-black-well plate in triplicate. Incubate plate in plate reader for 24 hours. Every hour, measure OD600, GFP, and mCherry.

TEST:

Results:

V004 had a lag phase of about 10 hours, which is longer than expected of a high-copy plasmid. This could be due to the fact that V004 is constantly producing GFP in the absence of an inducing agent. As a result of this longer lag phase, V004 does not reach the stationary phase during the 22 hour experiment.

V005 had a lag phase of about 3 hours, which is significantly shorter than V004 because in the absence of any inducing agent, it should not be producing any fluorescent molecules.

![](https://static.igem.wiki/teams/5875/experiments/experiments-3.webp)

Figure 3. Growth curve of V004 and V005 in 96-well plate over 22 hours

LEARN:

From this growth curve, we learned that in future experiments we should plan for V004 to take about 10 hours to grow from a starting OD of 0.01.

#### IPTG Dose Response

DESIGN:

Goal:

Determine the optimal concentration of IPTG for maximum biosensor response.

Notes:

Based on Selim et al (1), 0.5mM IPTG induction produced the most optimal results for the genetic design in their paper. Our genetic design was slightly different, so we hypothesized that we may have a different optimal concentration of IPTG. Thus, we tested a range of concentrations from 0 mM to 1 mM to optimize specifically for our design.

BUILD:

Procedure:

Make overnight cultures of V004 and V005 in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Pellet and resuspend the overnight culture in fresh LB media + amp such that OD = 0.1. Incubate in a baffle flask until OD reaches 1. Plate cultured cells at OD= 0.5 in a 96-black well plate and induce cultures with varying concentrations of IPTG (0mM, 0.1mM, 0.3mM, 0.5mM, 0.7mM, 1mM). Incubate plate in plate reader for 18 hours. Every 20 minutes, measure OD600, GFP, and mCherry.

TEST:

Results for V004:

We were interested in the OD of the cells induced with IPTG to determine if the addition of IPTG increased the metabolic burden on the cells, and thus slowed the growth rate. The graph of OD600 shows that the growth of V004 cells was not negatively affected by the induction of IPTG.

![](https://static.igem.wiki/teams/5875/experiments/experiments-4.webp)

Figure 4. OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

In V004, GFP is under the control of a synthetic promoter that is repressed by PuuR, a protein under control of a T7 promoter. We expect with no IPTG, GFP to be produced, and with increasing concentrations of IPTG, GFP to be repressed. Instead, what we observed was the opposite, with 0M IPTG having the lowest signal and the other varying concentrations of IPTG to have a slightly larger GFP signal .

mCherry is under control of a T7 promoter, thus, as the concentration of IPTG increases it is expected that mCherry fluorescence will proportionally increase. We observe that the addition of IPTG increases the fluorescent signal, as expected. Note, however, that the gain setting was too low to obtain reliable results.

![](https://static.igem.wiki/teams/5875/experiments/experiments-5.webp)

Figure 5. GFP values divided by corrected OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

mCherry is under control of a T7 promoter, thus, as the concentration of IPTG increases mCherry fluorescence should increase. We observe that the addition of IPTG (regardless of the concentration) increases the fluorescent signal an equal amount above the baseline of 0M IPTG. In the range of 0.1mM to 1mM of IPTG, there is no noticeable correlation between an increasing dose of IPTG and an increase in mCherry signal. We also observed high variation between technical replicates for each condition. This could be due to a lack of difference between concentrations of IPTG, as we did not use an exponential scale.

![](https://static.igem.wiki/teams/5875/experiments/experiments-6.webp)

Figure 6. mCherry values divided by corrected OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

Results for V005:

The graph of OD600 shows that the growth of V005 cells was not negatively affected by the induction of IPTG.

![](https://static.igem.wiki/teams/5875/experiments/experiments-7.webp)

Figure 7. OD600 measurements of V005 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

In V005, GFP is under the control of a T7 promoter that is induced by IPTG. We expect the addition of IPTG to increase the GFP signal. We observed a slight increase in GFP signal with the addition of IPTG concentrations ranging from 0.3mM to 1mM. Note there is high variation between technical replicates.

![](https://static.igem.wiki/teams/5875/experiments/experiments-8.webp)

Figure 8. GFP values divided by corrected OD600 measurements of V004 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

The mCherry coding sequence is under control of a T7 promoter, thus, as the concentration of IPTG increases it is expected that mCherry fluorescence will proportionally increase. We observed no significant change in mCherry expression with the addition of IPTG.

![](https://static.igem.wiki/teams/5875/experiments/experiments-8.webp)

Figure 9. mCherry values divided by corrected OD600 measurements of V005 during the IPTG dose response kinetic experiment. Raw OD600 data is corrected to account for plate reader specifics

LEARN:

From this dose response, we learned that the behavior of our constructs under IPTG induction did not align with our initial expectations based on the circuit design and previous literature.

For V004, we expected increasing concentrations of IPTG to repress GFP expression (since PuuR expression should increase under the T7 promoter, thereby repressing the synthetic promoter driving GFP). However, we observed the opposite trend where GFP expression slightly increased with IPTG induction. This suggests that (1) the PuuR repressor is not being expressed or folded as expected under the T7 system, (2) the T7 system is leaky even in the absence of IPTG, or (3) unintended design or regulatory effects may be influencing gene expression or promoter activity.

For V005, GFP expression was expected to positively correlate with IPTG concentration since it is directly under a T7 promoter. While a modest increase in GFP expression was observed between 0.3mM and 1mM, the response was weak and highly variable across technical replicates. Additionally, the lack of a proportional increase in mCherry expression (also under T7 control) further supports the possibility that T7-driven expression may not be functioning optimally in these constructs under our current conditions.

Overall, these results indicate that IPTG induction alone is insufficient to reliably drive or repress expression as predicted, implying that additional or confounding factors may be involved. To further understand the regulatory interactions within our system, our next step was to perform a dual-induction experiment with both IPTG and putrescine to assess if discrepancies are due to promoter leakiness, biosensor design, or conditional variables.

REFERENCES:

1. Selim, A. S., Perry, J. M., Nasr, M. A., Pimprikar, J. M., & Shih, S. C. C. (2022). A synthetic biosensor for detecting putrescine in beef samples.  *ACS Applied Bio Materials, 5(11), 5487–5496.* https://doi.org/10.1021/acsabm.2c00824

#### Putrescine Dose Response 1.0

DESIGN:

Goal:

Determine the biosensor response to varying concentrations of putrescine.

BUILD:

Procedure:

Make overnight cultures of V004 in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Pellet cells and resuspend overnight culture such that OD = 0.1 in fresh LB media and ampicillin. Incubate until OD is above 0.5. Pellet cells and resuspend such that OD = 0.5. Add resuspended cells to a 96-black-well plate. Induce wells with 0.5mM IPTG and varying concentrations of putrescine (0mM, 10mM, 100mM, 500mM). Incubate plate in plate reader for 24 hours. Every 20 minutes, measure OD600, GFP, and mCherry.

TEST:

Note: There was significant clumping of cells in wells with high concentrations of putrescine (500mM, 100mM). This greatly affected measurements performed by the plate reader, resulting in inaccurate
results for those treatment groups.

![](https://static.igem.wiki/teams/5875/experiments/experiments-10.webp)

Figure 10. Clumping observed in 96 well plate. In descending order, the rows were induced with 500mM, 100mM, and 10mM putrescine

Results:

Wells with significant clumping (100mM and 500mM) were not plotted due to significant variation and unreliability. After 10 hours, the cells with 10mM putrescine and 0.5mM IPTG began to produce more GFP than baseline cells without putrescine. This confirms our expectation that putrescine increases GFP signal.

![](https://static.igem.wiki/teams/5875/experiments/experiments-11.webp)

Figure 11. GFP divided by OD600 (corrected) for V004 induced with putrescine and IPTG. Wells with clumped cells not plotted

In the graph of mCherry fluorescent measurements over time, we see no difference between cells with or without putrescine. This is expected because mCherry is under the control of a T7 promoter, which should not be affected by putrescine.

![](https://static.igem.wiki/teams/5875/experiments/experiments-12.webp)

Figure 12. Raw mCherry fluorescent values for V004 induced with putrescine and IPTG. Wells with clumped cells not plotted

LEARN:

From this experiment the main takeaway was that putrescine in high concentrations causes cells to clump together in 96-well plates. Cell clumping is an indicator of changes in environmental condition, which aligns with the fact that it occurred in high putrescine conditions. We also observed that the biosensor responds as expected to GFP and mCherry under the influence of putrescine.

#### Putrescine Dose Response 2.0

DESIGN:

Goal:

Determine the biosensor response to varying concentrations of putrescine, and attempt to address the clumping issue by lowering the concentration of putrescine.

BUILD:

Procedure:

Make overnight cultures in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Pellet cells and resuspend overnight culture such that OD = 0.1 in fresh LB media and ampicillin. Incubate until OD is above 0.5. Pellet cells and resuspend such that OD = 0.5. Add resuspended cells to a 96-black-well plate. Induce wells with 0.5mM IPTG and varying concentrations of putrescine (0mM, 1mM, 10mM, 50mM). Incubate plate in plate reader for 14 hours. Every 20 minutes, measure OD600, GFP, and mCherry.

TEST:

Notes: There was still clumping observed at 50mM of putrescine (see columns 1, 5, and 6 in image). Columns 2 and 3 exhibit some signs of cells settling to the center of the well.

![](https://static.igem.wiki/teams/5875/experiments/experiments-13.webp)

Figure 13. Clumping in 96 well plate, observed after a 14 hour kinetic run with putrescine induction.]

Results:

We measured OD600 to understand the effect putrescine has on the growth of the cells. The effects of cell clumping is evident in the data with IPTG, 50mM of putrescine (blue dots). We observe that 10mM putrescine (with IPTG) inhibits the growth of V004.

We observe that without IPTG (graph on right) the growth of V004 has much more variation. Overall, there is a trend that putrescine inhibits the growth of V004 without IPTG addition.

![](https://static.igem.wiki/teams/5875/experiments/experiments-14.webp)

Figure 14. OD600 for V004 induced with putrescine and/or IPTG.

It is evident that 10mM putrescine (with IPTG) produced a noticeable increase in GFP, which aligns with our expectation. 50mM putrescine is not consistent due to the clumping in the well. 1mM of putrescine appears to have a slight increase in GFP beginning at hour 13.

![](https://static.igem.wiki/teams/5875/experiments/experiments-15.webp)

Figure 15. Raw GFP data for V004 induced with putrescine and IPTG

mCherry is under the control of a T7 promoter, and thus should only be active when IPTG is added. We observe that in the absence of IPTG, mCherry has a strong signal. This is the opposite of what is expected.

We also observe that putrescine has little impact on the production of mCherry, which aligns with expectations.

![](https://static.igem.wiki/teams/5875/experiments/experiments-16.webp)

Figure 16. Raw mCherry fluorescence of V004 induced with putrescine and/or IPTG

LEARN:

From this experiment testing the response of our biosensor to putrescine, we observed that 10mM putrescine results in a small increase in GFP signal. This aligns with our expectation that GFP increases in response to putrescine.

However, we also see some unexpected results, such as the observation that the mCherry signal is heightened without IPTG. To further investigate these observations, we proposed simpler “validation testing” to observe the basic response of our biosensor to the 4 permutations of induction (nothing, +putrescine, +IPTG, +both).

#### Validation Test 1.0

DESIGN:

Goal:

Observe response of the biosensor to four simple conditions (nothing, +putrescine, +IPTG, +both).

BUILD:

Procedure:

Make overnight cultures in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Pellet cells and resuspend overnight culture in culture tubes, such that OD = 0.1 in 5mL of fresh LB media and ampicillin. Incubate until OD reaches 0.5. Induce culture tubes with (A) nothing (B) 0.5mM IPTG (C) 10mM putrescine (D) 0.5mM IPTG and 10mM putrescine. Incubate culture tubes for 17 hours. At hours 3 and 17, measure OD600, GFP, and mCherry.

Note:

Cultures were incubated in culture tubes to avoid clumping issues observed in 96-well plates (see previous experiments for details).

Listed below are the expected responses from the V004 plasmid to each condition.

### Table 1: Expected Response of V004 to Specific Inducing Agents

| Condition | GFP | RFP |
| --- | --- | --- |
| Nothing | Yes, no repressor present | No |
| 0.5 mM IPTG | Repressed | Yes, repressor produced |
| 10 mM Putrescine | Yes, no repressor to interact with putrescine | No |
| 0.5 mM IPTG & 10 mM Putrescine | Yes, repressor alleviated by putrescine | Yes, repressor produced |

TEST:

Notes: There was still clumping observed at 50mM of putrescine (see columns 1, 5, and 6 in image). Columns 2 and 3 exhibit some signs of cells settling to the center of the well.

Results:

After 17 hours, we observe some differences in GFP expression between conditions. First, 0.5mM IPTG reduces GFP expression compared to nothing, as expected because IPTG induces expression of the PuuR repressor protein. Second, with only 10mM putrescine we also saw repression of GFP, although not as strong. This shows putrescine has an effect of GFP expression even without IPTG present, suggesting leaky expression of the T7 promoter controlling PuuR expression. Third, with both IPTG and putrescine we observe that GFP increases almost to the baseline (nothing) value. From this data, we can conclude that our biosensor response is as expected, with potential leaks from the T7 promoter.
![](https://static.igem.wiki/teams/5875/experiments/experiments-17.webp)

Figure 17. Raw GFP data of V004 at hour 17 during Validation Test #1

In the graph of mCherry, we expect conditions with IPTG to increase the production of the fluorescent protein. However, at hour 17 we do not observe this, instead we see that IPTG reduces expression of mCherry. This does not align with the graph of GFP data. One potential explanation is that the PuuR protein is being transcribed, and the mCherry protein that directly follows is not being transcribed. This would explain why the GFP signal is responsive to putrescine, and the mCherry signal does not increase with IPTG.

![](https://static.igem.wiki/teams/5875/experiments/experiments-18-real.avif)

Figure 18. Raw mCherry data of V004 at hour 17 during Validation Test #1

LEARN:

This experiment showed us that the GFP signal from the cells responds to putrescine, which is a promising result. Additionally, we uncovered that the T7 promoter may have a leaky expression of PuuR. Finally, we see that mCherry fluorescence unexpectedly decreases when IPTG is added.

The next step after this experiment is to redo it, but in a deep-well plate rather than in culture tubes. The protocol from the lab we operated in recommends all induction occur in plates, not in culture tubes. We hypothesized that correcting this mistake may lead to more typical expression of the fluorescent proteins.

#### Validation Test 2.0

DESIGN:

Goal:

Repeat the validation test experiment in a different culturing vessel.

BUILD:

Procedure:

Make overnight cultures in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Dilute overnight cultures 1:100 fresh LB media and ampicillin, and incubate until OD reaches 0.5. Pellet and resuspend cells such that OD = 0.5, and add 500uL to a 96-deep-well plate. Induce deep well plate with one of the following conditions per well (in triplicate)
(A) nothing (B) 0.5mM IPTG (C) 10mM putrescine (D) 0.5mM IPTG and 10mM putrescine.
The final concentration of cells in the plate is 0.5 .
Incubate for 17 hours, and at hours 3 and 17, measure OD600, GFP, and mCherry in a black-well plate.

TEST:

Results:

We see repression of GFP with the addition of IPTG alone. When putrescine and IPTG are added, the GFP signal should be strong, however the graph shows further decreased GFP expression in this condition. Additionally, the induction of putrescine alone drastically decreases the GFP signal, where it should have no effect. This could suggest some toxicity correlated with putrescine.
![](https://static.igem.wiki/teams/5875/experiments/experiments-19.webp)

Figure 19. Raw GFP data of V004 at hour 17 during Validation Test #2

We expect to see higher RFP in conditions with IPTG, however we observe no change in these conditions. Notably, putrescine alone decreases RFP expression. There are a few outliers, which encourages us to practice more sterile technique and potentially increase the number of replicates in future experimental designs.

![](https://static.igem.wiki/teams/5875/experiments/experiments-20.webp)

Figure 20. Raw mCherry data of V004 at hour 17 during Validation Test #2

LEARN:

In this experiment, we confirmed that IPTG has no apparent effect on mCherry. We hypothesized that inducing the cells in an earlier growth stage could have a different effect on protein expression.

#### Validation Test 3.0

DESIGN:

Goal:

Repeat the validation test experiment in a deep well plate and with a lower starting OD. Additionally, measure the cellular response up to 24 hours after induction.

BUILD:

Procedure:

Make overnight cultures in 5mL of LB and ampicillin. Incubate for 16-18 hours at 37 C shaking. Add cells to a 96-deep-well plate at an OD of 0.01. Induce deep well plate with each of the following conditions in triplicate: (A) nothing (B) 0.5mM IPTG (C) 10mM putrescine (D) 0.5mM IPTG and 10mM putrescine. Incubate for 24 hours, and at hours 2, 4, 8. 11, 14, 16 and 24 measure OD600, GFP, and mCherry in a black-well plate.

TEST:

Results:

The graph of raw GFP data from Validation Test #3 shows that the addition of IPTG strongly inhibits GFP signal, which is expected. Unfortunately, the data shows that 10mM putrescine (without IPTG) increases the GFP signal. Perhaps this is because there is some leaking from the T7 promoter that controls the repressor PuuR, so putrescine is interacting with this leaky expression of the repressor. The purple dots (both IPTG and putrescine) are not very visible as it follows the pattern of the red dot (IPTG only) and are covered. This means that putrescine has no effect on GFP expression.
![](https://static.igem.wiki/teams/5875/experiments/experiments-21.webp)

Figure 21. Raw GFP values over time for V004 during Validation Test #3

Once again in the graph of mCherry fluorescence over time, the purple dots (both IPTG and putrescine) are not very visible as they follow the pattern of the red dot (IPTG only) and are covered. We expect IPTG to increase the mCherry signal, however we see the opposite effect in the graph.

![](https://static.igem.wiki/teams/5875/experiments/experiments-22.webp)

Figure 22. Raw mCherry values over time for V004 during Validation Test #3.

LEARN:

From this experiment we can take away that IPTG has an effect on both mCherry and GFP expression. Unlike the previous two Validation Test experiments, we see no difference when putrescine is added in addition to IPTG.

Overall, after 24 hours the cell’s response to the different conditions is much more pronounced, which is potentially an effect of the slower growth observed during the growth curve experiment (see above).

Next, we hypothesized that the LB media we cultured the cells in could be having an effect on the biosensor, potentially dampening the effect of putrescine induction. We planned to investigate this by culturing cells in M9 media.

#### IPTG Dose Response in M9

DESIGN:

Goal:

Determine the response of the circuit to IPTG in M9 media, in a deep-well plate.

BUILD:

Procedure:

Make overnights of V004 and V005 in M9 media. Grow in the 37C shaking incubator in culture tubes for 16-18 hours. Spin down overnight culture to OD = 10 in volume of 400uL of fresh media. Make a series of IPTG dilutions at the following concentrations: 70mM, 50mM, 30mM, 10mM in filter sterilized water. Plate in deep well plate: 490uL M9 media, 5uL resuspended cells, 5uL respective IPTG stock solution. Measure OD600, GFP, mCherry at hours: 0, 4, 8, 16.

TEST:

Results:

We observe that in M9 media, GFP expression is repressed similarly by concentrations of 0.3mM to 1mM of IPTG. 0.1mM of IPTG also repressed GFP, but to a slightly lesser degree. This data aligns with our expectations of the biosensor, as IPTG induces PuuR expression, which represses PuuO and thus GFP.
![](https://static.igem.wiki/teams/5875/experiments/experiments-23.webp)

Figure 23. V004 GFP values subtracted by GFP values of blank media at hour 16 during IPTG dose response in M9 media

The biosensor did not produce very much mCherry protein in M9 media. This is shown by very low values (less than 15 RFU) when the mCherry fluorescence is subtracted from the fluorescence of the blank M9 media.

![](https://static.igem.wiki/teams/5875/experiments/experiments-24.webp)

Figure 24. V004 mCherry values subtracted by mCherry values of blank media at hour 16 during IPTG dose response in M9 media.

LEARN:

Overall from this experiment, we learned that IPTG represses GFP in V004.

After this experiment, we discussed conflicting results from this experiment, and previous ones, with our PI. During this discussion, we noticed that our plasmid had additional promoters leftover from the mCherry backbone we used. These promoters were under inducible control by IPTG, which may have had a very prominent effect on our biosensor.

#### Future Directions

We plan to modify our plasmid V004 to remove the additional undesired IPTG-inducible promoters. We hope that by removing the additional promoters, we will be able to characterize a clearer signal from the PuuR repressor protein.

![](https://static.igem.wiki/teams/5875/experiments/experiments-25-real-final.avif)

Figure 25. Snapgene plasmid map of V004 on the left, and V004 with modifications to remove additional promoters on the right.

We also plan to swap out the T7 promoter that controls PuuR and mCherry for pVeg. By accomplishing this, we will not need to add IPTG to induce expression of PuuR. Thus, GFP will always be repressed, and only turned on if putrescine is present. This will help to make our genetic circuit simpler.
![](https://static.igem.wiki/teams/5875/experiments/experiments-26.webp)

Figure 26. Snapgene plasmid map of V004 with modifications to remove additional promoters, and swap the T7 promoter for pVeg1.

# Hydrogel Experiments

#### Testing resuspension heat shock effect on *E. coli* viability

Depending on the concentration, agar hydrogels must be heated to high temperatures nearing 50C to become a liquid. For culture resuspension, these solutions must be heated before mixing, resulting in a heat shock effect. To test the effect of heated media on cells, we started by measuring the growth and expression of  *E. coli* constitutively expressing eGFP in regular LB media.

We followed our standard agar resuspension protocol but resuspended mid-log cultures in LB either at room temperature or 50C. Those resuspended cultures were then plated in a 96-well plate and measured in a plate reader at room temperature, shaking linearly at 452 rpm. The OD600 and eGFP fluorescence were measured over 63 hours.

#### Optimizing media conditions for hydrogel resuspension

The first step in evaluating hydrogels for biosensor longevity was determining the optimal hydrogel formulation. Mid-log phase  *E. coli*  constitutively expressing eGFP were resuspended following the above protocols in agar hydrogels with varied nutrient and matrix densities.

The nutrient density was varied by altering the concentration of LB in the media. A 10X LB stock was created by adding ten times the usual mass of LB powder to water and autoclaving. The concentrated stock was then diluted to 5X, 2X, 1X, and 0.1X. PBS was also tested as a resuspension media.

The hydrogel matrix density was altered by changing the concentration of agar in the media. A 5% (w/v) agar stock with the normal concentration of LB was made and diluted with regular liquid LB media to agar concentrations of 4%, 3%, 2%, 1%, and 0.5% (w/v). The solutions were kept at 50C in a heat block to prevent them solidifying before use.

The resuspended cultures were plated in a 96-well plate, covered with the lid, and placed in the plate reader at shaking at room temperature for 60 hours. The OD600 and eGFP fluorescence were measured every hour for 63 hours.

#### Evaluating long-term survival of *E. coli* in hydrogels

After optimizing the media conditions, we wanted to evaluate the ability of agar hydrogels to maintain  *E. coli*  viability for up to a week. We followed our agar resuspension protocol with the constitutive eGFP strain in 1% and 1.5% agar hydrogels. The cultures were plated in a 96-well plate, covered with the lid, and kept at room temperature for 8 days. The eGFP fluorescence was measured twice a day throughout that period.

#### Testing hydrogel resuspension in the 3D printed disk

To evaluate the performance of the agar hydrogels in our own device, we tested ‘plating’ resuspended cultures on a 3D printed model of the hydrogel disk holder.  *E. coli* constitutively expressing eGFP were prepared according to the agar resuspension protocol using 1% (w/v) agar hydrogels and aliquotted to the disk. The 3D printed disk was covered with a 3D printed lid and left at room temperature. The fluorescence of the hydrogels was measured using a gel imager twice a day while the gels were viable.

# Hardware Experiments

#### Testing photodiode sensitivity

To characterize photodiode sensitivity, we developed a simple circuit consisting of an op amp (Texas Instruments LMP7721), two one kiloohm resistors, and a 525 nm-specific photodiode sensor (Marktech Optoelectronics MTD5052D3) all in an non-invertising configuration to achieve a gain of two. The photodiode circuit was then put in a dark box to block out any ambient light and a 520 nm green LED (CHANZON 100F5T-YT-WH-GR) was set right above to shine light directly into the photodiode sensor. Starting at 3 V supplied to the LED, we scaled down the voltage in 0.1 V steps to decrease the brightness intensity the photodiode received. This gave us a plot of photodiode voltage (sensor sensitivity) versus LED voltage (brightness intensity).

![](https://static.igem.wiki/teams/5875/experiments/experiments-27.avif)

Figure 27. Circuit Diagram

![](https://static.igem.wiki/teams/5875/experiments/experiments-28.avif)

Figure 28. Photodiode output diagram

# Lab Protocols

#### Click on each panel to be taken to the full protocol

[### Standard Lab Materials

This page contains basic materials used throughout most protocols.](#materials)
[### Patch Plates

How to make patch plates.

Agar
Plates](#patch)
[### Sequencing

How to sequence with Plasmidsaurus.

Sequencing
Plasmidsaurus](#sequencing)
[### Stocking A Strain

How to stock a strain for later use

Stocking
Glycerol](#stocking)
[### Pouring Plates

How to pour a plate

Agar
Plates](#pouring)
[### Making Solid and Liquid Media

How to make both solid and liquid media appropriately

Media
Agar](#making)
[### Miniprep

How to miniprep for our samples

Miniprep
Plasmid](#miniprep)
[### Restriction Digest

How to cut DNA into smaller fragments using restriction enzymes

Digest
Enzymes](#restriction)
[### HiFi Assembly

How to assemble DNA using HiFi

DNA
Cloning](#hifi)
[### High Efficiency Transformation

How to efficiently transform bacteria

Transformation
Cells](#transformation)
[### Gel Electrophoresis

How to perform a gel electrophoresis

DNA
Electrophoresis](#gel)
[### Gel Extraction and Purification

How to extract DNA from the gel and purify it

DNA
Electrophoresis](#extraction)
[### gBlock gene fragment resuspension

How to resuspend a gBlock gene fragment

DNA
Electrophoresis](#gblock)
[### NanoQuant nanodrop

How to quantify nucleic acids

DNA](#nano)
[### Hydrogel Resuspension

How to resuspend cells in a hydrogel

Hydrogel
Cells](#hydrogell)

# Standard Lab Materials

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| UltraPure Water | Fisher Scientific | 10977023 |
| Milli-Q® Water | Millipore Sigma |  |
| PCR tubes | Fisher Scientific | 07-000-716 |
| 2 µL Pipette tips | Fisher Scientific | 12-111-119 |
| 10 µL Pipette tips | Fisher Scientific | 12-111-000 |
| 100 µL Pipette tips | Fisher Scientific | 12-111-003 |
| Sharpies | Sharpie | 30078 |
| Ziplock bags | Fisher Scientific | 01-816E |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Heat block | Fisher Scientific | 88860022, 88860103 |
| Bunsen burner | Fisher Scientific | S12813 |
| Bunsen burner hose | Fisher Scientific | NC0006926 |
| Striker | Fisher Scientific | NC1697588 |
| 2 µL Micropipette | Eppendorf | 136090025 |
| 10 µL Micropipette | Eppendorf | 13690026 |
| 100 µL Micropipette | Eppendorf | 13690029 |
| Centrifuge 5424 R | Fisher Scientific |  |
| Pipette Aid | Thermo Scientific | S1 Pipet Filler |
| 30°C Static Incubator | Thermo Scientific | 43160148 |
| 37°C Shaker Incubator | Multitron | BU-450648 |
| 37°C Static Incubator | Thermo Scientific | 43041093 |

# Patch Plates

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Poured plate | n/a |  |
| Liquid medium | n/a |  |
| UltraPure Water | Fisher Scientific | 10-977-023 |
| PCR strip tubes | Fisher Scientific | 07-000-716 |
| 14 mL Culture Tubes | Greiner Bio-One | 187262 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Multichannel pipette |  |  |

### Procedure

**Note:** The purpose of this protocol is to create a working supply of a single colony for further experimentation. It is generally only done after transformation of an assembly construct to create a plate and associated overnight that can be used for sequence confirmation through whole-plasmid sequencing.

1. After the transformation plate has fully grown, prepare to make a patch plate and start overnight cultures.
2. For the patch plate, choose an appropriate number of colonies (~8–48) from your transformation plate depending on confidence in your plasmid assembly and transformation:
   * **High confidence** → 8 colonies (1 PCR strip and row of patches)
   * **Lower confidence** → Select more colonies
3. For the overnight cultures:
   * With high-confidence single-fragment assemblies, choose ~4 colonies.
   * For low-confidence or multi-fragment assemblies, choose more colonies.
4. Take a PCR tube strip (or multiple if patching >8 colonies) and fill each tube with **20 µL ultrapure water**.
5. Pick a colony from your plate using a 10 µL pipette tip (hold it in your hand) and drop the tip into one of the PCR tubes with ultrapure water.
6. Mix the solution by shaking the pipette tip vigorously, then dispose of the tip in the sharps bin. Ensure the solution becomes cloudy.
7. Repeat steps 5–6 for each additional colony.
8. Use a multichannel pipette to transfer **4 µL** from each PCR tube to the appropriate agar plate:
   * “Stamp” the drops onto the plate by pressing the pipette plunger to the *first stop* and touching the drops to the plate surface.
   * **Do not** press to the second stop to fully eject droplets.
   * Wait for the drops to absorb into the agar.

![Stamping](https://static.igem.wiki/teams/5875/experiments/protocols1.webp)

![Stamping](https://static.igem.wiki/teams/5875/experiments/protocols2.webp)

9. Start overnight cultures by transferring **10 µL** from each PCR tube to a culture tube containing **5 mL of appropriate liquid media**.
10. Incubate:
    * Culture tubes: 37 °C, 275 RPM, overnight
    * Patching plate: 37 °C
11. The following day, use the overnight cultures to:
    * Make glycerol stocks
    * Perform minipreps
    * Send for plasmid sequencing

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Sequencing with Plasmidsaurus

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| PCR strip tubes | Fisher Scientific | 07-000-716 |
| UltraPure Water | Fisher Scientific | 10-977-023 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Standard |  |  |

### Procedure

1. Follow the Nanoquant protocol to get the concentration of the sample.
2. If concentration of the sample when nanodropping is >400 ng/uL, dilute with nuclease-free water to bring concentration down to ~250 ng/uL

* Do not dilute the entire sample, only dilute what is needed to send out for sequencing

3. Log in to Plasmidsaurus and follow given instructions to prep the sample.
4. Once sequencing is complete, align to reference in SnapGene.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Stocking A Strain

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Inoculation Loops | Fisher Scientific | 22-363-604 |
| Poured Plates | See ‘Pouring plates’ | n/a |
| Liquid medium | See ‘Making solid and liquid media’ | n/a |
| 14 mL Culture Tubes | Greiner Bio-One | 187262 |
| Serological Pipettes | Fisher Scientific | 13-678-11 |
| 50 mL Falcon Tubes | DAMP lab | n/a |
| 2.0 mL Cryovials | Corning | 430488 |
| Glycerol solution ≥ 99% | Sigma Aldrich | G6279 |
| Dry Ice | n/a | n/a |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| 37C Shaker Incubator | Multitron | BU-450648 |
| 37C static incubator | Thermo Scientific | 43041093 |
| -80C Freezer | Thermo Scientific | TSU600A |
| Glass media bottle (500 mL) | Fisher Scientific | FB-800-500 |
| Autoclave | Consolidated Sterilizer Systems |  |

### Procedure:

#### Day 1 - Streaking Out a Plate from Existing Stock

1. Turn on the Bunsen burner and set up the prepared plates and inoculation loops around it.
2. Label the plate with strain name, initials, and date.
3. Take sample from the glycerol stock source tube.

* Keep the glycerol stock on dry ice in an ice bucket at all times to prevent it from thawing out — **do not use wet ice.**
* Carefully remove inoculation loop from the bag.
* Scrape the glycerol stock using the loop.

4. Streak it out on appropriate media for strain.

* Gently swipe the loop on the plate to make a puddle. Be careful not to dig into the agar. Discard the inoculation loop.
* Using a new inoculation loop, swipe out of the puddle, following the curve labeled 2 in the figure below. Using another loop, repeat for the 3rd streak.

5. Incubate plates at 37°C for no more than 24 hours.

#### Day 2 - Picking a Colony & Overnight Culture

1. Look up the strain name on the ATCC for Colony Morphology (qualitative checks for picking a colony) and compare it to how your colonies look. General things to look for are: color, shape, translucency, smell, and size.
2. Turn on the Bunsen burner and set up all the materials around it—streaked plates, inoculation loops, culture tubes, media, serological pipettes, and the pipette gun.
3. Prepare culture tubes for each colony being picked.

* Label each culture tube with strain name, media, date, and your initials.
* Pipette 5 mL of the appropriate liquid medium and add antibiotic.

4. Using a fresh inoculation loop, dab the loop on a single colony to scoop some of that colony onto the loop. Be careful not to poke into the agar and try not to pick up the entire colony.

* Avoid picking colonies that look like multiple colonies growing very close to each other or are merged together.

5. Place the loop into the culture tube filled with media and mix it around. When capping the culture tube, only push down to the first stop—leaving the cap loose to allow free air exchange.
6. Incubate for 15-18 hours at 37°C and 280 RPM.

#### Day 3 - Glycerol Stocking

1. Check to make sure cultures are cloudy.
2. Turn on Bunsen burner and set up cryovials, 40% glycerol, pipette, pipette tips, and overnight liquid culture around it.
3. Make 40% glycerol if it has not been made already.

* Measure out 120 mL of Milli-Q water in a 500 mL glass bottle.
* Pipette 80 mL Glycerol solution ≥ 99% into the water. Pipette up and down to mix and dissolve any glycerol solution stuck to the inside of the pipette.
* Loosely cap bottle, add a strip of autoclave tape, and sterilize.

4. Label cryovials with strain information.
5. To the cryovial, first add 500 µL of 40% glycerol, then add 500 µL of the overnight culture and carefully pipette up and down to combine.

* Repeat for replicates.

6. Store cryovials in the -80°C freezer.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Pouring Plates

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Prepared Agar medium | See ‘Making solid and liquid media’ | n/a |
| Petri Dishes | Fisher Scientific | FB0875712 |
| Antibiotic stocks |  |  |
| 50 mL Falcon tubes | DAMP lab | n/a |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| BSC | Labconco |  |
| Microwave |  |  |
| Pipette aid/gun | Thermo Scientific | S1 Pipet Filler |

### Procedure:

1. Set up all materials next to the Bunsen burner.

* Stack plates and mark with Sharpie to indicate the antibiotic used based on your lab’s standards.
* Set up plates in a line with lids on.

2. Loosen cap of prepared agar media bottle. Microwave in 30-second intervals till fully melted, swirling each interval.
3. **Bunsen burner and sterilization techniques:**

* Light Bunsen burner, ensuring a pale blue inner cone and a larger outer cone flame.
* When opening the media bottle, sterilize by quickly passing the cap and bottleneck over the flame. Repeat before closing the bottle, and quickly pass the closed bottle over the flame again.

4. For each plate, pipette ~14 mL of LB agar + antibiotics into a 50 mL Falcon tube.

* This will give you extra media as a buffer when pipetting.
* Add antibiotics to working concentration for your strain.

5. Pipette up and down 2-3 times to mix, trying to avoid air bubbles.
6. Remove lids from your plates.
7. Pipette up media and transfer 12.5 mL to each plate. Gently rock plates to evenly distribute media.

* Avoid pulling up air bubbles; pull up an extra 2 mL to avoid dispensing air bubbles into the petri dish. Discard this extra volume at the end.

8. Allow plates to cool for 30 minutes on the bench, then dry for 30 minutes with lids off in the biosafety cabinet.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Making Solid and Liquid Media

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| LB broth, Lennox | BD | 240230 |
| Agar | BD | 214010 |
| Ampicillin | Millipore Sigma | A5354-10ML |
| 50 mL Falcon tube |  |  |
| Autoclave tape | Fisher Scientific | 15-901-111 |
| Weigh paper | Fisher Scientific | 09-898-12B |
| Weighboat | Fisher Scientific | 08-732-118 |
| Plastic scoopula | Fisher Scientific | 17211 |
| Milli-Q water |  |  |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Glass media bottle (500 mL) | Fisher Scientific | FB-800-500 |
| Gram balance |  |  |
| Autoclave |  |  |

### Procedure

1. Label 500 mL glass bottles with media type, initials, and date.
2. Rinse bottles and fill with Milli-Q water.

* **Metering procedure:** Check that the resistivity of the water is **18.18 mΩ·cm** at room temperature.
* Rinse both the bottle and its cap 3 times, ensuring the threads are also rinsed.

3. Set the Milli-Q to dispense:
   * **0.3 L** for the *solid medium* bottle
   * **0.5 L** for the *liquid medium* bottle
4. Refer to the table below for the amount of broth and agar to add.

* Carefully pour out the powder, keeping the lid mostly capped the entire time to reduce contamination risk.
* A 1–3% deviation from the required weight is acceptable.

### Medium Composition

| Type of Medium | Water Volume (mL) | Amount of Broth (g) | Amount of Agar (g) | Notes |
| --- | --- | --- | --- | --- |
| LB | 500 | 10 |  |  |
| LB Agar | 300 | 6 | 4.5 | Agar 1.5% w/v |

5. Tightly cap bottles and shake to coat all the powder in liquid. It does not need to be completely dissolved—chunks are okay.
Note: Some types of media must be heated prior to autoclaving. Check the media powder bottle for specific instructions before use.6. Add autoclave tape to each bottle and autoclave on the **liquid setting** for **15 minutes**.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Miniprep

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Miniprep Kit | Monarch | T1110L |
| Nuclease-free water | Fisher Scientific | 10-977-023 |
| Eppendorf Tubes | DAMP lab | n/a |
| 50 mL Falcon Tube | DAMP lab | n/a |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| AccuSpin Micro 17 | Fisher Scientific | 13-100-675 |

### Procedure

#### Notes/prep before starting

1. Check if RNase A has been added to Buffer B1 for a final concentration of **100 µg/mL**.
   * If yes, the check-box on the bottle should be marked.
   * If not, add **285 µL** of RNase A to Buffer B1.
2. Check if isopropanol has been added to Buffer BZ.
   * If yes, the check-box on the bottle should be marked.
   * If not, add **18 mL** of isopropanol to Buffer BZ.
3. Check if ethanol has been added to Buffer WZ.
   * If yes, the check-box on the bottle should be marked.
   * If not, add **104 mL** (4 volumes) of ethanol (95%) to Buffer WZ.
4. All centrifugation steps should be carried out at **16,000 x g (~13,000 RPM)**.
5. If precipitate has formed in Lysis Buffer (B2), incubate at **30–37°C**, inverting periodically to dissolve.
6. Store Plasmid Neutralization Buffer (B3) at **4°C** after opening, as it contains RNase A.
7. Empty all waste into a 50 mL Falcon tube. At the end of the experiment, empty the contents of the tube into the waste carboy. **Do not bleach.**

#### Miniprep

1. Pellet 3 mL bacterial culture by centrifugation for 1 minute. Discard the supernatant.
   * Ensure cultures are not overgrown (ideally 12–16 hours).
   * Either pellet 1.5 mL at a time in the same Eppendorf tube, discarding media in between, or centrifuge the culture tube and transfer to an Eppendorf tube in step 2.
2. Resuspend the pellet in **200 µL** of Buffer B1.
3. Add **200 µL** of Buffer B2, gently invert the tube 10 times, and incubate at room temperature for 1 minute. *Do not vortex.*
   * Color should change to dark pink, and solution will become transparent and viscous.
4. Add **400 µL** of Buffer B3, gently invert the tube until neutralized, and incubate at room temperature for 2 minutes. *Do not vortex.*

* The color should be uniformly yellow when neutralized.

5. Centrifuge lysate for 5 minutes.
   * Pellet should be compact; spin longer if needed.
6. Carefully transfer supernatant to the spin column.
   * Tilt the Eppendorf tube away from the pellet and carefully pipette up the supernatant, ensuring the pipette tip does not touch the pellet or debris stuck to the side of the tube.
7. Place spin column on a collection tube and centrifuge for 1 minute.
8. Discard flow-through.
9. Re-insert column in the collection tube and add **200 µL** of Buffer BZ. Centrifuge for 1 minute. Discard flow-through.
   * After discarding flow-through, dab the collection tube onto a Kimwipe to prevent reabsorption.
10. Add **400 µL** of Buffer WZ and centrifuge for 1 minute, then discard the buffer.
11. Spin the column empty for 1 minute to remove any remaining liquid.
12. Transfer column to a clean 1.5 mL Eppendorf tube.
    * Use care to ensure the tip of the column does not come into contact with the flow-through.
13. Add **30 µL** Nuclease-free water to the center of the matrix. Wait 1 minute, then spin for 1 minute to elute DNA.
14. Optional: transfer eluted solution onto the same spin column and spin for 1 minute. Heating water to 50°C before elution may increase yield.
15. Store extracted plasmids at -20°C.

*\*Original protocol sourced from NEB, and modified based on advice of mentors and testing.*

# Restriction Digest

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| UltraPure Water | Fisher Scientific | 10977023 |
| rCutSmart Buffer | New England Biolabs | B6004S |
| Plasmid | n/a | n/a |
| Restriction Enzymes | various | n/a |
| Eppendorf Tubes | Fisher Scientific | 05-408-129 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| C1000 Touch Thermal Cycler | BIORAD | BIO-C1000T |

### Procedure for Restriction Enzymes

1. Thaw and thoroughly mix restriction enzyme buffer (generally rCutSmart).
2. Calculate the amount of restriction enzyme and DNA needed for a 150 µL reaction.

* Target is ~3000 ng DNA.
* The restriction enzyme(s) should have a final concentration of 10 units/µg DNA.

3. Add all the components to an Eppendorf tube in the following order:
   * Nuclease-free Water
   * Restriction enzyme buffer
   * Plasmid
   * Restriction enzyme(s)
4. Incubate at the correct temperature for the enzyme. Cast a gel in the meantime and incubate until your gel is done setting (~30-45 min, see ‘Gel electrophoresis’ protocol for more).

* Incubation temperatures can be found on the information page of where you ordered the enzyme from.

5. Add loading dye to stop the reaction and run the digest on the prepared agarose gel.

* Treat the 6X loading dye as 10X and dilute down to 1X.

6. **Note:** You can put the stopped reaction in the freezer if you need to pause here.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# HiFi Assembly

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| HiFi master mix | New England Biolabs | E5520S |
| HiFi positive control | New England Biolabs | E5520S |
| Gel purified backbone fragment |  |  |
| gBlock insert |  |  |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| C1000 Touch Thermal Cycler | BIORAD | BIO-C1000T |

### Procedure

1. Thaw and vortex HiFi master mix and leave on ice.
2. Use calculator to determine component amounts using a target volume of 5 µL.
3. Add components to a PCR tube on ice.

* Add backbone, then insert, then HiFi master mix.

4. Set up a positive control using 2.5 µL of NEB positive control (NEBuilder) and 2.5 µL of master mix (1:1 ratio).
5. Mix well and spin down PCR tubes.
6. Incubate mixture in preheated thermal cycler at 50°C for 1 hour.
7. Store samples at -20°C or transform into competent cells.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# High Efficiency Transformation

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| LB plates with selection antibiotic |  |  |
| Assembled plasmid (See: HiFi assembly) |  |  |
| SOC media | New England Biolabs |  |
| NEB 5-alpha  *E. coli* competent cells | New England Biolabs | C2987 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Heat Block | Fisher Scientific | 88860022, 88860103 |

### Procedure

1. Set the heat block to 42°C.
2. Take SOC media out of fridge so that it warms up.
3. Thaw 2 + (2 × number of plasmids to transform) tubes of competent cells (C2987) on ice until the last ice crystals disappear (~15-20 minutes).
4. Aliquot out 50 µL of the competent cells into microcentrifuge tubes.
5. For each of your assemblies, perform a 4× dilution in PCR tubes using Ultrapure water:
   * 6 µL water + 2 µL HiFi assembly reaction
6. Add 2 µL of the following each into their own individual tube of cells:
   * PUC19
   * Positive Control
   * HiFi Assembly
   * 4x diluted HiFi Assembly
7. Carefully flick the tubes 4–5 times to mix. Do not vortex.
8. Place the mixtures on ice for 30 minutes. Do not mix.
9. Heat shock at 42°C for exactly 30 seconds. Do not mix.
10. Place on ice for 5 minutes. Do not mix.
11. Pipette 950 µL of room temperature SOC into each transformation tube and then transfer into a culture tube.
12. Incubate for one hour at 37°C, 280 RPM.

* About 20 minutes before the culture tube transformations are done, place selection plates in a static 37°C incubator to warm them up.

13. Spread 100 µL of each outgrowth onto their own plate using 5–8 rattler beads and incubate for 24 hours at 37°C.
14. Continue to Patch plates protocol the next day.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Gel Electrophoresis

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| UltraPure Water | Fisher Scientific | 10977023 |
| 10x TAE | Fisher Scientific | BP13354 |
| Agarose | Millipore Sigma | A9539-500G |
| SYBR safe | Fisher Scientific | S33102 |
| Purple Loading Dye | NEBiolabs | B7024S |
| 1 kb plus Ladder | NEBiolabs | N0550S |
| Eppendorf tubes | Fisher Scientific | 05-408-129 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Casting tray | BioRad | n/a |
| Well comb | BioRad | 3486-5512 |
| Voltage source | BioRad | 1645050 |
| Gel box | BioRad | B1A-BP |
| Microwave | Emerson | MW1338SB |
| Scale | Sartorius | BCE223-1S |

### Procedure

#### Pouring the Gel

1. Set up your gel station with a casting tray, gel tray, and level.
2. Place your gel tray inside the casting tray, making sure it is level and sealed tightly.
3. Make 1x TAE

* Dilute 10x TAE buffer to 1x. For a 500mL stock, add 50mL of 10x TAE to 450mL of milli-Q water

4. Weigh out the correct amount of agarose and add it to the 1x TAE in an Erlenmeyer flask.

* Microwave the mixture in 30-second intervals until the agarose is fully dissolved and no granules remain. Swirl carefully between intervals using a heat-safe glove or cloth.

### Gel Concentrations

| Tray type | Gel type | 1x TAE (mL) | Agarose (g) |
| --- | --- | --- | --- |
| Small | Routine Analytical | 50 | 0.5 |
| Small | Routine Preparatory | 50 | 0.4 |
| Small | Small fragment (<500 bp) analytical | 50 | 1 |
| Small | Large fragment (>10 kb) analytical | 50 | 0.25 |
| Large | Routine Analytical | 100 | 1 |
| Large | Routine Preparatory | 100 | 0.8 |
| Large | Small fragment (<500 bp) analytical | 100 | 2 |
| Large | Large fragment (>10 kb) analytical | 100 | 0.5 |

5. Add SYBR safe (10,000X) to 1X and swirl to mix.
6. Pour the solution into a level plastic tray. Use the comb to scoop any bubbles away.
7. Put the comb in and let the gel set for 30 min
8. Analytical gels should utilize the comb containing small teeth.
9. Preparative gels should utilize the comb containing wide teeth.

#### Running the Gel

1. Carefully remove the comb by pulling straight up with two hands.
2. Take the set gel out of the casting tray and place it into the gel box.
3. Add 1x TAE buffer to the fill line of the gel box.
4. Add loading dye to all your samples.
   * Treat the 6X loading dye as 10X and dilute down to 1X.
5. Load **5 µL of ladder** into the first well.
6. Carefully pipette your samples into each of the additional wells.
7. For each gel type:
   * **Analytical gels**: Pipette **10 µL** of the sample & loading dye mixture into each well.
   * **Preparative gels**: Pipette the full **165 µL** of sample and loading dye mixture into each well.
8. Run the gel at **120V** for the appropriate amount of time:
   * **Preparative gels**: 15 minutes (unless bands are very close)
   * **Analytical gels**: 25–45 minutes. Try a shorter time first, stop the power source, check in the transilluminator (leave gel in tray), and continue running in 5-minute increments as needed.
9. **Note:** Black is negative, red is positive. DNA is negatively charged and will run toward the red (positive) electrode. Always **Run to Red**.
10. Turn off the power, disconnect the electrodes from the power source, and carefully remove the gel from the gel box.
11. Place the gel into a large petri dish for imaging or further use.
12. TAE buffer liquid waste can be poured down the drain.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Gel Electrophoresis

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Monarch® spin DNA gel extraction kit | New England Biolabs | T1120L |
| Razor blade |  |  |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Blue-light transilluminator |  |  |

### Procedure

1. After running gel electrophoresis, remove the gel from the electrophoresis chamber and slide onto a large petri dish.
2. Place the petri dish on the blue-light transilluminator (with orange screen for protection) and turn the light on.
3. Carefully cut out the bands you want using a new sharp razor blade.

* Slice the gel vertically to separate the lanes one at a time, keeping track of which piece comes from which lane.
* Slice above and below the desired band to obtain a small sliver of gel.
* Flop the thin slice on it’s side and chop to cut away the gel between the bands
* remove all bits of gel with no DNA - err on side of losing DNA, only keep most concentrated piece of band

4. To collect your bands, first weigh an empty 1.5mL Eppendorf tube, then place the gel slice into the tube and measure again.

* your gel chunk should be about 0.1 g. Cut more gel away from the band if it is bigger.

5. Add 4 volumes of Buffer BY to 1 volume of gel slice of DNA gel.

* For example, add 400 µL Buffer BY per 100 mg agarose.

6. Incubate the sample at 55°C, vortexing periodically until the gel slice is completely dissolved (generally 5–10 minutes).
7. Insert the Spin Column into a Spin Collection Tube and load the sample onto the column. Note: Only load 800 µL max at a time onto the column
8. Spin for 1 minute, discard flow through
9. Re-insert column into collection tube. Add 200 μL Buffer WZ, spin for 1 minute, and discard flow-through

* After discarding the flow-through, dab the collection tube onto a Kimwipe to ensure the flow-through does not get reabsorbed by the collection tube

10. Repeat wash (step x)
11. Transfer column to a clean 1.5 mL microfuge tube

* Use care to ensure that the tip of the column does not touch the flow-through. If in doubt, re-spin for 1 minute

12. Add 6 μL UltraPure Water to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA

* Note: to maximize concentration, you can place the elution back onto the column, wait 1 minute, and re-spin

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# gBlock gene fragment resuspension

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| UltraPure water | Fisher Scientific | 10977023 |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| Heat block | Fisher Scientific | 88860022, 88860103 |
| Centrifuge 5424 R | Fisher Scientific |  |

### Procedure

1. Centrifuge tube in microcentrifuge at 16,000 × g for 10 seconds.
2. Add UltraPure Water to reach a final concentration of 20 ng/µL.
3. Vortex briefly.
4. Incubate at 50°C for 20 minutes.
5. Vortex briefly.
6. Centrifuge tube in microcentrifuge at 16,000 × g for 10 seconds.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Nanodrop

### Consumables

| Name | Vendor | Product Number |
| --- | --- | --- |
| Purified plasmid | - | - |
| Ultrapure water | Fisher Scientific | 10977023 |
| Kimtech wipe |  |  |

### Equipment

| Name | Manufacturer | Model Number |
| --- | --- | --- |
| NanoQuant Plate | Tecan |  |
| Plate reader | Tecan |  |

### Procedure

1. Clean the NanoQuant Plate adapter with a Kimwipe and gently blow with air to remove any fibers.
2. Open Tecan plate reader software. Navigate to applications, then open the nucleic acid quantification program.
3. Load 2 µL Ultrapure water blanks on the plate adapter as either group or individual blanks.
4. Insert adapter into plate reader.
5. Select correct wells and measure blanks.
6. Remove and clean plate, then load 2 µL of purified plasmid samples.
7. Insert adapter, select wells, and click the green triangle “run” button.
8. Remove and clean plate.

*\*Original protocol sourced from Boston University elMIG lab, and modified based on advice of mentors and testing.*

# Hydrogel Resuspension

### Procedure

1. Make overnight culture (37°C, 280 RPM, 12-16 hours) of selected strains in 5 mL LB media with appropriate concentration of antibiotic.
2. Refresh the overnight culture by diluting 1:100 into a 50 mL baffled flask.
3. Incubate at 37°C, 280 RPM to mid-log phase, checking the OD every hour until it is between 0.8 and 1.2.
4. While cells are incubating, prepare LB agar solutions in a 50 mL Falcon tube and place in the heat block at 50°C to keep the solution liquid.
5. Once cells reach mid-log phase, make 5 mL aliquots for each condition being tested. Spin down aliquots at 5,000 x g for 5 minutes to pellet the cells.
6. Pour off media and resuspend pelleted cells in 5 mL of respective LB agar solution.

* **Note:** The LB agar will begin to solidify as it cools so only resuspend one condition at a time and work through step 7 quickly.

7. Quickly and carefully pipette resuspended cells to a 96 well-plate, popping bubbles with a 2 µL tip as you go.
8. Once all conditions are plated, cover the plate with a lid.

*This protocol was created in collaboration with members of elMIG at Boston University based on Selim et. al and adapted for use in this project.*

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}
{% endblock %}

[![Back to top](https://static.igem.wiki/teams/5875/menu/vibi-arrows-totop.webp)](#top)

* [Introduction](#overview)
* [Putrescine Detection](#putrescine)
* [Hydrogel Suspension](#hydrogel)

![Results header](https://static.igem.wiki/teams/5875/results/results.webp)

# Introduction

---

BostonU iGEM 2025 set out to develop an at-home monitoring system for bacterial vaginosis (BV) using the tools of synthetic biology. After conversations with medical professionals and evaluating the current literature, the team identified putrescine as a relevant and measurable biomarker for vaginal microbiome health. A repressor circuit from the literature that is induced by putrescine was adapted for our use and built into a whole-cell biosensor in *E. coli*. That biosensor can then be suspended in a hydrogel for use with our combined hardware-biology platform. The device is able to monitor for bacterial vaginosis with better specificity and accuracy by encoding the logic of Amsel’s criteria, the current favored method for diagnosing BV, in the device using an electronic pH sensor.

Using this device, we can bring at-home vaginal microbiome testing to users at a relatively low cost. Our hardware and circuit design make this modular platform a jumping-off point for at-home vaginal health monitoring, enabling future iGEM teams to push forward in critical areas of women’s health research.

# Putrescine Detection

---

Using a circuit design inspired by Selim et al. (2022) [1], the team successfully designed, constructed, and cloned a putrescine sensing genetic circuit (Figure 1) in NEB® 5-alpha *Escherichia coli*, confirmed by whole-plasmid sequencing. An additional construct with a second constitutive promoter instead of the repressed pHyb(1B) promoter was also constructed as a positive control.

![Putrescine Circuit](https://static.igem.wiki/teams/5875/notebook/notebook1.webp)

Figure 1. Circuit Design.

The plasmids were then cloned into BL21(DE3) *E. coli* for testing and the sensing circuit was  able to detect levels of putrescine down to 10 mM from stock samples in LB media. The circuit produces a quantifiable response in 10 hours.

![Putrescine Results](https://static.igem.wiki/teams/5875/results/baseline-corrected-of-copy-of-gfp-v004.webp)

Figure 2. Kinetic view of putrescine dose response in V004, the genetic circuit with a putrescine-inducible promoter. At 10 hours, the cells begin to produce GFP, with the signal leveling off after 14 hours.

![Putrescine Results](https://static.igem.wiki/teams/5875/results/results4.webp)

Figure 3. Timepoint view at 16 hours of GFP divided by OD600

# Hydrogel Suspension

---

To deploy whole-cell biosensors in an at-home device, the team turned toward agar hydrogels as a medium for sustaining bacterial viability. Testing in the agar hydrogels was carried out in an internal *E. coli* strain that constitutively expresses eGFP under the strong pVeg1 promoter. We first tested the effect of resuspension in heated media, a requirement for the agar solids, on growth and fluorescence in regular LB liquid media. The results indicate that there was almost no effect on growth over 60 hours. However, there was a small effect on activity after around 20 hours, indicating a small loss in signal for our use case.

![Hydrogel Results](https://static.igem.wiki/teams/5875/results/results2.webp)

Figure 4. Growth-normalized fluorescence for M0065 in liquid LB media over 60 hours.

We tested different media conditions and found that standard LB nutrient concentration and 1% (w/v) agar were ideal for maintaining growth and signal strength. *E. coli* resuspended in PBS grew almost as well as cells in regular LB media, but their fluorescence expression remained lower than those in LB over the course of the experiment. The GFP signal strength was significantly lower in the hydrogel wells than in the liquid media, likely due to interfering effects of the solid agar matrix. Because of this, we stuck to 1% or 1.5% (w/v) agar for future experiments.

To test how well the agar hydrogel sustained *E. coli* viability over time, we took our optimal hydrogel conditions and suspended cultures for one week. After around 24 hours, the GFP signal strength plateaued and remained stable over the course of the week. This shows that agar hydrogels are a viable option for using whole-cell biosensors in deployable devices. There were some issues with agar hydrogels drying out over the course of the experiment. We found that covering the gels completely helped in most cases, but future testing is needed to optimize the size and conditions of the hydrogel.

![Hydrogel Results](https://static.igem.wiki/teams/5875/results/results3.webp)

Figure 5. GFP expression over time of differing LB Agar hydrogels.

## References

[1] Selim, A. S., Perry, J. M., Nasr, M. A., Pimprikar, J. M., & Shih, S. C. C. (2022). A synthetic biosensor for detecting putrescine in beef samples. *ACS Applied Bio Materials, 5(11), 5487–5496.* https://doi.org/10.1021/acsabm.2c00824

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}
{% endblock %}