iGEM 2024 — Team bolivia

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# Notebook

![Notebook](https://static.igem.wiki/teams/5257/project/notebook/banner-result.webp)

# Notebook La Paz

# Notebook Santa Cruz

# Notebook Cochabamba

{% endblock %}

{% 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. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

# Engineering Success

![Engineering Success](https://static.igem.wiki/teams/5257/project/engineering/banner-result.webp)

# Engineering Success

* Overview
* Design
* Build
* Test
* Lessons Learned
* References

# Overview

![overview](https://static.igem.wiki/teams/5257/project/engineering/overview-result-2-1.webp)
Our mercury biosensor aims to provide an innovative tool for the rapid and precise detection of mercury in
water, with a particular focus on organic mercury. By leveraging genetically engineered bacteria, we sought
to create a biosensor capable of identifying toxic concentrations of mercury in environmental settings,
specifically in aquatic ecosystems.

To achieve this, we employed the design-build-test-learn (DBTL)
cycle, a fundamental framework in synthetic biology that allows for continuous optimization throughout the
development process. This iterative approach was instrumental in refining each stage of the biosensor’s
evolution.

# Design

The iGEM Bolivia team developed two innovative strategies for creating a biosensor capable of selectively
detecting both organic and inorganic mercury. These designs incorporate genes and promoters optimized for
compatibility with E. coli DH5α, a derivative of E. coli K12 (Zhang, W., et al., 2023). Our choice of strain
was informed by prior success with similar bacterial systems, such as E.
coli BL21 (Amarelle, V., et al.,
2023), ensuring both efficiency and compatibility.

## Strategy 1: “NOT Logic Gate” for Selective Detection

This strategy utilizes a "NOT logic gate" mechanism to differentiate between organic and inorganic mercury,
ensuring that the biosensor is selectively responsive to organic mercury, while inhibiting responses to
inorganic mercury.

## Inorganic Mercury

Input
Processing
Output

## Organic Mercury

Input
Output

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/3-input-con-hg2-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/5-procesamiento-con-hg2-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/6-output-con-hg2-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/8-input-con-mehg-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/9-output-con-mehg-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy1/2-sin-nada-result-1.webp)

Dual-Plasmid System: The bacterium is transformed with constructs  #1 and #2.

In the Presence of Organic Mercury: The protein MerRm4-1, a mercury-sensitive transcription factor,
binds to organic mercury, activating the expression of the mRFP\_Magenta reporter, which emits magenta
fluorescence as an indicator of organic mercury.

In the Presence of Inorganic Mercury (Hg²⁺): Inorganic mercury binds to the merRtn501 transcription
factor, which triggers the expression of the mChartreuse reporter, emitting green fluorescence. While
inorganic mercury can also bind to MerRm4-1, the reporter is degraded due to a proteolysis mechanism
controlled by the TEV enzyme, this enzyme exposes a degradation tag. At the same time the expression of
the magenta reporter is suppressed by tetR, a repressor protein encoded by the tetR gene. TetR binds to
the tetO site, effectively preventing
mRFP\_Magenta expression and ensuring that no magenta signal is emitted in response to inorganic mercury.

This strategic design guarantees that the biosensor will exclusively emit a signal in the presence of organic
mercury, while any response to inorganic mercury either results in green fluorescence or is completely
blocked by TetR-mediated repression.

## Strategy 2: Selective Mercury Detection via Volatilization of Inorganic Mercury.

This strategy volatilizes inorganic mercury out of the cell before it enters the biosensor, ensuring that
the
biosensor responds selectively to organic mercury.

## Inorganic Mercury

Input
Output

## Organic Mercury

Input
Processing
Output

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/4-input-con-hg-2-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/5-output-con-hg2-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/7-input-con-mmhg-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/8-processing-con-mmhg-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/9-output-con-mmhg-result-1.webp)

![](https://static.igem.wiki/teams/5257/project/engineering/strategy2/2-input-sin-nada-result-1.webp)

Dual-Plasmid System: The bacterium is transformed with Constructs  #2 and #3.
In the Presence of Inorganic Mercury (Hg²⁺):Two key protein complexes, OmpTLOT-gdh and OmpTLOT-merA,
form on the bacterial membrane. Together, these complexes prevent inorganic mercury from entering the
cell. The OmpTLOT-merA complex reduces Hg²⁺ to Hg⁰ (elemental mercury), a volatile form that leaves the
system without eliciting a detection signal.

In the Presence of Organic Mercury The gomerB enzyme cleaves methylmercury, a form of organic mercury,
converting it into inorganic mercury. This is then detected by the merRtn501 transcription factor, which
activates the mChartreuse reporter, emitting green fluorescence. Additionally, TEV protease is expressed
to cleave MerA, preventing further reduction of inorganic mercury and ensuring proper detection..

This strategy is particularly effective in environments where both forms of mercury are present, as it allows
for the selective detection of organic mercury while volatilizing inorganic mercury to eliminate
interference.

# Build

## Developing the Mercury Biosensor

The build phase focuses on the construction and assembly of the necessary DNA constructs to create a
portable, sensitive, and selective system for detecting both organic and inorganic mercury. This phase is
critical for ensuring that the biosensor meets our rigorous standards for accuracy, modularity, and safety.

## Key Constructs and Components

Our biosensor design revolves around three distinct plasmid constructs, each serving a specific role in
mercury detection and detoxification:
Construct 1: Encodes the merRm4-1 transcription factor, which is sensitive to both methylmercury and
inorganic mercury. In the presence of organic mercury, this construct triggers the expression of
mRFP\_Magenta, producing magenta fluorescence.

### Construct #1 (1459 pb)

![Construct 1](https://static.igem.wiki/teams/5257/project/engineering/construct1-result-2.webp)

![Construct 1](https://static.igem.wiki/teams/5257/project/engineering/construct1-qr-result-1.webp)

### Scan me!

Construct 2: Contains merRtn501, a transcription factor that specifically responds to inorganic mercury
(Hg²⁺), inducing the expression of mChartreuse, which emits green fluorescence. TetR is also expressed to
suppress the magenta signal, ensuring clear differentiation between organic and inorganic mercury.

### Construct #2 (2978 pb)

![Construct 2](https://static.igem.wiki/teams/5257/project/engineering/construct2-result-2.webp)

![Construct2](https://static.igem.wiki/teams/5257/project/engineering/construct2-qr-result-1.webp)

### Scan me!

Construct 3: Comprises MerA, gomerB, and gdh for detoxification. MerA reduces Hg²⁺ to Hg⁰, while gomerB
breaks down methylmercury into Hg²⁺. gdh provides NADPH for the reduction process.

### Construct #3 (4603 pb)

![Construct 3](https://static.igem.wiki/teams/5257/project/engineering/construct3-result-2.webp)

![Construct 3](https://static.igem.wiki/teams/5257/project/engineering/construct3-qr-result-1.webp)

### Scan me!

These constructs were synthesized by IDT and Twist. They will be transformed into E. coli DH5α, with combinations of plasmids 1 with 2, and 3 with 2. Although
this requires the use of two selection markers, it avoids the need for molecular cloning, simplifying the
process.

### Construct  transformation.

Each construct will be introduced into E. coli DH5α, a strain known
for its stability and ease of transformation. The modular design allows us to assemble each plasmid
separately before combining them in the host bacterium. This staged approach enables individual
verification of each construct before proceeding to final assembly.

### Screening and validation

To confirm the correct assembly of our constructs, we will use plasmid selection markers and observe
colony growth on antibiotic media. Fluorescence assays will be conducted to verify the expression of
the fluorescent proteins in response to methylmercury and inorganic mercury. If any inconsistencies
arise, we will reassess the assembly strategy, paying particular attention to restriction sites and
potential chassis toxicity.

### Final testing and transition to the next phase of the DBTL.

After verifying the constructs, we will assess functionality by introducing methylmercury and
inorganic mercury into the biosensor. Fluorescence will be measured to confirm the system’s ability
to differentiate between the two mercury species. Upon successful validation, the system will
advance to the next phase, undergoing rigorous testing in real-world conditions in accredited
laboratories, guided by heavy metal experts.

# TEST

## Sensitivity Evaluation

To ensure the selective detection of organic mercury, we will define the maximum allowable concentration of
methylmercury at 1.6 µg/L or 1.6 ppb, aligning with the World Health Organization (WHO) guidelines for 2021.
The sensitivity of our biosensor, designed according to the promoter characteristics outlined by Zhu et al.
(2023), has demonstrated a detection limit (LOD) of 0.098 µg/L for methylmercury—significantly below the
WHO's recommended threshold. We will conduct tests using standard solutions of methylmercury at
concentrations ranging from 0 to 100 ppb (0, 20, 40, 60, 80, 100). Before these tests, we will evaluate cell
densities at OD600 as per the methodology of Guo et al. (2021). This approach is designed to capture data at
concentrations close to the expected detection limits and will be supported by the development of a
calibration curve using these varied standard solutions.

![Sensitivity evaluation](https://static.igem.wiki/teams/5257/project/engineering/sensitivity-evaluation-result-1.webp)

#### Evaluation of specificity to mercury and different standard metal solutions.

## Detection of Methylmercury in Environmental Samples.

Upon establishing the necessary parameters for effective function, the biosensor will be employed to detect
methylmercury within environmental water samples. We will utilize GFP fluorescence as an indicator within
these samples, employing both positive and negative controls with predefined methylmercury concentrations to
affirm the biosensor's accuracy. Exposure to real sample environments will allow us to derive mercury
concentrations from established standard curves. Additionally, for a thorough comparative analysis, we will
measure the total mercury concentration present in these samples using atomic absorption spectroscopy,
ensuring a comprehensive understanding of mercury levels in natural water bodies.

![](https://static.igem.wiki/teams/5257/project/engineering/detection-methylmercury-1-result-1.webp)
![](https://static.igem.wiki/teams/5257/project/engineering/detection-methylmercury-2-result-1.webp)

#### Detection of heavy metals and measurement of gfp protein fluorescence.

## Biosensor characterization

The fluorescence of each biosensor, exposed to varying concentrations of organic and inorganic mercury, will
be measured to evaluate its performance.

### Strategy 1: Utilizes a NOT logic gate for selective detection Strategy 2: Employs cell surface display with mercuric reductase

#### Strategy #1: Use of NOT logic gate

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat1-1-result.webp)

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat1-2-result.webp)

#### Strategy #2: Cell surface display with mercuric reductase

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat2-1-result.webp)

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat2-2-result.webp)

## New composite part characterization

The new composite part expresses both mercuric reductase (MerA) and glucose-1-dehydrogenase (GDH) on the
bacterial surface. MerA requires NADPH as a cofactor, which will be provided by GDH. Characterization will
occur in two phases:

* Phase 1.- Each enzyme, MerA and GDH, will be characterized individually and in combination.
* Phase 2. - The entire enzyme system will be characterized together.

## Phase 1: Enzyme Characterization

Expected results. - GDH activity should increase absorbance by generating NADPH, while MerA will decrease
absorbance by consuming NADPH. If both enzymes are balanced in the bacterial cultures, there should be no
significant change in absorbance, indicating equilibrium between NADPH production and consumption.

#### Enzymatic activity of GDH-expressing E. coli

![Enzymatic activity of GDH-expressing <span class=](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-1-result.webp)

#### Enzymatic activity of MerA-expressing E. coli

![Enzymatic activity of MerA-expressing <span class=](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-2-result.webp)

#### Combination of GDH-expressing and MerA-expressing E. coli

![Combination of GDH-expressing and                     MerA-expressing <span class=](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-3-result.webp)

## Phase 2: Complete System Characterization

The full enzyme system will be assessed for its ability to reduce inorganic mercury in the presence and
absence of NADPH. After culturing, aliquots will be collected and mercury concentrations in the supernatant
will be quantified.

![Complete System Characterization](https://static.igem.wiki/teams/5257/project/engineering/complete-system-characterization-result.webp)

Expected results. -  E. coli
expressing the composite part, when
cultured with glucose, should show maximum inorganic mercury reduction. Comparable results are expected with
NADPH supplementation alone. No reduction should occur in the absence of glucose or NADPH, as MerA will
degrade intracellularly.

## NADPH recycling using surface displayed GDH and MerA on E. coli

![NADPH recycling using surface displayed GDH and MerA](https://static.igem.wiki/teams/5257/project/engineering/nadph-recycling-using-surface-result-2.webp)

# Lessons Learned

Our most complex part (BBa\_K5257023) performs the most important task for strategy 2, volatilizing inorganic
mercury Hg2+ to prevent it from entering the bacteria and giving it the ability to selectively target
organic mercury, without the need for NADPH cofactors.
Due to its complexity, we propose 3 redesign approaches for our new composite part (BBa\_K5257023):

1. Redesign RBS30 and 31.- The stronger RBS (RBS30) controls
   translation of the MerA enzyme and RBS31
   controls GDH. The MerA\_2D variant used has a better affinity for mercury and better catalytic
   activity,
   so it consumes much more NADPH than other MerA enzymes. We believe that reversing the strengths of
   the
   RBSs could result in higher GDH production and more efficient NADPH recycling.
2. Redesign of gdh dehydrogenase. -  GDH provides NADPH cofactors to
   the MerA enzyme, however, it makes use
   of glucose to function, which may hinder the use of other promoters such as PBAD by catabolic
   repression, it is also worth mentioning that GDH is a huge homotetramer of more than 100kDa which
   may
   hinder its anchoring. We consider that other alternatives such as glutamate dehydrogenase (Gldh,
   Liang
   et al. 2015) or alcohol dehydrogenase (ADH, Tian et al. 2022) result viable options to fulfill the
   same
   role without the use of glucose.
3. Redesign of MerA anchor. -  MerA is on the bacterial surface,
   another alternative would be to secrete the
   MerA enzyme by replacing DogTag with a signal peptide, this would not limit the amount of proteins
   that
   are located within the outer membrane boundaries (Liu et al. 2016).

# References

* Amarelle, V., Roldán, D. M., & Fabiano, E. (2023). Identification of novel broad host-range promoter
  sequences functional in diverse Pseudomonadota by a promoter-trap approach. Brazilian Journal of
  Microbiology, 54(1), 1-12.
  <https://doi.org/10.1007/s42770-023-00840-7>

* Fernandez-Rodriguez, J., & Voigt, C. A. (2016). Post-translational control of genetic circuits using
  Potyvirus proteases. Nucleic acids research, 44(13), 6493-6502.
  <https://doi.org/10.1093/nar/gkw537>

* Gallus, S., Peschke, T., Paulsen, M., Burgahn, T., Niemeyer, C. M., & Rabe, K. S. (2020). Surface
  Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction. ChemBioChem, 21(15), 2126-2131.
  <https://doi.org/10.1002/cbic.202000102>

* Guo, M., Chen, X., Su, H., Liu, H., Xie, G., & Sun, B. (2024). Replacing manual operation with
  bio-automation: A high-throughput evolution strategy to construct an integrated whole-cell biosensor
  for simultaneous detection of methylmercury and mercury ions. Journal of Hazardous Materials, 465,
  133492.
  <https://doi.org/10.1016/j.jhazmat.2024.133492>

* Hui, C. Y., Guo, Y., Liu, L., Zheng, H. Q., Wu, H. M., Zhang, L. Z., & Zhang, W. (2019). Development
  of a novel bacterial surface display system using truncated OmpT as an anchoring motif.
  Biotechnology letters, 41, 763-777.
  <https://doi.org/10.1007/s10529-019-02676-4>

* IGEM Technology. (s. f.).
  <https://technology.igem.org/engineering/introduction>

* Keeble, A. H., Yadav, V. K., Ferla, M. P., Bauer, C. C., Chuntharpursat-Bon, E., Huang, J., ... &
  Howarth, M. (2022). DogCatcher allows loop-friendly protein-protein ligation. Cell chemical biology,
  29(2), 339-350.
  <https://doi.org/10.1016/j.chembiol.2021.07.005>

* Kostylev, M., Otwell, A. E., Richardson, R. E., & Suzuki, Y. (2015). Cloning Should Be Simple:
  Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies. PLoS
  ONE, 10(9), e0137466.
  <https://doi.org/10.1371/journal.pone.0137466>

* Lian, P., Guo, H. B., Riccardi, D., Dong, A., Parks, J. M., Xu, Q., & Guo, H. (2014). X-ray
  structure of a Hg²⁺ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical
  study of Hg²⁺ transfer between the C-terminal and buried catalytic site cysteine pairs.
  Biochemistry, 53(46), 7211-7222.
  <https://doi.org/10.1021/bi500608u>

* Liang, B., Zhang, S., Lang, Q., Song, J., Han, L., & Liu, A. (2015). Amperometric L-glutamate
  biosensor based on bacterial cell-surface displayed glutamate dehydrogenase. Analytica Chimica Acta,
  884, 83-89.
  <https://doi.org/10.1016/j.aca.2015.05.012>

* Liu, Z., Ho, S. H., Sasaki, K., Den Haan, R., Inokuma, K., Ogino, C., . & Kondo, A. (2016).
  Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic
  biofuel production. Scientific reports, 6(1), 24550.
  <https://doi.org/10.1038/srep24550>

* Tian, H., Furtmann, C., Lenz, F., Srinivasamurthy, V., Bornscheuer, U. T., & Jose, J. (2022). Enzyme
  cascade converting cyclohexanol into ε‐caprolactone coupled with NADPH recycling using surface
  displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli. Microbial Biotechnology,
  15(8), 2235-2249.
  <https://doi.org/10.1111/1751-7915.14062>

* Zhang, G., Hu, S., & Jia, X. (2021). Highly Sensitive Whole-Cell Biosensor for Cadmium Detection
  Based on a Negative Feedback Circuit. Frontiers in Bioengineering and Biotechnology, 9.
  <https://doi.org/10.3389/fbioe.2021.799781>

* Zhang, W., Tang, L., Xie, J., & Liu, Y. (2023). Systems metabolic engineering of Escherichia coli
  for hyper-production of 5-aminolevulinic acid. Biotechnology for Biofuels and Bioproducts, 16(1),
  1-20.
  <https://doi.org/10.1186/s13068-023-02195-4>

* Zhu, X., Bai, M., Qin, Y., Zhang, X., Tang, J., Liu, Z., & Gao, J. (2023). Integrated pathway
  analysis reveals a systemic response to airborne particulate matter in the lung. Environmental
  Science & Technology.
  <https://pubs.acs.org/doi/10.1021/acs.est.3c04915>

* World Health Organization (WHO). (2021). Estándares de calidad del agua potable: Directrices de la
  OMS para la calidad del agua potable.
  <https://iris.who.int/bitstream/handle/10665/341450/9789240025134-spa.pdf>

* Guo, J., Zhuang, Q., Zheng, Y., Shi, Y., & Zhang, W. (2021). Engineered whole-cell biosensors for
  heavy metal detection and bioremediation. Frontiers in Bioengineering and Biotechnology, 9, 799781.
  <https://doi.org/10.3389/fbioe.2021.799781>

* Zhang, Y., Wang, Q., & Liu, W. (2021). The development of mercury biosensors based on synthetic
  biology for environmental monitoring. Scientific Reports, 11, 92878.
  <https://doi.org/10.1038/s41598-021-92878-6>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

* Protocol for competent cells
* Bacterial transformation protocol
* Protocol for Handling Mercury Compounds

# Protocol for competent cells

### Objective:

To generate competent E. coli  DH5α cells, enabling them to uptake genetic material through chemical methods
(heat shock method).

### Materials:

### Biological Materials:

* E. coli DH5α in an Eppendorf tube or on an LB agar Petri dish

### Reagents:

* LB broth
* Calcium chloride (CaCl2), 150 mM, kept at 4°C (on ice)
* 100% glycerol

### Labware:

* Eppendorf tubes
* Falcon tubes
* Polystyrene cuvette
* Sterile tips (1000 µL)
* Micropipette (100 µL to 1000 µL range)
* Crushed ice ≈ 4°C (refrigerator)

### Equipment:

* Eppendorf tube centrifuge
* UV-Vis spectrophotometer
* Temperature-controlled centrifuge
* Incubator with shaking capability
* Ultralow freezer (-80°C)
* Laminar flow hood
* Autoclave

### Procedure:

Day 1 (afternoon/evening): Prepare an overnight culture

1. Place the sterile LB broth (10 ml in a tube) and the E. coli DH5α bacteria (either in the Eppendorf
   tube
   or on the LB agar plate) in a rack.
2. Add 0.1 ml (100 µL) of E. coli DH5α to the 10 ml of sterile LB broth.
3. Incubate the tube overnight (approximately 12 hours) at 37°C with shaking at 200 rpm.

Day 2 (morning): Ensure you have crushed ice for the culture and CaCl2.

1. Remove the overnight culture and measure the OD600 (should be >1). To do this, add 10 µL
   of
   the E. coli DH5α culture to a spectrophotometer cuvette and 900 µL of LB broth. Record the reading
   and
   correct for dilution.
2. Calculate the necessary dilution to reach an OD600=0.1. Transfer the calculated volume to the tube
   with
   10 ml of LB broth using a micropipette.
3. Incubate the tube at 37°C with shaking at 200 rpm until an OD600=0.5-0.7 is reached (approximately 2
   hours).
4. Transfer 1 ml of the culture to an Eppendorf tube and place on crushed ice (4°C) for 20 minutes.
5. Centrifuge the Eppendorf tubes for 1 minute at 13,000 rpm. Remove the tube, discard the supernatant,
   and
   place it back on crushed ice.
6. Slowly add 500 µL of cold CaCl2 (150 mM) to the tubes. Gently invert the tubes 4 times to resuspend
   the
   pellet.
7. Incubate on ice for 20 minutes.
8. Centrifuge the Eppendorf tubes for 1 minute at 13,000 rpm. Remove the tube, discard the supernatant,
   and
   place it back on crushed ice.
9. Slowly add 50 µL of cold CaCl2 (150 mM) to the tubes. Gently invert the tubes 4 times to resuspend
   the
   pellet.
10. Store at 4°C for immediate use or at -20°C for use within the week.

### Notes:

The cells are now ready for use in the transformation protocol. If long-term storage at -80°C is required,
prepare CaCl2 (150 mM) with 20% glycerol and use it in step #12.

### Reference

Green, M. Richard, & Sambrook, J. Frank. (2012). Molecular cloning : A laboratory manual. 4th ed. Cold Spring
Harbor (N.Y.): Cold Spring Harbor Laboratory Press.

## Competent Cells Protocol

1

Day 1 (afternoon/evening): Prepare an overnight culture
1. Add 0.1 ml E. coli DH5a to 10 ml LB broth.
2. Incubate overnight at 37°C, 200 rpm.

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-1-result.webp)

2

Day 2 (morning): Measure OD600 of culture (>1).
Dilute to OD600-0.1 in 10 ml LB.

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-2-result.webp)

3

Incubate at 37°C with
shaking until
OD600=0.5-0.7 (~2 hrs).

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-3-result.webp)

4

Transfer 1 ml of culture to an Eppendorf tube and place on crushed ice (4°C) for 20 minutes.
Centrifuge at 13,000 rpm for 1 min, discard supernatant.

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-4-result.webp)

5

Add 500 μL cold CaCl2 (150 mM), invert to resuspend. Incubate on ice for 20 min, centrifuge again,
discard the supernatant.
20 min

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-5-result.webp)

6

Add 50 μL cold CaCl2 (150 mM), resuspend. Store at 4°C (short-term) or -20°C (1 week).
CaCl2 (150 mM)

![](https://static.igem.wiki/teams/5257/project/experiments/competent-cells-protocol-6-result.webp)

# Bacterial Transformation Protocol

### Objective:

To transform DH5-alpha Thermo ScientificⓇ cells with mercury-detecting plasmids.

### Prerequisites

* Plasmids
* Sterile Eppendorf tubes
* Competent cells
* Ice
* LB broth
* LB agar
* Drigalski spatula
* Antibiotic (Kanamycin)
* Bunsen burner
* White and yellow tips
* Large Petri dishes
* 37°C incubator
* 42°C heater (hot water bath and thermometer)

### Procedure

1. Autoclave Materials: Prepare LB liquid medium.
2. FlasAfter autoclaving, gently swirl the medium to mix the agar evenly. Cool the medium to around 50°C,
   then add kanamycin/ampicillin at a concentration of 0.1 mg/ml. Mix well without creating bubbles, and
   pour approximately 15 ml of the medium into each plate.ks
3. Once the medium has fully solidified, the plates are ready for use. Alternatively, invert the plates and
   store them at 4°C. To prevent contamination, perform the entire process using a Bunsen burner or within
   a laminar flow hood.
4. For bacterial cell transformation with a plasmid, use competent E. coli DH5 cells, previously prepared
   with CaCl₂ and stored at -70°C in 100 μl aliquots.
5. Thaw the cells on ice and add 50 μl to a ster
6. ile Eppendorf tube.Add 1 μl of plasmid DNA and incubate on ice for 25-30 minutes.
7. Subject the mixture to heat shock at 42°C for 90 seconds, then immediately place on ice for 2 minutes.
8. Add 400 μl of LB broth without antibiotic and incubate at 37°C for 15 minutes in a water bath or
   incubator.
9. Plate the cells on LB agar plates containing ampicillin (selection antibiotic) and spread them evenly
   with a Drigalski spatula. Perform plating with and without DNA on plates with and without antibiotic (4
   plates in total).
10. Incubate overnight at 37°C.
11. The next day, check for the growth of ampicillin-resistant bacterial colonies.
12. Calculate transformation efficiency, expressed as the number of transformants per microgram of DNA.

### References

Green, M. Richard, & Sambrook, J. Frank. (2012). Molecular cloning : A laboratory manual. 4th ed. Cold Spring
Harbor (N.Y.): Cold Spring Harbor Laboratory Press.

## Transformation Protocol

1

Prepare LB agar plates:

1. Autoclave materials.
2. Cool LB medium to 50°C, add 0.1 mg/ml antibiotic (Kanamycin/Ampicillin), and pour ~15 ml into
   each plate.
3. Let plates solidify and store at 4°C (if not using mmediately).

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-1-result.webp)

2

Thaw competent cells:

1. Thaw 50 μl of DH5-alpha cells on ice
2. Add 1 μl plasmid DNA to the cells and incubate on ice for 25-30 minutes.

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-2-result.webp)

3

Heat shock:
Heat shock at 42°C for 90 seconds, then place on ice for 2 minutes.

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-3-result.webp)

4

Recovery: Add 400 μl LB broth
(no antibiotic) and incubate at
37°C for 15 minutes.

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-4-result.webp)

5

Plate cells:

* Plate cells on LB agar
  with antibiotic, using a
  Drigalski spatula to
  spread evenly.
* Prepare 4 plates: with and without DNA, with and
  without antibiotic.

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-5-result.webp)

6

Incubate: Incubate plates overnight at 37°C.
Results:

* Check for growth of antibiotic-resistant colonies the next day.
* Calculate transformation efficiency (transformants/μg DNA)

![](https://static.igem.wiki/teams/5257/project/experiments/transformation-protocol-6-result.webp)

# Protocol for Handling Mercury Compounds

### Objective:

Ensure the proper handling of mercury compounds used during the validation of the mercury biosensor,
including their storage, minimizing handling, contamination, and disposal of residuals.

### Prerequisites

* Obtain the Safety Data Sheets (SDS) for mercury compounds.
* Have prior knowledge of the use of personal protective equipment (PPE).
* Have received first aid training.

### Materials:

* Beakers
* Flasks

### Reagents:

| Mercury (II) chloride (CAS 7487-94-7) | |
| --- | --- |
| Density | 5,44 g /cm³ at 20 °C |
| Color | white |
| Melting point/freezing point | 277 °C |
| Water solubility | 74 g /l at 20 °C |
| Danger label(s) | ![](https://static.igem.wiki/teams/5257/project/experiments/dangers-1-result.webp) |

| Methylmercury(II) chloride (CAS 115-09-3) | |
| --- | --- |
| Physical State | Liquid |
| Danger label(s) | ![](https://static.igem.wiki/teams/5257/project/experiments/dangers-2-result.webp) |

### Equipment:

* Fume hood
* Balance

### Personal Protective Equipment

* Nitrile gloves
* Safety goggles
* Lab coat
* N95 mask

### Storage of Reagents

* Store in glass containers with an acid-resistant coating or in high-density polyethylene (HDPE) or
  polypropylene (PP) plastic containers with a completely airtight seal.
* Do not store with compounds containing: acetylene, fulminic acid, ammonia.

### Storage of Reagents

* Ensure that the fume hood is functioning properly.
* Have a neutralizing solution readily available in case of spills. A sodium sulfide (Na₂S) 0.5M solution
  can be used (weigh 39.02 g of Na₂S, dissolve in a small amount of distilled water, then transfer to a 1
  L volumetric flask and fill with distilled water; work under a fume hood).
* Ensure the laboratory temperature is controlled to prevent the volatilization of compounds.

## Handling of Mercury Compounds

### a) Weighing

* Weighing should be done within a fume hood, using a glass or Teflon container with a lid; close
  immediately after weighing.
* Clean the balance surface with a towel impregnated with a sodium thiosulfate solution, disposing of the
  towels as hazardous waste. Subsequently, clean the surface with towels impregnated with distilled water
  and isopropyl alcohol.
* Finally, recalibrate the balance.

### b) Solution Preparation

* Dissolve the required amount of mercury compound in distilled water to prepare the appropriate quantity,
  using glassware dedicated exclusively to preparing inorganic or organic mercury solutions.
* Use Teflon containers for storing methylmercury solutions for up to one month. If glass bottles are
  used, acidify the solution to pH 1 with concentrated HCl or HNO₃.

### c) Cleaning the Work Area

* Clean the work area with towels impregnated with sodium thiosulfate, followed by distilled water.
* Dispose of the towels in sealed hazardous material containers.

### d) Spill Procedure

* Establish a safety perimeter around the spill.
* Neutralize with sodium thiosulfate.
* Use mercury sponges.
* Place waste in a single container and label it.
* Clean the area with distilled water and detergent.
* Finally, ventilate the area.

## Procedure in Case of Contact with Compounds

The first step in the event of a laboratory accident is to implement established biosafety and containment
protocols for mercury, and inform the responsible supervisor of the incident.

### a) Mercury Salts Spilled on Skin

* Wash and rinse immediately with abundant running water for 15 minutes. Safety showers installed in
  laboratories should be used if the affected area is large and sink washing is insufficient.
* Remove contaminated clothing and wash the affected area as soon as possible. Prompt washing is crucial
  to reduce the severity and exposure to mercury.
* Seek medical assistance. The affected individual should seek medical evaluation and receive further
  instructions if necessary.

### b) Inhalation of Mercury Salts

* Leave the contaminated area where mercury vapor exposure occurred.
* Seek fresh air: immediately move the affected person to an area with fresh air.
* Seek medical assistance as soon as possible. At the first sign of respiratory difficulty, initiate
  artificial respiration. Oxygen should be administered only by trained personnel.

### References

Leermakers, M., Lansens, P., & Baeyens, W. (1990). Storage and stability of inorganic and methylmercury
solutions. Fresenius' Journal of Analytical Chemistry, 336, 655-662. <https://doi.org/10.1007/BF00331410.>
Ramírez, A. V. (2008, March). Intoxicación ocupacional por mercurio. In Anales de la Facultad de Medicina
(Vol. 69, No. 1, pp. 46-51). UNMSM. Facultad de Medicina.
Basu, N., Bastiansz, A., Dórea, J. G., Fujimura, M., Horvat, M., Shroff, E., Weihe, P., & Zastenskaya, I.
(2023). Our evolved understanding of the human health risks of mercury. Ambio, 52(5), 877–896.
<https://doi.org/10.1007/s13280-023-01831-6>

## Mercury Compounds Handling Protocol

1

Prerequisites

* Obtain Safety Data Sheets (SDS) for mercury compounds.
* Wear Personal Protective Equipment (PPE): gloves, goggles, lab coat, N95 mask.
* Complete First Aid Training.

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-1-result.webp)

2

Work Area Preparation

* Ensure fume hood is functioning.
* Prepare neutralizing solution (Sodium Sulfide Na2S 0.5M).
* Maintain controlled lab temperature to prevent volatilization

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-2-result.webp)

3

 Handling & Weighing Mercury Compounds

* Weigh compounds in a fume hood, using glass or Teflon containers.
* Clean balance with sodium thiosulfate solution.
* Recalibrate balance after cleaning.

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-3-result.webp)

4

Solution Preparation

* Use glassware exclusively for mercury solutions
* Store methylmercury in Teflon containers for up to one month

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-4-result.webp)

5

Spill Procedure

* Neutralize with sodium thiosulfate.
* Prepare 4 plates: with and without DNA, with and
  without antibiotic.
* Use mercury sponges to clean up.
* Label and dispose of waste.

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-5-result.webp)

6

Emergency Protocol

* Skin Contact: Wash for 15 minutes; use safety showers for large areas.
* Inhalation: Move to fresh
* air and seek medical

![](https://static.igem.wiki/teams/5257/project/experiments/mercury-compounds-6-result.webp)

# Encapsulation

### Objective:

To encapsulate recombinant bacteria that detect inorganic and organic mercury in alginate gels to enhance
their functionality and viability.

### Reagents:

* High-density sodium alginate
* Low-density sodium alginate
* CaCl₂

### Materials:

* 250 mL beaker
* 100 mL beaker

### Equipment:

* Autoclave
* Stirrer
* Peristaltic pump

### Personal Protective Equipment:

* Nitrile gloves
* Lab coat
* Balance
* Face mask
* Safety goggles

### Alginate Encapsulation Protocol

* Weigh the high and low-viscosity sodium alginate to prepare a 2% (w/v) concentration in
  demineralized
  water.
* Sterilize the solution at 117°C for 15 minutes.
* Add the necessary amount of alginate to a bacterial culture with an approximate concentration of 10⁹
  CFU/mL to achieve a final concentration of 10% (v/v).
* Discharge the mixture of sodium alginate and bacterial culture through a peristaltic pump with a 0.5
  mm
  diameter tubing at a flow rate of 3 mL/min into a 0.2 M CaCl₂ solution with continuous stirring (100
  rpm).
* Allow the mixture to stand in the 0.2 M CaCl₂ solution for 20 minutes or until firm structures are
  formed.
* Wash the beads and suspend them in saline solution (0.9% w/v) until use.

### References

Hernández-Gallegos, M., Javier Solorza-Feria, M., Velázquez-Martínez, J., Rodríguez-Huezo, M.,
Gutiérrez-López, M. & Hernández-Sánchez, H.( 2023) "Protective Effect of Alginate Microcapsules with
Different Rheological Behavior on Lactiplantibacillus plantarum 299v" Gels 9, no. 9: 682.
<https://doi.org/10.3390/gels9090682>

## Mercury Compounds Handling Protocol

![](https://static.igem.wiki/teams/5257/project/experiments/encapsulation-result.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

![Results](https://static.igem.wiki/teams/5257/project/results/banner-result.webp)

The iGEM Bolivia team proposes a workflow to characterize and validate the organic mercury biosensor

![](https://static.igem.wiki/teams/5257/project/results/1-results.webp)

General scheme of the steps to characterize and validate our biosensor.

## Characterization of each construct

Expected results. - Expected results: Each construct has a
HisTag-tagged protein with a size of 30 kDa, will
be purified with a kit (His-Spin Protein Miniprep, #P2001, zymoresearch) and SDS page electrophoresis will
be performed, bands are expected to be present in the presence of mercury (construct #1 & #2) and IPTG
(construct #3). Only construct #3 should not be induced by mercury because it does not present mercury
sensors, while construct #1 and #2 will exhibit fluorescence in the presence of mercury.

![](https://static.igem.wiki/teams/5257/project/results/2-results.webp)

Expected results of purification and electrophoresis of the proteins of each construct.

## Characterization of each strategy

The fluorescence of each biosensor,
exposed to varying concentrations of organic and inorganic mercury, will
be measured.

#### Strategy #1: Use of NOT logic gate

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat1-1-result.webp)

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat1-2-result.webp)

#### Strategy #2: Cell surface display with mercuric reductase

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat2-1-result.webp)

### Expected response curve

![](https://static.igem.wiki/teams/5257/project/engineering/expected-response-curve-strat2-2-result.webp)

Curves of expected responses for each strategy.

## Characterization of the new composite part of strategy 1

Expected results. - The new composite part BBa\_K5257025 makes use of
the magenta reporter coupled to a NOT logic gate circuit, allowing control of transcription and the presence
of the reporter. Other iGEM teams have already characterized regulation by TetR and TEV, so we set out to
demonstrate that the TEV protease is capable of cutting the magenta reporter and allowing its rapid
degradation.

![](https://static.igem.wiki/teams/5257/project/results/4-results.webp)

New composite part for strategy 1, BBa\_K5257025

Purification and electrophoresis of the reporter will be carried out, it is expected that in the presence of
TEV protease 2 bands will be generated, while in its absence there will be only 1 band. At the same time,
construct #1 will be transformed into an E. coli DH5α with a plasmid expressing the TEV protease, it is
expected that at higher concentrations of TEV there will be a lower fluorescence.

#### Cleavage of mRFPmagenta by TEV protease

![](https://static.igem.wiki/teams/5257/project/results/5-1-results.webp)

#### Controlled degradation of mRFPmagenta by TEV protease

![](https://static.igem.wiki/teams/5257/project/results/5-2-results.webp)

Expected results of the NOT logic gate for magenta reporter

## Characterization of the new composite part of strategy 2

The proposed new composite part (BBa\_K5257023), visualizes MerA and GDH enzymes on the bacterial surface,
therefore we employ the LOT anchor motif which is suitable for large molecular size proteins (Liang et al.
2021), LOT can posttranslationally couple with both enzymes thanks to the Dogtag/DogCatcher system within
minutes (Keeble et al. 2022). To avoid the need to add NADPH cofactors we expect to generate an NADPH
recycling system via GDH (Tian et al. 2022). Therefore, we will first measure the enzymatic activity of MerA
and GDH, then evaluate the ability to recycle NADPH.

![](https://static.igem.wiki/teams/5257/project/results/6-results.webp)

Theoretical basis of the new composite part for strategy 2

Expected results. - The enzymatic activity of GDH is expected to cause
an increase in absorbance, while MerA
will consume NADPH, resulting in a decrease in absorbance. If the two bacterial strains are grown in the
correct proportions, no significant change in absorbance should occur, indicating a balance between NADPH
production and consumption.

#### Enzymatic activity of GDH-expressing E. coli

![Enzymatic activity of GDH-expressing E. coli](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-1-result.webp)

#### Enzymatic activity of MerA-expressing E. coli

![Enzymatic activity of MerA-expressing E. coli](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-2-result.webp)

#### Combination of GDH-expressing and MerA-expressing E. coli

![Combination of GDH-expressing and                     MerA-expressing E. coli](https://static.igem.wiki/teams/5257/project/engineering/enzymatic-activity-mera-expressing-3-result.webp)

Expected results. - E. coli cells expressing the new composite part,
when cultured with glucose, should demonstrate the maximum rate of inorganic mercury reduction. Similarly,
the presence of NADPH alone should yield comparable results. In the absence of glucose or NADPH, no
reduction of inorganic mercury is expected, as MerA will degrade intracellularly.

NADPH recycling using surface displayed GDH and MerA on E. coli

![NADPH recycling using surface displayed GDH and MerA on E. coli](https://static.igem.wiki/teams/5257/project/engineering/nadph-recycling-using-surface-result-2.webp)

Expected results of NADPH recycling of the new composite part of strategy 2

## Biosensor validation

Traditionally MerA and MerB enzymes were used as a bioremediation system, our strategy 2 proposes to use the
MerA enzyme on the surface to volatilize Hg2+ and reduce its bioavailability, favoring only organic mercury
to generate a measurable fluorescent response. In contrast, strategy 1 mutes the signal emitted by organic
mercury when inorganic mercury is present. Therefore, we will subject each biosensor to different similar
heavy metals such as cadmium or lead to see their selectivity to organic mercury, we do not expect
fluorescence against other metals, because merR from Tn501 and the m4-1 mutant exhibit high selectivity
(Wang et al. 2020).

## Validate with environmental samples

We will validate our biosensors by determining the amount of organic mercury in real samples with
concentrations already known by atomic absorption spectroscopy (AAS), we expect to obtain a high correlation
between both methods. We expect a high sensitivity of both strategies (ppb), because organic mercury is
more liposoluble than inorganic mercury.

#### Cleavage of mRFPmagenta by TEV protease

![](https://static.igem.wiki/teams/5257/project/results/9-1-results.webp)

#### Controlled degradation of mRFPmagenta by TEV protease

![](https://static.igem.wiki/teams/5257/project/results/9-2-results.webp)

Expected correlation between both strategies of our biosensor with atomic absorption spectroscopy (AAS).

## On-Hardware Validation

We propose to deploy our biosensors in a portable, cheap and easy to use platform. To keep the bacteria
alive
and active we will use lyophilization or alginate encapsulation (see [lab-experiments](https://2024.igem.wiki/bolivia/experiments) ), our biosensor will be
inside autoclavable glass tubes with a low volume requirement (1-2ml) of sample.

While incubation is required, the mChartreuse reporter is fast maturing and will provide results in less
time than the mRFPmagenta reporter (t= 18 to 24h, Bao et al. 2020 & Fraikin et al. 2024). Therefore, we
expect that strategy 2 of our biosensor will give results in less time than strategy 1, thus reducing the
requirements before measuring fluorescence in the hardware.

A calibration curve will be performed with known mercury concentrations before determining the concentration
of mercury present in the samples. The excitation wavelength of the mChartreuse is λex=487nm and the
emission wavelength is λem=510nm, while mRFPmagenta has λex=588nm and λem=616nm. After data collection, the
bacteria will be inactivated by taking them to the autoclave and will be followed as presented in “Waste and
Disinfection Protocol for the Mercury Biosensor Device” ([Safety](https://2024.igem.wiki/bolivia/safety) ).

![](https://static.igem.wiki/teams/5257/project/results/10-results.webp)

Sequence of steps to validate our biosensor in hardware.

# References

* Bao, L., Menon, P. N. K., Liljeruhm, J., & Forster, A. C. (2020). Overcoming chromoprotein
  limitations by engineering a red fluorescent protein. Analytical biochemistry, 611, 113936.
  <https://doi.org/10.1016/j.ab.2020.113936>

* Fraikin, N., Couturier, A., & Lesterlin, C. (2024). A palette of bright and photostable monomeric
  fluorescent proteins for bacterial time-lapse imaging. bioRxiv, 2024-03.
  <https://doi.org/10.1101/2024.03.28.587235>

* Gallus, S., Peschke, T., Paulsen, M., Burgahn, T., Niemeyer, C. M., & Rabe, K. S. (2020). Surface
  Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction. ChemBioChem, 21(15), 2126-2131.
  <https://doi.org/10.1002/cbic.202000102>

* Keeble, A. H., Yadav, V. K., Ferla, M. P., Bauer, C. C., Chuntharpursat-Bon, E., Huang, J., &
  Howarth, M. (2022). DogCatcher allows loop-friendly protein-protein ligation. Cell chemical biology,
  29(2), 339-350.
  <https://doi.org/10.1016/j.chembiol.2021.07.005>

* Liang, B., Liu, Y., Zhao, Y., Xia, T., Chen, R., & Yang, J. (2021). Development of bacterial
  biosensor for sensitive and selective detection of acetaldehyde. Biosensors and Bioelectronics, 193,
  113566.
  <https://doi.org/10.1016/j.bios.2021.113566>

* Tian, H., Furtmann, C., Lenz, F., Srinivasamurthy, V., Bornscheuer, U. T., & Jose, J. (2022). Enzyme
  cascade converting cyclohexanol into ε‐caprolactone coupled with NADPH recycling using surface
  displayed
  alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli. Microbial Biotechnology, 15(8),
  2235-2249.
  <https://doi.org/10.1111/1751-7915.14062>

* Wang, D., Zheng, Y., Wei, L., Wei, N., Fan, X., Huang, S., & Xiao, Q. (2020). A signal-amplified
  whole-cell biosensor for sensitive detection of Hg2+ based on Hg2+-enhanced reporter module. Journal
  of Environmental Sciences, 96, 93-98.
  <https://doi.org/10.1016/j.jes.2020.03.020>

{% endblock %}