iGEM 2021 — Team Bolivia

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Bolivia/Notebook

Bolivia

* HOME(current)
* PROJECT

  Description
  Engineering
  Model
* PARTS

  Parts
  Contributions
  Improvements
* HUMAN PRACTICES

  [Human practices](https://2021.igem.org/Team%3ABolivia/Human_Practices)
  Education
  Inclusion

# Bolivia team

![arsenito](https://static.igem.org/mediawiki/2021/a/a4/T--Bolivia--arsenito1.png)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Bolivia/Engineering

Engineering

* [HOME](https://2021.igem.org/Team%3ABolivia)
* AWARDS
  + [INCLUSIVITY](https://2021.igem.org/Team%3ABolivia/Inclusivity)

- [EXCELLENCE](https://2021.igem.org/Team%3ABolivia/Excellence)
- [EDUCATION](https://2021.igem.org/Team%3ABolivia/Education)
- TEAM
  * [COLLABORATIONS](https://2021.igem.org/Team%3ABolivia/Collaborations)
  * [TEAM](https://2021.igem.org/Team%3ABolivia/Team)
  * [ATTRIBUTIONS](https://2021.igem.org/Team%3ABolivia/Attributions)
- PROJECT
  * [DESCRIPTION](https://2021.igem.org/Team%3ABolivia/Description)
  * [DESIGN](https://2021.igem.org/Team%3ABolivia/Design)
  * [ENGINEERNING](https://2021.igem.org/Team%3ABolivia/Engineering)
  * [CONTRIBUTION](https://2021.igem.org/Team%3ABolivia/Contribution)
  * [LAB PROTOCOLS](https://2021.igem.org/Team%3ABolivia/LaboratoryProtocols)
  * [HARDWARE](https://2021.igem.org/Team%3ABolivia/Hardware)
- HP
  * [HUMAN PRACTICES](https://2021.igem.org/Team%3ABolivia/Human_Practices)
  * [PROPOSED IMPLEMENTATION](https://2021.igem.org/Team%3ABolivia/Implementation)
  * [COMMUNICATION](https://2021.igem.org/Team%3ABolivia/Communication)

[ ]

#### TEAM BOLIVIA

# ENGINEERING

Tracing
the route to get to work

![](https://static.igem.org/mediawiki/2021/f/fa/T--Bolivia--description_fig1.png)

[![](https://static.igem.org/mediawiki/2021/7/7d/T--Bolivia--engen_icono1.png)

EXPERIMENTAL

DESIGN](#experimentalDesing)
[![](https://static.igem.org/mediawiki/2021/a/a8/T--Bolivia--engen_icono2.png)

EXPECTED
RESULTS](#expectedResult)

## EXPERIMENTAL DESIGN

![](https://static.igem.org/mediawiki/2021/a/a2/T--Bolivia--experimental_Plasmido_morado.png)

The objective of the experiment is to characterize and
validate the semi-quantitative and quantitative
response of our biosensor. The proposed experimental
design consists of 5 phases.

![...](https://static.igem.org/mediawiki/2021/9/96/T--Bolivia--experimentaldesign_fig0a1.png)

![...](https://static.igem.org/mediawiki/2021/9/99/T--Bolivia--experimentaldesign_fig0a2.png)

![...](https://static.igem.org/mediawiki/2021/1/10/T--Bolivia--experimentaldesign_fig0a3.png)

![...](https://static.igem.org/mediawiki/2021/f/fe/T--Bolivia--experimentaldesign_fig0a4.png)

![...](https://static.igem.org/mediawiki/2021/9/95/T--Bolivia--experimentaldesign_fig0a5.png
)

Previous

Next

The quantitative validation of the biosensor will be carried out by comparing
interlaboratory results. [1] The results reported by the biosensor will be
compared to those of a traditional method: "Atomic absorption spectrometry
with hydride generation". [2]  **In other words, the environmental
samples
will be analyzed in parallel.**

## Other details for the sampling process

Thanks to previous studies, carried out by Bolivian scientists in 2014 and
2019, we identified 18 contaminated wells with arsenic in the municipalities of
Cercado and Colcapirhua in the city of Cochabamba, designated as Base
Territorial Organizations (TBOs). In these studies, the arsenic concentrations
(ppb) in each well were determined by atomic absorption. [3][4] **For
our
project, we selected six of the eighteen wells considering the
concentration interval established for the operation of our biosensor (0
- 100 ppb As).**

![](https://static.igem.org/mediawiki/2021/2/2b/T--Bolivia--experimentaldesign_tabla1.png)

**Table 1.**  Arsenic concentrations of the
selected
wells. 2019 updated data.
Provided by the scientist Ph.D Ramiro Escalera.

![...](https://static.igem.org/mediawiki/2021/c/c8/T--Bolivia--experimentaldesign_fig1general.png)

![...](https://static.igem.org/mediawiki/2021/6/6d/T--Bolivia--experimentaldesign_fig1.1.png)

![...](https://static.igem.org/mediawiki/2021/1/11/T--Bolivia--experimentaldesign_fig1.2.png)

![...](https://static.igem.org/mediawiki/2021/c/cf/T--Bolivia--experimentaldesign_fig1.3.png)

Previous

Next

**Figure
1.**  Geographical
location of the selected
wells.

##### Each well sample will be subjected to different determinations:

* **Total
  arsenic
  content :** Universidad Privada Boliviana,
  Cochabamba, Bolivia.
* **Physicochemical parameters :** C.A.S.A facilities.
  Cochabamba, Bolivia.
* **Determination of total arsenic content by
  biosensor:**
  Universidad Franz Tamayo Cochabamba, Bolivia.

**Well water sampling will follow the protocol elaborated by our team.**

## EXPECTED RESULTS

![](https://static.igem.org/mediawiki/2021/f/fc/T--Bolivia--result_plasmido_verde.png)

**COVID-19 presented a challenge to our team by limiting our
access to facilities, reagents, and sampling campaigns. For this reason
we were not able to materialize the biosensor yet.** However, with the help of
the gathered literature we next describe the expected
results for each of the 5 phases of our experimental
design with their corresponding differences with the biosensor designed by Wang et al.
[3]

## I. Arsenic toxicity and fitness cost of genetic constructs over bacterial chassis

**We expect that the toxicity exerted on the biosensor will be
minimal, since the concentrations we plan to use are several orders of magnitude lower
than the reported concentrations as toxic to the chassis.** Nadra et al.
tested a biosensor that uses chromoproteins in concentrations of arsenic as high as 1000 ppb
withoutreporting a significant toxicity [1], the same way, Zhuang et al. reported that the
bacterial chassis started to be affected by toxic effects of arsenic on concentrations above
1874 ppb. [2]

![](https://static.igem.org/mediawiki/2021/e/e4/T--Bolivia--expectedresults_fig1.png)

**Figure 1.** Arsenic toxicity over
bacterial chassis development.

**We expect that in our design the As0 and As2 constructs
will present a higher metabolic load for the bacteria in comparison with As4 and As5**
The metabolic load that genetic constructs will have is closely related to their complexity
and to the length of the amplification cascade. Wang et al. noticed a high metabolic
load when the cascade was composed of three amplifiers. In contrast, by reducing the use of
one amplifier, the construct will represent a lower load. It was also stated that a certain
combination of amplifiers (i.e HrpRS-RinA) will result in lower metabolic effort for the
microorganism. [3]

![](https://static.igem.org/mediawiki/2021/0/09/T--Bolivia--expectedresults_fig2.png)

**Figure 2.** Metabolic load of our
genetic constructs over bacterial
chassis represented as a OD600
decrease.

## II. Characterization of the biosensor's semi-quantitative response

The theoretical limit of detection (LOD) of the constructs are **≥ 0.5 ppb for As0, ≥ 3 ppb for As2, ≥ 10 ppb for As4 and ≥ 50 ppb for As5.**
At this point and without the use of any instrument, the experiments will give us semi-
quantitative results. **we expect that the reported LODs will be
considerably higher than the theoretical ones, and the data collected will be helpful for
later tests.**
We also expect that the background noise levels to be higher than
those reported by Wang et al. since our design does not
contain additional ArsR binding sites (ABS) for mitigation [3].
To inmobilize microorganisms, we will perform the lyophilization of bacteria on paper strips
along protective solutions that will ensure the viability up to 2 months
after processing, **we expect minimal loss of their sentitivity to
arsenic under the appropriate storage conditions.** [4]

![](https://static.igem.org/mediawiki/2021/2/29/T--Bolivia--expectedresults_fig3.png)

**Figure 3.** Paper strip
with the 4 biosensors
lyophilized over the
surface generating
traffic light patterns
after arsenic exposure.

## III. Characterization of the biosensor's quantitative response

At this stage, we will work with the constructs that present an optimal
response at concentrations **≥ 5 - 20 ppb**  of arsenic, being
 **As2 and As4 possible candidates that fulfills this criterion.**
As mentioned before, **we expect that the background noise will be
considerably high in comparison to constructs without amplifiers,
especially those with transcriptional amplifiers** due to the
absence of additional ABS sites [3], **As2 and As4 output is
expected to be linearly correlated to arsenic concentration within the range of 5 - 100
ppb.** We expect the sensitivity at this point will be lower than the one that
traditional methods present, but the repeatability is yet to be confirmed by the
generation of calibration curves.

![](https://static.igem.org/mediawiki/2021/8/8f/T--Bolivia--expectedresults_fig4.png)

**Figure 4.** Expected calibration curve for As2/As4 construct response

## IV. Biosensor quantitative response validation

In the attempt to test the specificityto arsenic of the respons, we
will expose the biosensor to three different metals (Fe, Hg, Mg) looking for
any type of non-specific response.
 **We expect no response above the background noise against these 3
metals. [3]**  The only reported case of non-specific results in this type of biosensor
is caused by antimony that could generate responses even higher than arsenic itself depending
on the design employed. [5]

![](https://static.igem.org/mediawiki/2021/4/44/T--Bolivia--expectedresults_fig5.png)

**Figure 5.** Specific
arsenic response,
reporter induction by
other metals is
negligible.

## V. Biosensor response with real samples

Initially, we expect that the concentrations reported by the biosensor will
be lower than those reported by spectrometric methods because our biosensor points to
bioavailable arsenic in contrast with traditional methods that detect total arsenic.[6]
But bioavailability of arsenic depends on several intrinsic physicochemical factors of the
sample and the culture medium resulting in possible biased results related to the nature of
the matrix. For instance, **we expect the bioavailability to be lower
in samples with high concentration of dissolved organic matter. Similarly, if the
phosphate concentration is high, the uptake of As(V) will also be significantly reduced [7]**, which would result in a considerably lower reported concentration than the
spectrometric methods.

On the other hand, an advantage that our biosensor has is the speciation of arsenic in
drinking water collected from wells.  **This water contains mainly As
(III) because of the reduced contact of the well with oxygen in contrast to surface waters,
whole-cell biosensors are especially sensitive to this species because it
doesn't need an extra reduction step. [7]**

## VI. Tackling the background noise problem:

As mentioned before, one of the principal expected drawbacks is the background
noise (leakage) of our biosensor, especially in constructs with transcriptional amplifiers. We
propose 2 potential solutions:

* **Adding an extra ABS sequence into the inducible promoter pArs:** using specially designed primers in a PCR and considering
  that the ABS sequences are relatively short (24 bp).
* **Coupling with a degradation tag-TEV protease system:** which is a special approach that
  takes the advantage of natural protein degradation systems of the bacterial chassis.
  A short TEV cleavage site and a degradation tag is added at the end of the reporter (mRFPViolet) coding sequence. Additionally, an arsenic responsive construct, responsible for the
  TEV protease expression, is coupled. Without arsenic in the medium the reporter is
  immediately degraded by bacterial proteases because it still has the degradation tag
  lowering the background noise. With arsenic in the medium, the TEV protease is
  expressed and cuts the degradation tag allowing normal expression of the reporter.

## References

##### Experimental Design

[1] B. Magnusson and U. Örnemark (eds.) (2016) Eurachem Guide: The Fitness for Purpose
of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics,
(2nd
ed. 2014). ISBN 978-91-87461-59-0. Available from

<https://www.eurachem.org>

[2] Morand, E.; Giménez, M.; Benitez, M. & Garro, O. (2021). Determinación de arsénico
en
agua por espectrometría de absorción atómica con generación de hidruro (HG-AAS).

<https://www.researchgate.net/publication/228700959_Determinacion_de_arsenico_en_agua_por_espectrometria_de_absorcion_atomica_con_generacion_de_hidruro_HG-AAS>

[3] Escalera, R, & Ormachea, M. (2017). Hidroquímica de la presencia natural de
arsénico
en aguas subterráneas de Cochabamba-Bolivia y evaluación de la viabilidad técnica de
procesos de remoción. Investigación & Desarrollo, 1(17), 27-41. Recuperado en 15 de
septiembre de 2021, de.

<http://www.scielo.org.bo/scielo.php?script=sci_arttext&pid=S2518-44312017000100004&lng=es&tlng=es>

[4] Escalera, R.; Ormachea, M.;, Ormachea, O. & Heredia, M. (2014). Presencia de
arsénico
en aguas de pozos profundos y su remoción usando un prototipo piloto basado en
colectores solares de bajo costo. Investigación & Desarrollo, 2(14), 83-91.
Recuperado
en
15 de septiembre de 2021, de

<http://www.scielo.org.bo/scielo.php?script=sci_arttext&pid=S2518-44312014000200006&lng=es&tlng=es>

##### Expected Results

[1] Barone, F., Dorr, F., Marasco, L. E., Mildiner, S., Patop, I. L., Sosa, S.,
Vattino,
L. G.,
Vignale, F. A., Altszyler Lemcovich, E. J., Basanta, B., Carlotto, N., Gasulla, J.,
Giménez, M., Grande, A. V., Nieto Moreno, N., Bonomi, H. R., & Nadra, A. D. (2017).
Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor
based on standard iGEM parts.

<https://doi.org/10.1093/synbio/ysx006>

[2] Barone, F., Dorr, F., Marasco, L. E., Mildiner, S., Patop, I. L., Sosa, S.,
Vattino,
L. G.,
Vignale, F. A., Altszyler Lemcovich, E. J., Basanta, B., Carlotto, N., Gasulla, J.,
Giménez, M., Grande, A. V., Nieto Moreno, N., Bonomi, H. R., & Nadra, A. D. (2017).
Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor
based on standard iGEM parts.

<https://doi.org/10.1093/synbio/ysx006>

[3] Hu, Q., Li, L., Wang, Y., Zhao, W., Qi, H., & Zhuang, G. (2010). Construction of
WCB-
11: A novel phiYFP arsenic-resistant whole-cell biosensor. Journal of Environmental
Sciences, 22(9), 1469-1474.

[https://doi.org/10.1016/S1001-0742(09)60277-1](https://doi.org/10.1016/S1001-0742%2809%2960277-1)

[4] Wan, X., Volpetti, F., Petrova, E., French, C., Maerkl, S. J., & Wang, B. (2019).
Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic
metals.
Nature Chemical Biology, 15(5), 540-548.

<https://doi.org/10.1038/s41589-019-0244-3>

[5] Stocker, J., Balluch, D., Gsell, M., Harms, H., Feliciano, J., Daunert, S., Malik,
K.
A., &
van der Meer, J. R. (2003). Development of a Set of Simple Bacterial Biosensors for
Quantitative and Rapid Measurements of Arsenite and Arsenate in Potable Water.
Environmental Science & Technology, 37(20), 4743-4750.

<https://doi.org/10.1021/es034258b>

[6] Kaur, H., Kumar, R., Babu, J. N., & Mittal, S. (2015). Advances in arsenic
biosensor
development – A comprehensive review. Biosensors and Bioelectronics, 63, 533-545.

<https://doi.org/10.1016/j.bios.2014.08.003>

[7] Pothier, M. P., Hinz, A. J., & Poulain, A. J. (2018). Insights Into Arsenite and
Arsenate
Uptake Pathways Using a Whole Cell Biosensor. Frontiers in Microbiology, 9.

<https://doi.org/10.3389/fmicb.2018.02310>

![...](https://static.igem.org/mediawiki/2021/0/04/T--Bolivia--experimentaldesign_fig1.JPG)

* [HOME](https://2021.igem.org/Team%3ABolivia)
* AWARDS
  + [INCLUSIVITY](https://2021.igem.org/Team%3ABolivia/Inclusivity)
  + [EXCELLENCE](https://2021.igem.org/Team%3ABolivia/Excellence)
  + [EDUCATION](https://2021.igem.org/Team%3ABolivia/Education)
* TEAM
  + [COLLABORATIONS](https://2021.igem.org/Team%3ABolivia/Collaborations)
  + [TEAM](https://2021.igem.org/Team%3ABolivia/Team)
  + [ATTRIBUTIONS](https://2021.igem.org/Team%3ABolivia/Attributions)

* PROJECT
  + [DESCRIPTION](https://2021.igem.org/Team%3ABolivia/Description)
  + [DESIGN](https://2021.igem.org/Team%3ABolivia/Design)
  + [ENGINEERNING](https://2021.igem.org/Team%3ABolivia/Engineering)
  + [CONTRIBUTION](https://2021.igem.org/Team%3ABolivia/Contribution)
  + [LAB
    PROTOCOLS](https://2021.igem.org/Team%3ABolivia/LaboratoryProtocols)
  + [HARDWARE](https://2021.igem.org/Team%3ABolivia/Hardware)
* HP
  + [HUMAN PRACTICES](https://2021.igem.org/Team%3ABolivia/Human_Practices)
  + [PROPOSED IMPLEMENTATION](https://2021.igem.org/Team%3ABolivia/Implementation)
  + [COMMUNICATION](https://2021.igem.org/Team%3ABolivia/Communication)

![](https://static.igem.org/mediawiki/2021/1/19/T--Bolivia--sponsorAniaki.png)

![](https://static.igem.org/mediawiki/2021/3/38/T--Bolivia--Bago.png)

![](https://static.igem.org/mediawiki/2021/4/46/T--Bolivia--IDT.png)

![](https://static.igem.org/mediawiki/2021/1/15/T--Bolivia--ZymoResearch.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Bolivia/Experiments

Bolivia

* HOME(current)
* PROJECT

  Description
  Engineering
  Model
* PARTS

  Parts
  Contributions
  Improvements
* HUMAN PRACTICES

  [Human practices](https://2021.igem.org/Team%3ABolivia/Human_Practices)
  Education
  Inclusion

# Bolivia team

![arsenito](https://static.igem.org/mediawiki/2021/a/a4/T--Bolivia--arsenito1.png)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Bolivia/Results

Bolivia

* HOME(current)
* PROJECT

  Description
  Engineering
  Model
* PARTS

  Parts
  Contributions
  Improvements
* HUMAN PRACTICES

  [Human practices](https://2021.igem.org/Team%3ABolivia/Human_Practices)
  Education
  Inclusion

# Bolivia team

![arsenito](https://static.igem.org/mediawiki/2021/a/a4/T--Bolivia--arsenito1.png)

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)