iGEM 2023 — Team hkust

# Notebook

Notebook

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* [Overview](#overview)
* [Cycle 0](#cycle0)
* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)
* [References](#references)

## Overview

The creation of a biosensor for detecting cereulide levels in food samples is a big step
forward in food safety and quality control. Cereulide is a poisonous chemical produced
by certain strains of Bacillus cereus bacteria that causes health problems when
consumed. This biosensor is intended to provide a quick and accurate means of detecting
cereulide contamination in food products. To develop this biosensor, we went through
three engineering cycles.

## Engineering cycles

Cycle 0 The design of mechanism

Research

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/research_design.png)

To create a biosensor that could detect cereulide levels in
food samples, we searched for detectable physical changes that
could
be used to trigger our circuit.

It has been discovered that cereulide can act as a membrane
channel-forming potassium ionophore, damaging mitochondria
and
blocking oxidative phosphorylation. With this discovery,
past
researchers have conducted experiments using boar semen to
carry
out a spermatozoon toxicity test[[1]](https://doi.org/10.1128/AEM.64.4.1338-1343.1998).
Unfortunately, this method
is
ethically questionable and not viable commercially.

In light of this, we created a biosensor to detect cereulide
levels in food samples by sensing the potassium ion efflux
that
is caused by cereulide.

##### Potassium-ion-selective Ionophore Characteristics of Cereulide

In nature, cereulide binds and inserts itself into the
inner
mitochondrial membrane, acting as an ionophore to allow
potassium ion diffusion. This disrupts the potassium ion
concentration gradient, resulting in mitochondrial
transmembrane
potential disturbance, which impairs ATP synthesis and
cell
survival.

The ionophore property of cereulide is due to its inner
cyclic
dodecadepsipeptide that contains amino acids and hydroxy
acid
residues, which enhance the complex formation with
potassium
ions, thus allowing the binding with and diffusion of
potassium
ions[[2]](https://doi.org/10.3390/foods12040833).
Our circuit utilizes
this property to detect cereulide.

##### The Endogenous Potassium Sensing Pathway in *B. subtilis*

KinC is a transmembrane sensor histidine kinase. It has
a PAS-PAC domain that is responsible for sensing
potassium
ion leakage from the cell. The potassium ion leakage
activates KinC to phosphorylate Spo0A, a protein
involved in the
regulation of biofilm formation[[3]](https://doi.org/10.1016/S0076-6879%2810%2971013-2).
Our
circuit also utilizes this pathway to sense cereulide.

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Design & Build

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/build.png)

##### Cycle 0 Sensing Module

Our sensing module makes use of the
potassium-ion-selective ionophore property of cereulide
and the endogenous potassium sensing pathway in B.
subtilis to convert the cereulide signal to an
intracellular signal of phosphorylated Spo0A.

At a high cereulide level, cereulide binds to the cell
membrane of *B. subtilis*, resulting in potassium ion
leakage. This, in turn, activates KinC to phosphorylate
Spo0A.

##### Cycle 0 Processing and Output Module

Our processing module utilizes the promoter PsinI, which
is under the control of Spo0A. When Spo0A is
phosphorylated, it will bind to the Spo0A box in PsinI,
resulting in PsinI transcription[[4]](https://doi.org/10.1111/j.1365-2958.2007.06040.x)
and, thus, the
downstream expression of RFP.

At a high cereulide level, our sensing module responds
to the signal, leading to an increase in the
intracellular phosphorylated Spo0A level. This, in turn
activates Psinl and thus the expression of RFP, which
acts as an output signal to inform users that the sample
contains cereulide.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-1.png)

Figure 1
Cycle 0 genetic circuit diagram

Our cycle 0 circuit has a PsinI promoter which is active when
cereulide is present. Downstreams of the PsinI promoter are an
RFP gene which will give our red fluorescence signal when
cereulide is present.

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Test

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/Test.png)

The construct is tested by exposing the transformed *B.
subtilis* to different concentrations of cereulide and
its structurally similar ionophore, valinomycin[[5]](https://pubmed.ncbi.nlm.nih.gov/21357430/).

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle0-test-1.png)

Figure 2
RFP fluorescence on cycle 0 upon exposure to 3-9
μM valinomycin, fold change when compared to no
valinomycin added, em:584 nm ex:607 nm

In our first trial, we tested the circuit with 3 μM, 6 μM, and 9
μM of valinomycin. All three concentrations triggered RFP
production at around 4 hours and showed a 60-fold increase in
RFP fluorescence after 6 hours when compared to no valinomycin
added.

After our first trial, we wanted to find out the lower detection
limit of our circuit, so we tested the circuit with 0.03 μM, 0.3
μM, 3 μM, and 4.5 μM of valinomycin in our second trial.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle0-test-2.png)

Figure 3
RFP fluorescence on cycle 0 upon exposure to
0.03-4.5 μM valinomycin, fold change when compared
to no valinomycin added, em:584 nm ex:607 nm

In our second trial, concentrations of 0.3 μM above triggered
RFP production at around 4 hours and showed a 20-fold increase
in RFP fluorescence after 6 hours when compared to no
valinomycin added. At the same time, we found out that 0.03 μM
of valinomycin failed to trigger the cycle 0 circuit.

Due to a limited stock of cereulide, we previously used
valinomycin to test our cycle 0 circuit and assumed that
valinomycin and cereulide would cause the same effect on our
circuit as they have very similar structures and effects on
gram-positive cell[[5]](https://pubmed.ncbi.nlm.nih.gov/21357430/).
To validate our assumption, in the third
trial, 0.3 μM of cereulide and valinomycin were used,
respectively, to test our circuit. The intervals of sample
collection are also shortened to 30 minutes to get more
comprehensive data.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle0-test-3.png)

Figure 4
RFP fluorescence on cycle 0 upon exposure to 0.3
μM valinomycin and cereulide, fold change when
compared to no valinomycin added, em:584 nm ex:607
nm

The results of the third trial showed that the same
concentration of cereulide and valinomycin cause very similar
effects on our cycle 0 circuit, and therefore our assumption is
validated.

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Learn

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/learn.png)

1. 0.3 uM of cereulide is enough to trigger our circuit and
   cause a significant increase in red fluorescence.
2. Our circuit takes 4-5 hours to trigger in liquid medium.
3. Cereulide and valinomycin can cause the same effect on cycle
   1 and can be used interchangeably.
4. From a user experience standpoint, a “safe signal” (i.e.,
   GFP should be expressed when there is no cereulide) should
   be added to our circuit so users of the test kit can be sure
   that there is no cereulide in the sample and eliminate the
   possibility of cells dying during transit or due to improper
   storage.

Cycle 1

Research & Lessons from Cycle 0

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/research_design.png)

From cycle, 0 we found out that our circuit was able to produce
a red signal when there is 0.3 μM or above valinomycin or
cereulide, however, the emission of a red signal with the
presence of cereulide would not be enough. The absence of RFP
shall not be taken as a safe indicator; the absence of RFP may
be caused by reasons other than the absence of cereulide, like
cells dying during transit or due to improper storage.
Therefore, to make our biosensor more user-friendly, GFP should
be expressed when there is no cereulide as a negative control to
prevent false negative results.

To achieve this purpose, we employ a degradation system that
makes use of inducible protein degradation in *B.
subtilis*. It
works by adding a modified ssrA degradation tag to the terminus
of the protein of interest. SspB, an adaptor protein that
delivers the modified ssrA-tagged protein to protease ClpXP, is
essential for protein degradation. In the absence of SspB, the
tagged protein is not degraded[[6]](https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06467.x).

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Design & Build

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/build.png)

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-5.png)

Figure 5
Cycle 1 genetic circuit diagram

Cycle 1 genetic circuit is composed of a cereulide-activated and
cereulide-repressed component.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-6.png)

Figure 6
Cycle 1 cereulide-activated component
diagram

The cereulide-activated component is our cycle 0 circuit. It has
a PsinI promoter which is active when cereulide is present.
Downstreams of the PsinI promoter are an RFP gene, SspB gene,
and LacI gene. The SspB gene expresses SspB protein which will
degrade ssrA-tagged GFP. The LacI acts as the repressor for the
cereulide-repressed component so that the component is repressed
when there is cereulide, and vice versa.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-7.png)

Figure 7
Cycle 1 cereulide-repressed component
diagram

The cereulide-repressed component has a hyper-spank promoter,
which is repressed by LacI. When there is cereulide, the LacI
expression would result in the repression of this promoter and
thus the downstream gene expression, and vice versa. Downstream
of the hyper-spank promoter is a ssrA-tagged GFP gene. The
ssrA-tagged GFP is expressed when cereulide is absent so that
the user is informed that the food samples do not contain
cereulide. Additionally, a His-Tag is added to the GFP for
validation of our circuit through western blotting.

|  |  |
| --- | --- |
| In the absence of cereulide,   1. PsinI promoter is inactive 2. LacI is not expressed 3. SspB is not expressed 4. RFP is not expressed 5. Hyper-spank promoter is active (not    repressed by LacI) 6. GFP is expressed at high level   Eventually, there will be a high level of GFP. | When cereulide present,   1. PsinI promoter is active 2. LacI is expressed 3. SspB is expressed 4. RFP is expressed 5. Hyper-spank promoter is inactive (repressed    by LacI) 6. GFP is not expressed   Eventually, there will be a high level of RFP. |

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Test

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/Test.png)

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle1-test-1.png)

Figure 8
RFP fluorescence on cycle 1 upon exposure to 0.3-6
μM valinomycin, Fluorescence/OD, em:584 nm ex:607
nm

The rise in RFP after valinomycin is added is not quite
prominent if we look at the RFP fluorescence/OD data. Figure 8
shows that the RFP fluorescence/OD when 0 μM of valinomycin is
added (i.e., basal expression) is at the same level as when
0.3-6 μM of valinomycin is added at 0 hours. Since the basal
expression is relatively high, we will have to address this in
the subsequent engineering cycle.

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle1-test-3.png)

Figure 9
GFP fluorescence on cycle 1 upon exposure to 0.3-6
μM valinomycin, fluorescence/OD , em:504 nm ex:515
nm

For the GFP, it is found that the production of GFP is slower
than anticipated, and the degradation of GFP also takes a
considerable amount of time. Therefore, further tuning of the
circuit is needed.

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Learn

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/learn.png)

1. The basal expression of RFP is rather high. This has to be
   addressed in cycle 2.
2. The degradation of GFP is not satisfactory; therefore, we
   are planning to make a degradation tag library and evaluate
   its effectiveness in cycle 2.
3. Further tuning of our circuit is needed, we will try
   different combinations of RBS to optimize the responsiveness
   of our circuit to different valinomycin concentration(check
   dry lab). We can also try to use a positive feedback loop in
   PsinI for RFP to shorten the time needed for the red
   fluorescence signal to show.

Cycle 2

Research & Background

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/research_design.png)

The results from cycle 1 show that the GFP fails to fluoresce.
We suspect that it may be because we added a degradation tag and
a His tag at both termini, distorting the structure of GFP and
rendering it unable to fluoresce. However, without the
experimental data, we can use the modeling result from the dry
lab ([Check Circuit Modeling
Cycle 1](https://2023.igem.wiki/hkust/circuit_modeling#circuit-modelling)).

In addition, the results from the previous experiments also show
that the PsinI promoter may be leaky. As a result, there may
still be red signals even when the sample does not contain
cereulide. Additionally with the interpretation from dry lab
modeling, the dry lab proposed a possible dual conditional
degradation system for our circuit, which is a degradation
system to regulate the level of RFP as well as that of GFP
([Check Circuit Modeling Cycle
2](https://2023.igem.wiki/hkust/circuit_modeling#circuit-modelling)).

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle2-research-1.png)

Figure 10
Modeling of cycle 2, Cereulide = 0.3uM (
< toxicity threshold), time in hour

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/cycle2-research-2.png)

Figure 11
Modeling of cycle 2, Cereulide = 0.5mM (
> toxicity threshold), time in hour

From the graph above, we can see that it is possible to reduce
the RFP signal from the basal expression and is possible to set
a threshold where the GFP would always be higher than RFP or
vice versa.

Our degradation system makes use of an inducible protein
degradation in *B. subtilis*. It works by adding a modified
ssrA
degradation tag to the terminus of the protein of interest.
SspB, an adaptor protein that delivers the modified ssrA-tagged
protein to the protease ClpXP, is essential for protein
degradation. In the absence of SspB, the tagged protein is not
degraded [[6]](https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06467.x)

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/arrow-down-solid.svg)

Design & Build

![](https://gitlab.igem.org/2023/hkust/-/raw/main/img/Engineering_img/build.png)

For our design, we use dual degradation to make sure the
appearance of GFP and RFP will follow the order under two
cereulide concentrations orderly.

When there is no cereulide, the phosphorylation level of spo0A
is low, resulting in a low activation of the sinI promoter. This
leads to very low expression of SspB (*E. coli*), lacI, and
RFP.
These three proteins are attached with a ssrA (*C.
crescentus*)
tag.

Since lacI serves as a repressor of the hyper-spank promoter,
with a low lacI expression level, the hyper-spank promoter is
not repressed, enabling its activation. Consequently, the
downstream SspB (*C. crescentus*) and GFP will exhibit high
expression levels. The two proteins are tagged by ssrA (*E.
coli*)
tag.

Importantly, degradation occurs only when the ssrA tag and SspB
are of the same bacterial protein type, that is, proteins with
ssrA (*E. coli*) tag will be degraded by SspB (*E.
coli*) and
proteins with ssrA (*C. crescentus*) tag will be degraded
by SspB
(C. crescentus). Therefore, the high level of SspB (*C.
crescentus*) targets only the proteins tagged with ssrA
(*C. crescentus*) and delivers it to ClpXP for degradation
rather than
the GFP tagged with ssrA (*E. coli*) tag.

Eventually, there will be no RFP but a high level of GFP.([Check circuit modeling](https://2023.igem.wiki/hkust/circuit_modeling#circuit-modelling))

1. Very low expression of RFP-(Cc)ssrA, lacI-(Cc)ssrA and
   (Ec)SspB-(Cc)ssrA
2. Promoter hyper-spank is active[[7]](https://pubmed.ncbi.nlm.nih.gov/20863884/)
3. High expression of GFP-(Ec)ssrA and (Cc)SspB-(Ec)ssrA
4. High level of (Cc)SspB targets RFP-(Cc)ssrA, lacI-(Cc)ssrA
   and (Ec)SspB-(Cc)ssrA to degradation
5. No RFP, high level of GFP

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-13.png)

Figure 12
The portion of circuit that will be activated when
there is no cereulide

On the other hand, in the presence of cereulide, the
phosphorylation level of spo0A is elevated, leading to a higher
activation of the sinI promoter. This leads to very high
expression of SspB (*E. coli*), lacI, and RFP. These three
proteins are attached with a ssrA (*C. crescentus*) tag.

As a repressor itself, the high expression of lacI strongly
inhibits the hyper-spank promoter. Consequently, the downstream
SspB (*C. crescentus*) and GFP will express at very low
levels. In
the same manner, the ssrA (*E. coli*) is tagged to GFP.

As a result, the remaining GFP tagged with ssrA (*E. coli*)
is
targeted by the high level of SspB (*E. coli*) and sent to
ClpXP
for degradation. In the end, there will be no GFP but a high
level of RFP.

1. High expression of RFP-(Cc)ssrA, lacI-(Cc)ssrA and
   (Ec)SspB-(Cc)ssrA
2. Promoter hyper-spank is repressed[[7]](https://pubmed.ncbi.nlm.nih.gov/20863884/)
3. Very low expression of GFP-(Ec)ssrA and (Cc)SspB-(Ec)ssrA
4. High level of (Ec)SspB targets GFP-(Ec)ssrA and
   (Cc)SspB-(Ec)ssrA to degradation
5. No GFP, high level of RFP

![](https://static.igem.wiki/teams/4725/wiki/engineering-img/eng-14.png)

Figure 13
The portion of circuit that will be activated when
there is cereulide

We have ordered the gBlocks from IDT and have tried to clone
them into *E. coli*. Unfortunately, due to time
limitations, we
did not have time to remove the His tag through Gibson assembly
and test the circuit in *B. subtillis*. We plan to test the
circuit in the future after we have successfully removed the His
tag from the GFP and cloned the circuit into *B. subtilis*.

Check
Hardware Engineering

## References

---

[1] M. A. Andersson, “A Novel Sensitive Bioassay
for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide
Ionophores,” Applied and environmental microbiology,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106152/ (accessed Oct. 11, 2023).

[2] S. Yang et al., “Cereulide and emetic bacillus
cereus: Characterizations, impacts and public precautions,” MDPI,
https://www.mdpi.com/2304-8158/12/4/833 (accessed Oct. 11, 2023).

[3] D. López, E. A. Gontang, and R. Kolter,
“Potassium Sensing Histidine Kinase in Bacillus subtilis,” Methods in
enzymology, https://doi.org/10.1016/S0076-6879(10)71013-2 (accessed Oct. 11,
2023).

[4] Y. Chai, F. Chu, R. Kolter, and R. Losick,
“Bistability and biofilm formation in bacillus subtilis,” Molecular
microbiology, https://pubmed.ncbi.nlm.nih.gov/18047568/ (accessed Oct. 11,
2023).

[5] M. H. Tempelaars , S. Rodrigues, and T. Abee,
“Comparative analysis of antimicrobial activities of valinomycin and cereulide,
the bacillus cereus emetic toxin,” Applied and environmental microbiology,
https://pubmed.ncbi.nlm.nih.gov/21357430/ (accessed Oct. 11, 2023).

[6] K. L. Griffith and A. D. Grossman, “Inducible
protein degradation in Bacillus subtilis using heterologous peptide tags and
adaptor proteins to target substrates to the protease ClpXP,” Molecular
microbiology,
https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06467.x (accessed
Oct. 11, 2023).

[7] L. Vavrová, K. Muchová, and I. Barák,
“Comparison of different bacillus subtilis expression systems,” Research in
microbiology, https://pubmed.ncbi.nlm.nih.gov/20863884/ (accessed Oct. 11,
2023).

# Team Member

# Team Member

#### Wet Lab Training

# Team Member

#### Wet Lab Training

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## What should this page contain?

---

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).

I describe the experiments my team has done as very detailed and repeatable.

## Inspirations

---

* [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)

{% 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 %}

## 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.

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## Inspirations

---

* [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)

{% endblock %}