iGEM 2023 — Team hongkong-jss

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{% 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 %}
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# Notebook

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# **Engineering cycles** ![](https://static.igem.wiki/teams/4813/wiki/engineering-cartoon.jpg)

During the experimental phase of our project, we applied the principles of the engineering cycle to structure
our project flow, experimental design, and problem-solving approach. This systematic framework greatly aided
us in tackling challenges encountered during the cloning process. In the following section, we will describe
how we effectively implemented our project by incorporating the key components of engineering design
thinking.

---

**## 1. Design**

**### Inspiration of the project**
![](https://static.igem.wiki/teams/4813/wiki/engineering-success/randomphoto.png)

In recent local news, there has been a growing concern about indoor air quality (IAQ) and the need to study
the health effects of indoor air pollution. The Hong Kong Environmental Protection Department has identified
various significant air pollutants present in homes, with formaldehyde being one of the most prevalent.
Research studies have revealed that formaldehyde can originate from multiple sources, including the
combustion of fuels in gas stoves, building and furnishing materials, renovation activities, new wooden
furniture, and even the food we consume. This highlights the importance of investigating the impact of
formaldehyde on indoor air quality and its potential health implications.

After consulting with our science teachers, we have gained valuable insights and developed a preliminary idea
and direction for designing our project. While we are incorporating synthetic biology concepts into our
work, it is crucial to consider that our project's target audience is the general public. Therefore, our
main focus should be on creating products that are user-friendly, accessible, and do not require any
specialized equipment. We aim to develop solutions that can be easily understood and embraced by the public,
emphasizing simplicity and practicality in our design approach. By considering the needs and preferences of
the general population, we can ensure that our project resonates with a wider audience and has the potential
for real-world impact.

Consequently, our objective is to develop a device that offers an accessible and user-friendly solution for
individuals to monitor formaldehyde levels in their homes. Our aim is to create a device that enables users
to easily assess the presence of formaldehyde by simply looking at the device.

**### The Designs of constructs**
**#### Choice of formaldehyde sensing promoters/parts**

To explore the potential of formaldehyde-inducible promoters and reporters for creating a household
formaldehyde sensing device, we turned to the iGEM Parts Registry. We decided to focus on two
well-documented parts: the *pFrmR* promoter ([BBa\_K2728001](http://parts.igem.org/Part%3ABBa_K2728001)) (Fig. 1) designed by iGEM team
BGIC-Global in 2018, and the *HxlR* operon ([BBa\_K1334002](http://parts.igem.org/Part%3ABBa_K1334002)) designed by iGEM team WHU-China in
2014.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f1.png)

Our objective was to compare these two parts' effectiveness and determine which would better serve our
purpose. By examining their performance and characteristics, we aimed to identify the most suitable part for
our household formaldehyde sensing device.

#### The designs of *pFrmR* constructs

The graphic illustration below shows the designs of *pFrmR* constructs that we planned to use for formaldehyde
sensing.

![](https://static.igem.wiki/teams/4813/wiki/newresult20231012.png)

**#### The designs of *HxlR* constructs**

The other design is based on a HxlR protein which is a formaldehyde-responsive transcription factor from
Bacillus subtilis located upstream of the *hxlAB* operon (Fig 4). In the presence of formaldehyde, wild-type
HxlR protein binds to two specific DNA binding sites (BRH1&2) and activates the expression of the downstream
*AB* operon in Bacillus subtilis (Zhu et al., 2021 & Yurimoto et al., 2005)

![](https://static.igem.wiki/teams/4813/wiki/engineering-success/enginering3.png)

When formaldehyde is present, *HxlR* is triggered to bind to BRH1&2, activating the promoter of our *dTomato*
reporter gene. This subsequently promotes the transcription and expression of our reporter gene in the host
*E. coli*, allowing us to detect the presence of formaldehyde simply by observing if there is a red-colour
change in our engineered *E. coli* cells.

**#### K13A mutation in *HxlR***

According to the research by Zhu (2021), when the 13th amino acid in *HxlR* protein, lysine, was replaced by
alanine, the mutated K13A version of *HxlR* showed a more optimal conformation for DNA binding activity and a
higher transcription activation than the wild-type *HxlR* protein.

Therefore, besides testing the wild-type *HxlR*, we also incorporate *HxlR-K13A* into our construct designs to
examine whether *HxlR-K13A* can lead to an enhanced expression of the reporter genes to give *E. coli* cells a
more obvious red-colour change upon formaldehyde detection. If yes, *HxlR-K13A* can be used in our project as
an improved design from the wild-type *HxlR*, by allowing a higher sensitivity in detecting formaldehyde.

**#### To compare the effectiveness of different *HxlR* designs**

Afterwards, we designed four distinct composite parts with the goal of comparing the effectiveness of various
*HxlR* variants. This allowed us to evaluate and determine which variant worked best for our intended purpose.

In our first construct ([BBa\_K4813014](http://parts.igem.org/Part%3ABBa_K4813014)) as shown in Fig.5
, we utilized Part BBa\_K3332042 which was obtained from a previous iGEM team and contains several parts such
as the *HxlR* gene, a b constitutive reverse promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), two *HxlR* binding sites, and the original
*HxlR* activating promoter and RBS sequence for the reporter genes. Notably, the expression of the wild-type
*HxlR* protein occurs in Bacillus subtilis, is driven by a reverse promoter as reported by Yurimoto et al. in
2005.

We made modifications to this construct by introducing our desired reporter gene, the *dTomato* coding
sequence, right after the *HxlR* activating promoter.

![](https://static.igem.wiki/teams/4813/wiki/engineering-success/engineering4.png)

In our second construct (Fig. 6), we incorporated the same components as the first construct, except for one
significant difference. Instead of the wild-type HxlR protein coding sequence, we substituted it with the
K13A mutated sequence. This particular mutation has been reported to enhance the binding efficiency and
activation of the reporter gene promoter, making it more effective.

Similar to the first construct, both of these constructs were designed to be expressed using the widely
utilized plasmid pUC19. This choice was made for convenience and compatibility with standard laboratory
procedures.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/eingeering-fig-6.png)

To explore the potential impact of increased HxlR protein expression on our device's sensitivity to
formaldehyde, we proceeded with the development of our third and fourth *HxlR* constructs.

Our aim was to enhance *HxlR* expression by using the T7 promoter, a very common b promoter known for its
efficient protein expression capabilities. To accommodate this change, we planned to clone our constructs
into the pET28A(+)-FGF2 plasmid.

It is important to note that the wild-type *HxlR* involves backward transcription driven by a reverse promoter
in Bacillus subtilis while there is limited prior research on the feasibility of forward transcription for
*HxlR*. Therefore, we specifically designed constructs 3 (Fig. 7) and 4 (Fig. 8) to investigate whether
forward transcribing *HxlR* could still be successfully expressed while retaining its activity. This was
achieved by incorporating a forward T7 promoter from the pET28A(+)-FGF2 plasmid to drive HxlR protein
expression. Moreover, We chose pET28A(+)-FGF2 plasmids because they have modifications in the T7 promoter
and RBS site. These modifications have been shown to increase protein production yield. (Shilling et al.,
2020)

The figures below illustrate the designs of our third and fourth constructs.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/eingeering-fig-7.png)

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/eingeering-fig-8.png)

**#### Choice of reporter genes**

In order to create a user-friendly device that can be easily used by individuals without specific knowledge
or equipment, we recognized the importance of choosing a reporter that would provide a clear and easily
observable signal. Among the various options available, we decided that a chromoprotein reporter would be
the simplest and most direct choice.

After conducting a literature review, we found that red chromoproteins were particularly vivid and easy to
express. Given our team's prior experience with the red chromoprotein mCherry, we decided to explore the use
of *dTomato* as it was reported to be an even more intense red chromoprotein compared to mCherry. Further
research revealed that *tdTomato* is an enhanced version of *dTomato*, although this claim specifically referred
to its fluorescence properties rather than its chromoprotein color (Shaner, 2004).

To determine whether *tdTomato* also exhibited a more intense color than *dTomato* when expressed in bacterial
colonies, we planned to compare the two red proteins as part of our project's objectives. To facilitate this
comparison, we obtained the native coding sequences of *dTomato* and *tdTomato* from NCBI and codon-optimized
the sequence specifically for *E. coli* expression. We then incorporated these sequences, along with a
commonly used b constitutive promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), into
two constructs. These constructs were ordered as gBlocks from IDT.

Additionally, we also ordered constructs that contained the *pFrmR* promoter and *HxlR* operon driving the
expression of *dTomato* and *tdTomato*, respectively, from IDT. These constructs would enable us to examine the
performance of *dTomato* and *tdTomato* under the control of different promoters.

**#### Choice of biological system**

We decided to use *E. coli* as our main organism for this project because it is easily accessible in our
laboratory and we are familiar with working with it. An additional advantage is that the *pFrmR* promoter,
which we are using, naturally functions in *E. coli* since it was discovered in this bacterium. This
simplifies our experimental setup as we don't need to introduce an additional protein.

Now that we have chosen *E. coli* as our host organism, we can move on to the building phase of our project.

---

**## 2. Build**

**### Cloning of constructs**

We chose to use the HiFi-assembly method from NEB (New England Biolabs) for our cloning. This method allows
us to create the constructs without leaving any scars.
![](https://static.igem.wiki/teams/4813/wiki/individual-photos/engineering-success/engineering-pic-for-2.png)
Thanks to NEB's sponsorship, we received a free HiFi assembly kit, making the process easier for us. We
designed 20-base pair overlapping regions ([BBa\_K4813007](http://parts.igem.org/Part%3ABBa_K4813007), [BBa\_K4813008](http://parts.igem.org/Part%3ABBa_K4813008)) in our constructs, which were
successfully assembled into the EcoRI restriction site of the pUC19 vector. This confirmed the effectiveness
of the HiFi-assembly method for our cloning purposes.

We encountered difficulties in cloning our formaldehyde sensing constructs at first. However, by using
engineering thinking skills, we were able to solve the problem systematically. We identified the issue,
analyzed the process, troubleshooted, made improvements, and validated our modified approach.

**### Cloning of *pFrmR* constructs ([BBa\_K4813002](http://parts.igem.org/Part%3ABBa_K4813002),[BBa\_K4813000](http://parts.igem.org/Part%3ABBa_K4813000)) and reporter gene comparison ([BBa\_K4813005](http://parts.igem.org/Part%3ABBa_K4813002),[BBa\_K4813006](http://parts.igem.org/Part%3ABBa_K4813006)) constructs**
**#### 1st attempt**

This was our first time using the HiFi assembly kit and method, and we faced a few challenges during the
process. Since we didn't have a spectrophotometer to measure vector concentration, we had to estimate it
from gels.

Initially, we believed our cloning attempt was successful because the HiFi assembly positive controls and the
colonies transformed with J23100 constructs ([BBa\_K4813005](http://parts.igem.org/Part%3ABBa_K4813005) and [BBa\_K4813006](http://parts.igem.org/Part%3ABBa_K4813006)) showed red colors (Fig. 9). Encouraged
by this, we proceeded with the double enzyme digestion check of our clones (please refer to the detailed
results and experiment page for more information).

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f9.png)

The gel electrophoresis results revealed two bands, indicating the presence of an insert in the digested
plasmids. Thus, we conducted the first round of functional assay with formaldehyde, but none of the colonies
demonstrated a color change. Further testing and team discussions revealed an important observation: even
the negative control plate (with digested pUC19) showed a few colonies. This suggested that the digestion
may not have been completed properly, and the colonies appearing on the *pFrmR* constructs ([BBa\_K4813002](http://parts.igem.org/Part%3ABBa_K4813002), [BBa\_K4813004](http://parts.igem.org/Part%3ABBa_K4813002)) plates might be false positives.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f10.png)

To address this issue, we decided to repeat the cloning process and adopt a faster and higher-throughput
screening method called colony PCR. For this purpose, we ordered M13 primers from the TechDragon company.
This change in strategy would allow us to more efficiently identify positive clones and expedite our
progress.

Based on our observations from this cloning attempt, we have noticed that *dTomato* exhibits a higher level of
coloration compared to *tdTomato*, (will be further discussed in the test section). Due to the limited time
remaining until the deadline, we have decided to focus solely on cloning constructs using *dTomato*.

This decision was made to optimize our time and resources, allowing us to concentrate our efforts on a single
construct that has demonstrated more desirable coloration properties. By narrowing our focus, we aim to
streamline our work and increase the chances of meeting our project objectives within the given timeframe.

**#### Several other attempts:**

After the initial cloning attempt failed, we suspected that the incorrect vector to insert ratio might have
been the cause. To address this, we adjusted the vector concentration accordingly. Additionally, to reduce
the chances of incomplete digestion and the formation of false-positive colonies, we increased the digestion
time.

Despite these adjustments, when we performed colony PCR on all the resulting colonies, none of them contained
our desired construct inserts. This outcome indicates that there may be other factors contributing to the
cloning failure, requiring further investigation and troubleshooting to pinpoint the root cause and find a
solution.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f11.png)

**#### Final attempt:**

To address the challenges we faced in our previous cloning attempts, we made some important changes. Firstly,
we increased the digestion time of the pUC19 vector to 4 hours to ensure complete digestion. We also
performed gel purification to obtain purified DNA fragments before proceeding with the assembly. These
adjustments improved the quality of our DNA samples by ensuring only digested vectors were extracted and
removing impurities.

Additionally, we increased the molar ratio of insert to vector to around 5:1. This means we had more insert
DNA compared to the vector, which increased the chances of successful cloning.

Thanks to these modifications, we achieved a significant improvement in our cloning success. We obtained a
large number of colonies, indicating a higher chance of successful transformation.

After confirmation by colony PCR, we are excited that we have finally successfully cloned both the *pFrmR*
constructs (BBa\_K4813002) and the *HxlR* constructs ([BBa\_K4813025](http://parts.igem.org/Part%3ABBa_K4813025)) this time. This progress marks a
significant advancement in our research project, bringing us closer to achieving our goals just before the
deadline. It's an exciting moment as we can now move forward to the next phase, which involves conducting
functional assays.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f12.png)

**### Cloning of *HxlR* constructs ([BBa\_K4813014](http://parts.igem.org/Part%3ABBa_K4813014), [BBa\_K4813025](http://parts.igem.org/Part%3ABBa_K4813025), [BBa\_K4813022](http://parts.igem.org/BBa_K4813022) and [BBa\_K4813024](http://parts.igem.org/BBa_K4813024))**

**#### 1st attempt:**

We have tried to clone all of our 4 constructs carrying the *HxlR* gene into plasmids using NEBuilder HiFi DNA
Assembly. We designed 20 base pair 5’ or 3’ overlap sequences in constructs 1 and 2 to insert them into
EcoRI-digested pUC19 plasmids and 20 base pair 5’ or 3’ overlap sequences in constructs 3 and 4 to insert
them into XbaI- and EcoRI-digested pET28A(+)-FGF2 plasmids. After assembling the constructs into
corresponding plasmids, we transformed the recombinant plasmids into host cells, DH5 alpha and BL21 star
*E. coli*. However, there were no colonies grown after overnight incubation.

As in this first attempt, we have digested the plasmids overnight and it was likely that the restriction
enzyme digestion time had been too long degrading our plasmids.

**#### 2nd attempt:**

After the first cloning attempt failed, we repeated the experiment and shortened the restriction enzyme
digestion time to 20 minutes. In the hope of increasing the successful rate, we also used a higher
concentration of pUC19 plasmid this time for cloning. Unfortunately, the pET28a(+)-FGF2 plasmids stock had
been used up, therefore, in our second cloning attempt, we only performed the NEBuilder HiFi DNA Assembly to
insert our constructs 1 and 2 with *HxlR* into EcoRI-digested pUC19 plasmids. The assembled constructs into
pUC19 plasmids were transformed into DH5 alpha *E. coli* cells and spread on LB agar plates with Ampicillin
added. However, there were still no colonies appearing on the plates for all the cells transforming with our
constructs 1 and 2 and even the NEBuilder positive control. The cells were proven viable when they were
grown on normal LB agar plates without antibiotics.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/eingeering-fig-13.png)

We suspected that there might be some problems with the transformation process and the cells could not take
up the DNA. Due to lacking time and running out of chemicals in the high school lab, we at the end chose to
continue with cloning *pFrmR* constructs ([BBa\_K4813002](http://parts.igem.org/BBa_K4813002)) and
the *HxlR* constructs ([BBa\_K4813025](http://parts.igem.org/BBa_K4813025)) which were proved
successful with colony PCR as stated above.

---

**## 3. Test**

**### Comparison of Color Intensity: *dTomato* ([BBa\_K4813005](http://parts.igem.org/BBa_K4813005)) vs. *tdTomato* ([BBa\_K4813006](http://parts.igem.org/BBa_K4813006)) in Colonies**
![](https://static.igem.wiki/teams/4813/wiki/individual-photos/engineering-success/engineering-pic-for-3.png)

In our experiments with the J23100-*dTomato* ([BBa\_K4813005](http://parts.igem.org/BBa_K4813005))
and J23100-*tdTomato* ([BBa\_K4813006](http://parts.igem.org/BBa_K4813006)) constructs, we made an
interesting observation. The colonies formed from these constructs displayed varying intensities of red
color. In Figure 11, it is evident that the plates containing colonies expressing *dTomato* exhibited a
significantly deeper pink to red color compared to the plates with colonies expressing *tdTomato*.

We have formulated a hypothesis to explain the differences we observed in the expression levels and color
intensity of the *dTomato* and *tdTomato* proteins. Our hypothesis suggests that the larger size of *tdTomato*
compared to *dTomato* makes it more challenging for *E. coli* to express it effectively. This difficulty in
expression may hinder the growth of *E. coli* and ultimately result in a lower production of red color
proteins. Supporting this idea, we also noticed that the colonies expressing *tdTomato* appeared smaller in
size compared to those expressing *dTomato* under the same growth time and growth condition.

However, it is important to remember that our hypothesis requires further investigation.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f14.png)

Based on our findings, we have decided to continue testing the formaldehyde sensing constructs using *dTomato*.
This choice is due to our initial goal of creating a device with a signal that can be easily seen by the
naked eye. We observed that *dTomato* displayed a ber and more obvious coloration compared to *tdTomato*, making
it a preferable option for our project.

Moreover, we learned that the fluorescence signal of a protein does not necessarily directly relate to its
color or chromoprotein properties. This realization highlights the complexity of protein behavior and
reminds us to consider various factors when selecting fluorescent or chromoprotein markers for specific
applications.

### Formaldehyde functional assay on *pFrmR*-*dTomato* constructs ([BBa\_K4813002](http://parts.igem.org/BBa_K4813002)) and *HxIRK13A-dTomato* ([BBa\_K4813025](http://parts.igem.org/BBa_K4813025)) constructs

Due to the approaching deadline, we made the decision to focus our testing efforts on the *pFrmR*-*dTomato* and
*HxlR-K13A-dTomato* constructs, leaving out other designs.

During our testing, we encountered a problem known as leaky expression in both the *pFrmR* and *HxlR* constructs.
We noticed that a few colonies on the plate showed a pink color even in the absence of formaldehyde. While
this confirmed the presence of our gene of interest, it raised concerns about potential false positive
results in our formaldehyde testing. To address this issue, we carefully selected a colony with a white
color and performed colony PCR to verify its composition for further analysis.

For our formaldehyde testing, we added formaldehyde to LB broth along with our engineered *E. coli* cells. We
then cultured them for 12 hours in the presence of ampicillin. After the incubation period, we used
centrifugation to collect cell pellets, making it easier to observe any color changes in the cells.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/engineering-f15.png)

The figures above show that our engineered *pFrmR*-*dTomato* *E. coli* cells responded as expected to formaldehyde,
but the cells expressing the *HxIRK13A-dTomato* constructs did not. We need to figure out why this happened by
examining our experiment and considering possible factors that influenced the results.

Now, let's move on to the learning phase of our project. We will discuss and interpret our results to plan
for the next steps.

---

**## 4. Learn**

Through the experimental engineering cycles, we have gained valuable insights. We learned that using an
effective and systematic problem-solving approach helped us successfully clone our constructs and genes.

Moreover, from our experimental results, we discovered that the *pFrmR* promoter effectively detects the
presence of formaldehyde and triggers the production of a chromoprotein. We also observed that colonies
containing the *dTomato* red chromoprotein exhibited a more noticeable color signal when observed without any
equipment, compared to colonies containing *dTomato*.

While these preliminary results confirm the validity of our design concept, we are aware that there is still
room for improvement and further investigation. Additionally, our hardware team has developed a prototype
device to integrate with our engineered biological system. Now, our next step is to combine our system with
the device, which will require further collaboration and integration efforts.

There are several major considerations that we need to address in our project:

* **Investigating the concentration effect:** In addition to detecting the presence of formaldehyde, we
  also aim to determine its concentration. We need to explore the relationship between formaldehyde
  concentration and the intensity of the color signal. This investigation will provide us with valuable
  information on quantifying formaldehyde levels using our system.
* **Sensitivity to low concentrations:** The standard safety limit for formaldehyde exposure to humans
  is 0.75 ppm (24.98 uM), which is much lower than our experimental conditions. We need to assess if our
  device and hardware system can detect such low concentrations. It may require improving the sensitivity
  of our system through synthetic biology approaches to ensure it meets safety standards.
* **Investigating *HxlR*'s ability to sense formaldehyde:** *HxlR* is well-documented for its response to
  formaldehyde. To evaluate its suitability for real-world applications, we need to compare the
  effectiveness of *pFrmR* and *HxlR* in sensing formaldehyde. This investigation will help us determine which
  regulatory element is more suitable for our specific needs.
* **Testing on agar plates:** Besides using broth, we also plan to test the growth of our engineered
  cells on agar plates. Solid media offer durability and ease of monitoring, which are advantageous for
  practical applications. By evaluating the performance of our system on agar plates, we can assess its
  viability in different conditions.
* **Ensuring safety measures:** Safety is a crucial aspect of our project. We need to consider
  implementing safety measures such as incorporating a kill switch or containment mechanisms when working
  with our engineered organisms. Additionally, exploring the use of a cell-free expression system can
  reduce concerns regarding the safety of genetically modified organisms (GMOs).
* **Integration into hardware and real-world situations:** We recognize the need to investigate how our
  biological system can be effectively integrated into the hardware for detecting airborne formaldehyde.
  While our current experiments involve using formaldehyde solutions, our ultimate goal is to detect
  formaldehyde in the air, which reflects real-world scenarios and applications.

Therefore, we're beginning a new round of the engineering cycle to tackle these important points. By
combining our biological system with the device, we want to make our product better in how it works, where
it can be used, and how safe it is. It's crucial for us to investigate everything thoroughly to make sure
our system meets the standards and rules for real-life use.

---

**### References**

1. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., & Tsien, R. Y. (2004).
   Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red
   fluorescent protein. Nature Biotechnology, 22(12), 1567–1572. <https://doi.org/10.1038/nbt1037>
2. Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved
   designs for pet expression plasmids increase protein production yield in escherichia coli.
   Communications Biology, 3(1). <https://doi.org/10.1038/s42003-020-0939-8>
3. Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N., & Sakai, Y. (2005). HXLR, a member of the
   duf24 protein family, is a DNA-binding protein that acts as a positive regulator of the
   formaldehyde-induciblehxlaboperon inbacillus subtilis. Molecular Microbiology, 57(2), 511–519. <https://doi.org/10.1111/j.1365-2958.2005.04702.x>
4. R., Zhang, G., Jing, M., Han, Y., Li, J., Zhao, J., Li, Y., & Chen, P. R. (2021a). Genetically encoded
   formaldehyde sensors inspired by a protein intra-helical crosslinking reaction. Nature Communications,
   12(1). <https://doi.org/10.1038/s41467-020-20754-4>

[Design](#title1)
[Build](#title2)
[Test](#title3)
[Learn](#title4)

{% extends "layout.html" %}
{% block title %}Experiment Protocols{% endblock %}
{% block page\_content %}
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# **Experiment protocols**

---

## Preparation of pUC19 vector for Hifi Assembly of gBlock fragments

### Transformation of pUC19

Materials:

pUC19 positive control DNA (Invitrogen), One Shot™ TOP10 Chemically Competent *E. coli* (ThermoFisher, Cat# C404003), S.O.C Medium (ThermoFisher, Cat# 15544-034), LB agar (ThermoFisher, Cat# 22700-025) plates with ampicillin added

Procedures:

1. Mix 5 μL of DNA with 1 mL of competent cells in a polypropylene tube stored at -80°C.
2. Incubate the tube on ice for 5 minutes.
3. Heat shock the tube by exposing it to 42°C for 45 seconds.
4. Return on ice for 2 minutes.
5. Add 1 mL of pre-warmed SOC medium to the tube.
6. Incubate the tube at 37°C for 30 minutes for recovery.
7. Take 100 μL of the solution and spread it evenly on an ampicillin LB plate.
8. Incubate the plate at 37°C overnight.

### Preparation of pUC19 plasmids

Materials:

PureLink™ Quick Plasmid Miniprep Kit (ThermoFisher, Cat# K210010)

Procedures:

Refer to manufacturer’s manual.

### Restriction digestion of pUC19

Materials:

EcoRI-HF (NEB, Cat# R3101S), CutSmart Buffer (NEB, Cat# B6004S)

Procedures:

Components

1X (µl)

EcoRI-HF

2

DNA (~0.5 ug/µl)

2

10X CutSmart Buffer

5

dH2O

41

Total Volume

50

### Gel purification of digested pUC19

Materials:

PureLink™ Quick Gel Extraction Kit (ThermoFisher, Cat# K210012)

Procedures:

Refer to the manufacturer’s manual.

---

# Cloning recombinant plasmids and introduce into host cells.

### Hifi assembly

Materials:

NEBuilder® HiFi DNA Assembly Master Mix (NEB, Cat# E2621S)

Procedures:

Refer to manufacturer’s manual.

### Heat shock transformation of recombinant plasmid

Materials

One Shot™ TOP10 Chemically Competent *E. coli* (ThermoFisher, Cat# C404003), S.O.C Medium (ThermoFisher, Cat#15544-034)

Procedures:

1. Combine 5 μl of recombinant plasmid with 1 ml of competent cells in a polypropylene tube stored at -80°C.
2. Place the polypropylene tube on ice and incubate for 5 minutes.
3. Conduct a heat shock by exposing the tube to 42°C for 45 seconds.
4. Add 1 ml of SOC medium to the polypropylene tubes.
5. .Incubate the tubes at 37°C for 30 minutes to facilitate recovery.
6. Add 100 μl of the solution to an ampicillin plate.
7. Spread the solution evenly across the plate and incubate at 37°C to promote bacterial growth.

## Verification of the assembly

### Double enzyme digestion

Materials:

EcoR1-HF enzyme(NEB, #R3101S) , Xbal enzyme(NEB, #R0145S), CutSmart Buffer (NEB, Cat# B6004S)

Procedures:

1. Prepare reaction mixture according to the table below:

   Components

   1X (µl)

   EcoRI-HF

   0.5

   XbaI

   0.5

   DNA (~0.5 ug/µl)

   2

   10X CutSmart Buffer

   2.5

   dH2O

   19.5

   Total Volume

   25
2. Incubate at 37oC for at least 1 hour.

### Colony polymerase chain reaction (PCR)

Materials:

M13 forward and reverse primer (100uM, Tech Dragon),
OneTaq® 2X Master Mix with Standard Buffer (NEB, Cat# M0482S)

Procedures:

1. Prepare mastermix of PCR reaction according to the table below:

   Components

   1X (µl)

   Forward Primer (10 µM)

   0.5

   Reverse Primer (10 µM)

   0.5

   Template DNA

   5

   OneTaq 2X MasterMix

   12.5

   dH2O

   6.5

   Total Volume

   25
2. Run the PCR with the condition below for 25-30 cycles:

   Stage

   Time (s)

   Initial Denaturation (94°C)

   300

   Denaturation (94°C)

   25

   Annealing (55°C)

   55

   Extension (68°C)

   90

   Final extension (68°C)

   300

### Gel electrophoresis

Materials:

Gel Loading Dye Purple (6X) (NEB, #B7024S),
Agarose (invitrogen ,#LOT0000695970) ,
50 X TAE buffer (ThermoFisher, #LOT 00671729),
Quick-Load® Purple 1kb Plus DNA ladder (NEB,#LOT10034620)

Procedures:

1. Add 5μl of loading dye (LD) into each tube of DNA
2. Add 25μl of distilled water into a new tube and 5μl of LD into the tube (negative control)
3. Set up the gel tank:
4. Prepare 1X TAE buffer for gel forming and gel tank buffer
5. Prepare 1% agarose gel with 1X TAE / TBE buffer in a conical flask
6. Microwave the gel for 1 min to make the agarose dissolve completely
7. Cool down the conical flask with running water
8. Add Gel Stain into agarose gel for DNA visualization (i.e.2 uL in 20mL of gel)
9. After the gel formed, transfer the gel into the gel tank and add buffer to just cover the whole gel
10. Add the DNA and ladder into the wells.

---

## Functional assay of formaldehyde

Materials:

40% Formaldehyde solution (methanal)
LB broth (ThermoFisher)

Procedures:

1. Dilute 40% formaldehyde to desired concentrations with LB broth solution
2. Inoculate 10 mL of LB/Ampicillin broth with *E. coli* with formaldehyde solution to concentrations specified (500 uM and 1000 uM)
3. Incubate for 12 hours
4. Centrifuge the culture for 3 minutes at 6,000 g
5. Observe any color change in the pellet

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

[pUC19 preparation](#title1)
[Cloning](#title2)
[Failed attempts](#title3)
[Successful attempts](#title4)
[Comparison of RFP](#title5)
[Functional assay](#title6)

↑

# **Experimental Results**

---

### **Preparation of pUC19 vectors for *pFrmR* constructs**

**Amplification of pUC19 vectors**
![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/studnet-1-1.png)

We transformed pUC19 positive controls into TOP10 competent cells using the heat shock method, following the given protocols at the protocol page.
Unfortunately, without a spectrophotometer, we couldn't determine the precise yield of the pUC19 vectors obtained. As a solution,
we conducted two sets of restriction digestions using the EcoRI enzyme to convert varying concentrations of the pUC19 vectors into a linearized form.

**Linearization of pUC19 by EcoRI restriction digestion**

Digestion is performed to cut the vector by EcoRI. As we had unknown concentration of pUC19,
we performed two sets of digestion which assumed there were 5 µg and 10 µg of pUC19 respectively.

|  | 5 µg pUC19 (µl) | 10 µg pUC19 (µl) | No enzyme control (µl) |
| --- | --- | --- | --- |
| pUC19 DNA | 15 (1µg) | 7.5 (1µg) | 15 |
| CutSmart Buffer | 5 | 5 | 5 |
| EcoRI-HF | 2 | 2 | 0 |
| dH2O | 28 | 35.5 | 30 |

Then we check the linearization by gel.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/result-1.png)

The digestion process appeared to be successful since the digested samples look different compared to the undigested control,
so we moved forward with the assembly of the pUC19 vector using our gBlock fragments.

The table below shows the constructs we have in the form of gBlock fragments. One challenge we encountered was related to the *pFrmR-tdTomato* construct ([(BBa\_K4813004)](http://parts.igem.org/Part%3ABBa_K4813004)),
which contains regions with a high GC content that proved difficult to synthesize as a single fragment. As a result,
we had to redesign the construct and divide it into three separate fragments that would be ordered from IDT gBlock.
This approach was necessary to overcome the synthesis difficulties associated with the high GC content regions.

| Fragment name | Description | Part registry entry |
| --- | --- | --- |
| J23100-dTomato | RFP dTomato expression driven by a strong constitutive promoter | ([(BBa\_K4813005)](http://parts.igem.org/Part%3ABBa_K4813005)) |
| J23100-tdTomato-1 | RFP tdTomato expression driven by a strong constitutive promoter | ([(BBa\_K4813006)](http://parts.igem.org/Part%3ABBa_K4813006)) |
| J23100-tdTomato-2 |
| J23100-tdTomato-3 |
| *pFrmR-dTomato* | RFP dTomato expression driven by a formaldehyde inducible promoter | ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) |
| *pFrmR-tdTomato* | RFP tdTomato expression driven by a formaldehyde inducible promoter | ([(BBa\_K4813004)](http://parts.igem.org/Part%3ABBa_K4813004)) |

Next, we followed the manufacturer's instructions to dilute these fragments to a concentration of 100 ng/µl.
Subsequently, we performed HiFi assembly using the linearized pUC19 vectors.

---

**Preparation of pUC19 and pET28a(+)-FGF2 vectors for *HxlR* constructs**

For our other 4 constructs containing HxlR protein to activate expression of our reporter gene in *E. coli* cells,
constructs 1 and 2 will be inserted into pUC19 plasmids and constructs 3 and 4 will be inserted into pET28a(+)-FGF2 plasmids.
Unfortunately, the precise yield of the pUC19 vectors obtained from miniprep could not be obtained.
The concentration of DNA obtained from miniprep is around 1-4 μg DNA, so we assumed the concentration of our pUC19 vectors to be 1μg DNA in 75μl volume.

**Linearization of pUC19 by EcoRI restriction digestion**

|  | pUC19 (µl) |
| --- | --- |
| pUC19 DNA | 16.5 (220 ng) |
| CutSmart Buffer | 2 |
| EcoRI-HF | 1 |
| dH2O | 0.5 |

**Linearization of pET28a(+)-FGF2 by Xbai and EcoRI restriction digestion**

|  | pET28a(+)-FGF2 (µl) |
| --- | --- |
| pET28a DNA | 1.1 µl (500ng) |
| CutSmart Buffer | 2 |
| EcoRI-HF | 0.5 |
| XbaI | 0.5 |
| dH2O | 15.9 |

| Fragment name | Description | Part registry entry |
| --- | --- | --- |
| Reverse *HxlR-dTomato* | Reverse *HxlR-dTomato formaldehyde* sensing chromoprotein reporter | ([(BBa\_K4813014)](http://parts.igem.org/Part%3ABBa_K4813014)) |
| Reverse *HxlR-K13A-dTomato* | Reverse *HxlR-K13A-dTomato* formaldehyde sensing chromoprotein reporter | ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) |
| Forward *HxlR-dTomato* | Forward *HxlR-dTomato* formaldehyde sensing chromoprotein reporter | ([(BBa\_K4813022)](http://parts.igem.org/Part%3ABBa_K4813022)) |
| Forward *HxlR-K13A-dTomato* | Forward *HxlR-K13A-dTomato* formaldehyde sensing chromoprotein reporter | ([(BBa\_K4813024)](http://parts.igem.org/Part%3ABBa_K4813024)) |

Next, we followed the manufacturer's instructions to dilute the above 4 gblock fragments to a stock concentration of 10 ng/µl.
Subsequently, we performed HiFi assembly using the linearized pUC19 and pET28a(+)-FGF2 vectors.

---

### **Cloning of constructs**

**HiFi assembly of *pFrmR* constructs into pUC19**
DNA Hifi assembly was conducted to assemble digested pUC19 and our gene constructs to obtain recombinant plasmid.

We make several setups with controls to ensure the success of our HiFi assembly:

| Assembly Mix | Content |
| --- | --- |
| A | linearized pUC19 with *pFrmR-dTomato* |
| B | linearized pUC19 with J23100-dTomato |
| C | linearized pUC19 with J23100-tdTomato |
| D | linearized pUC19 with *pFrmR-tdTomato* |
| E | linearized pUC19 without assembly mix |

To ensure the success of the HiFi assembly, it is crucial to calculate the molar ratio of the inserts to the vector.
According to the manufacturer's protocol, for assembly involving 2-3 fragments, a recommended molar ratio of 2:1 for inserts to vector should be used.
On the other hand, for assembly involving 4-6 fragments, a 1:1 molar ratio is suggested.

Based on these recommendations, we need to carefully calculate the assembly mix by considering the number of fragments involved and
the concentrations and mole of the fragments. However, it is important to note that since we don't have access to a nanodrop machine,
the concentration of the vector will be estimated rather than precisely measured.
This estimation may introduce some uncertainty in the molar ratio calculations.

The individual mix ratio is shown in the table below:

For assembly mix A, B and C:

|  | A (µl) | B (µl) | C (µl) |
| --- | --- | --- | --- |
| Fragment | 4.2 (0.06pmol) | 3.6 (0.06pmol) | 6.3 (0.06pmol) |
| dH2O | 10.3 | 10.9 | 8.2 |
| pUC19 (diluted to) | 0.03 pmol | 0.03 pmol | 0.03 pmol |
| HiFi Assembly Mix | 15 | 15 | 15 |

**HiFi assembly of *HxlR* constructs into pUC19 and pET28a(+)-FGF2**

DNA HiFi assembly was conducted to assemble digested pUC19 and pET28a(+)-FGF2 vectors and our gene constructs to obtain recombinant plasmid.

We make several setups with control to ensure the success of our HiFi assembly:

| Assembly Mix | Content |
| --- | --- |
| 1 | linearized pUC19 with Reverse *HxlR-dTomato* |
| 2 | linearized pUC19 with Reverse *HxlR-K13A-dTomato* |
| 3 | linearized pET28a(+)-FGF2 with Forward *HxlR-dTomato* |
| 4 | linearized pET28a(+)-FGF2 with Forward *HxlR-K13A-dTomato* |
| 5 | NEBuilder HiFi DNA assembly positive control |

To ensure the success of the HiFi assembly, it is crucial to calculate the molar ratio of the inserts to the vector.
According to the manufacturer's protocol, for assembly involving 2-3 fragments, a recommended molar ratio of 2:1 for inserts to vector should be used.

The individual assembly mix ratio is shown in the table below:

|  | Reverse *HxlR-dTomato* (µl) | Reverse *HxlR-K13A-dTomato* (µl) |
| --- | --- | --- |
| Fragment | 5.5 (0.06pmol) | 5.5 (0.06pmol) |
| dH2O | 0 | 0 |
| pUC19 (diluted to) | 0.03 pmol | 0.03 pmol |
| HiFi Assembly Mix | 10 | 10 |

|  | Forward *HxlR-dTomato* (µl) | Forward *HxlR-K13A-dTomato* (µl) |
| --- | --- | --- |
| Fragment | 2.7 (0.03pmol) | 2.7 (0.03pmol) |
| dH2O | 5.3 | 5.3 |
| pUC19 (diluted to) | 0.016 pmol | 0.016 pmol |
| HiFi Assembly Mix | 10 | 10 |

**Transformation of recombinant plasmid of *pFrmR* constructs**

Subsequently, the assembled products of *pFrmR* constructs (A to E) are transformed into TOP10 competent cells using the heat shock transformation method.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/resultcrop2.png)

Based on the results obtained, we can see that the colonies expressing the J23100-dTomato construct ([(BBa\_K4813005)](http://parts.igem.org/Part%3ABBa_K4813005))
and J23100-tdTomato construct ([(BBa\_K4813006)](http://parts.igem.org/Part%3ABBa_K4813006)) displayed different colors. The colonies expressing dTomato appeared to have a
deeper and sharper red color compared to the colonies expressing tdTomato. This difference led us to decide that, if time allows,
we will focus our further investigate on the dTomato construct. By choosing dTomato, we expect to obtain more noticeable and distinct results for our study.

After conducting the assembly of the *pFrmR* constructs, we observed the appearance of colonies on the plate,
leading us to proceed with the next step for functional assay. Initially, we believed that the assembly process
was successful based on the presence of these colonies. Nevertheless, we were also aware that colonies appeared
on the control plates without the assembly mix. This raised concerns that the colonies observed on the *pFrmR* plates
might be false positives. In order to validate the results and confirm the presence of the desired constructs,
we decided to conduct a double enzyme digestion.

**Double enzyme digestion for assembly verification**

After picking and culturing the colonies, we performed a mini-prep to extract the plasmids. To confirm the successful insertion of the gene of interest,
we carried out a double enzyme digestion using XbaI and EcoRI. This enzymatic process cleaves the recombinant plasmid into two fragments.

To visualize and analyze the results of the digestion, we conducted gel electrophoresis.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/for-result.png)

| Lane | Content |
| --- | --- |
| 1 | NEB 1kbp+ DNA Ladder |
| 2 | No digestion control of colony 1 |
| 3 | Colony 1 |
| 4 | Colony 2 |
| 5 | Colony 3 |
| 6 | Colony 4 |
| 7 | Colony 5 |

**Transformation of recombinant plasmid of *HxlR* constructs**

We generated Mix & Go chemically competent cells from TOP10 and BL21 *E. coli* strains for DNA transformation. Subsequently,
the assembled products of *HxlR* constructs into pUC19 and pET28a(+)-FGF2 vectors are transformed into competent cells using the
Mix & Go. *E. coli* transformation kit and buffer set we bought from ZYMO research.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/image6.png)

Based on the results obtained, no colonies can be grown for our two constructs of *HxlR* (Reverse *HxlR-dTomato* and Reverse *HxlR-K13A-dTomato*)
transformed in both TOP10 and BL21 *E. coli* strains. In the positive controls of TOP10 and BL21 *E. coli* grown on LB agar without antibiotics added,
cells were proved to be viable. This showed that the DNA transformation into TOP10 and BL21 *E. coli* cells had not been successful.

---

### Unsuccessful functional assay

**First attempt of formaldehyde functional assay**

We prepared desired concentrations of formaldehyde solution by diluting the 40% formaldehyde solution (methanal) . Following the protocol provided in the protocol page,
we incubated our engineered cells with the formaldehyde solution. We selected the range of concentrations based on previous reports from other iGEM teams,
where a concentration of 100 μM of formaldehyde was reported to have a stimulation effect on *pFrmR*.

The specific concentrations used are outlined in the table below:

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/tableforresult20231012.png)

We performed the same procedures and experiments for both the *pFrmR-dTomato* ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) and *pFrmR-tdTomato* constructs ([(BBa\_K4813004)](http://parts.igem.org/Part%3ABBa_K4813004)).

An example of our setups is shown in the photo below:

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/result-tubes.png)

After the incubation, we harvest the cells and centrifuge them to form pellets and examine if any red colour is being produced.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/for-result-as-well.png)

However, in all the setups, there is no colour change observed when compared with the control setups.

We tried conducting the experiment several times using different concentrations of formaldehyde and adjusting the incubation conditions.
However, even after these changes, we still didn't see any noticeable differences in the results. It seems that the formaldehyde treatment
didn't have the expected effects on our constructs, despite our efforts to modify the experimental setup.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/for-result-also.png)

In our experiments, we made an interesting discovery regarding high formaldehyde concentrations (> 1000 µM) and their impact on *E. coli* growth.
We observed that when the formaldehyde concentration exceeded this threshold, there was no growth of the *E. coli* cells.
This indicates that high concentrations of formaldehyde have a noticeable inhibitory effect on *E. coli* growth.

However, it is important to note that formaldehyde solutions are typically stabilized with methanol. Therefore, in order to draw a definitive conclusion,
further confirmation is required to determine whether the observed effect is solely exerted by formaldehyde or if it is influenced by the presence of methanol.

After noticing false-positive results on the plates, it became apparent that the colonies formed might only contain the vector instead of the desired recombinant plasmids.
To address this issue and improve the efficiency of our colony screening, we sought guidance from our teachers and engaged in discussions.

Following these consultations, we have decided to order M13 primers for conducting colony PCR.

**Colony PCR**

Despite multiple attempts to repeat the assembly process on the *pFrmR-dTomato* constructs ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) and utilizing colony PCR for screening,
we encountered difficulties and did not achieve the desired results. The colony PCR results showed that our attempts were unsuccessful
in obtaining the correct clones.
The below gel photos shows one the results obtained from our attempts as example:

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/needed-for-result.png)

| Lane | Content |
| --- | --- |
| 1 | NEB 1kbp+ DNA Ladder |
| 2-9 | PCR products from different colonies |

The small band size seen in the colony PCR results suggests that there is no insert present in the colonies.
Since our inserts are expected to be around 1.1 kbp in size,
we should have seen a band of approximately that size.

---

### Successful cloning

After numerous trials and adjustments to our assembly conditions, including extending the digestion time to 4 hours and performing gel purification
to improve the purity of vectors, we have successfully cloned the *pFrmR-dTomato* constructs ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)). Additionally, using these optimized conditions,
we were also able to clone the *HxlR-K13A-dTomato* construct ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)).

By increasing the digestion time and adjusting the insert to vector molar ratio to approximately 5:1, we improved the efficiency of our cloning process.
These modifications allowed us to obtain the desired constructs successfully.

The gel image presented below shows the colony PCR results showing the expected band size of the colonies containing the successful recombination plasmids.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/resultcrop.png)

As mentioned earlier, the expected band size for the *HxlR-K13A-dTomato* ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) colony PCR is approximately 1.5 kbp,
while for the *pFrmR-dTomato* ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) colony PCR, it is around 1.1 kbp. The gel diagrams provided above confirm that the
observed band sizes align with these expectations.

We were able to successfully proceed with the functional assay using the correct clones just before the deadline.

---

### Colour comparison of dTomato and tdTomato by naked eyes

In our initial cloning attempt, while we were unable to clone the formaldehyde-sensing devices, we successfully cloned the
J23100-dTomato ([(BBa\_K4813005)](http://parts.igem.org/Part%3ABBa_K4813005)) construct and J23100-tdTomato ([(BBa\_K4813006)](http://parts.igem.org/Part%3ABBa_K4813006)). The colonies expressing these constructs were used to
compare their color through direct visual observation.

After 12 hours of incubation, we observed a noticeable variation in color expression between the two constructs. The colonies
expressing dTomato exhibited a significantly stronger red color compared to those expressing tdTomato. This indicates that dTomato
would be more suitable for our intended purpose of creating a sensing device for easy color detection.

![](https://static.igem.wiki/teams/4813/wiki/20231012-uploads/result-new.png)

Additionally, by examining the colonies, we noticed that the average size of the dTomato-expressing colonies was larger than that of
the tdTomato-expressing colonies. This led us to hypothesize that the lighter color in the tdTomato colonies could be attributed to
the larger size of the tdTomato protein. The expression of this protein likely consumes more resources from the bacterial cells,
potentially hindering their growth rate and resulting in lower color expression.

---

### Formaldehyde functional assay

However, during our experiments, we encountered a problem with leaky expression in both the *pFrmR-dTomato* ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002))
and *HxlR-K13A-dTomato* ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) constructs.
Some colonies on the plates exhibited a pink color even in the absence of formaldehyde. To address this issue and avoid potential false-positive results,
we carefully selected only the white colonies for further analysis in the functional assay with formaldehyde.

In the functional assay, we incubated the engineered *E. coli* cells (containing the *pFrmR*([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002))and *HxlR* ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) constructs, respectively)
with different concentrations of formaldehyde as indicated in the table below:

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/tableforresult12.png)

After harvesting and centrifuging the cells into pellets, we made exciting observations. In the 500 µM formaldehyde concentration setups,
the *E. coli* expressing the *pFrmR-dTomato* constructs ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) displayed an observable color change in the pellet compared to the control
group without formaldehyde treatment. However, all other setups remained white, indicating no significant response.

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/also-needed-for-result.png)

In the 1000 µM formaldehyde concentration setup, no turbidity was observed in the *pFrmR* ([(BBa\_K4813002)](http://parts.igem.org/Part%3ABBa_K4813002)) setups, and no pellet formed after centrifugation,
suggesting that the growth of *E. coli* was inhibited. On the other hand, in the *HxlR* ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) setups, the 1000 µM formaldehyde concentration setup still
exhibited pellet formation and growth. This leads us to hypothesize that the *HxlR* operon may enhance the resistance of *E. coli* cells to formaldehyde.
However, further investigation is needed to confirm this hypothesis.

In summary, our investigation revealed the following key findings:

1. The *pFrmR* promoter is capable of detecting the presence of formaldehyde and activating the production of a chromoprotein.
2. Colonies containing the dTomato red chromoprotein ([(BBa\_K4813005)](http://parts.igem.org/Part%3ABBa_K4813005)) exhibit a more obvious color signal visible to the naked eye compared to colonies \ containing tdTomato ([(BBa\_K4813006)](http://parts.igem.org/Part%3ABBa_K4813006)).
3. The *HxlR* operon shows potential for enhancing *E. coli*'s resistance to formaldehyde solution.
   Within the tested conditions, the *HxlR* device ([(BBa\_K4813025)](http://parts.igem.org/Part%3ABBa_K4813025)) was unable to detect the presence of formaldehyde.
4. Both the *pFrmR* and *HxlR* devices displayed leaky expressions.

Based on these findings, we conducted discussions and developed future plans for the next steps of our project.

For more details, please refer to the [engineering page](https://2023.igem.wiki/hongkong-jss/engineering)

![](https://static.igem.wiki/teams/4813/wiki/resultsupdated/for-result-and-is-needed.png)

{% endblock %}