iGEM 2022 — Team jlu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}In Notebook, we recorded the details of our experiments as well as the important events of the day in a
chronological order.{% endblock %}
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# Lab Notebooks

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}In the face of negative emotions of sadness and anxiety, our project aims to combine Artificial
Intelligence with synthetic biotechnology to construct an effective emotion recognition and regulation device.{%
endblock %}
{% block page\_left\_content %}

* [Overview](#1)
* [Inspiration](#2)
* [Target 1](#3)
* [Target 2](#4)
* [Target 3](#5)

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{% block page\_content %}

# Overview

In the face of negative emotions of sadness and anxiety, our project aims to combine Artificial Intelligence with
synthetic biotechnology to construct an effective emotion recognition and regulation device.

In the recognition part, we use Convolutional Neural Network for facial emotion
recognition judgment, and combine with
bracelet heart rate
sampling, and finally realize multimode emotion judgment through software. From there, the recognition result is
transformed into a corresponding light source activation signal to activate the engineered bacteria to synthesize and
release the corresponding odor substance.

In the regulation part, we take the emotion of sadness as the recognition subject
and convert it into a blue light
signal through the
recognition judgment module, thus stimulating the conformations change of LOV structural domain of E. coli EL222
photosensitive protein, which initiates the downstream KdcA and Adh1 enzymes to catalyze the synthesis of PPA into
phenylethanol and effectively release it through the designed aromatherapy system to achieve emotional calming.

On the basis of the overall project to achieve the closed loop of emotion regulation, we further explored more light
sources and extended possibilities of corresponding emotion regulation, such as red light system to capture anxiety
and release linalool regulation. In addition, we also extended the design of some other emotion regulation functions,
such as the activation of specific music and lights after the completion of emotion recognition, in order to better
complement the odor regulation function and regulate individual emotions in a comprehensive and multi-sensory manner.

In this regard, in order to better illustrate the iteration and upgrading of the engineering cycle of the project, we
will divide the following into the overall inspiration and three major cycles: 1. how to effectively identify
emotions, 2. how to design a light-activated expression system from emotions to smells, and 3. how to ensure the
biosafety of the product. The three major cycles, with a total of nine minor cycles, illustrate the gradual
improvement and iterative upgrading process of our engineering goals

# Inspiration

### Social background

With the accelerated industrialization of society, it also brings a lot of new problems and worries to people. The
accumulation of negative emotions, such as sadness and anxiety, can also have a long-term impact on physical and
mental health. This trend is especially prominent among teenagers and working youth, and it also deeply touched us as
a group of teenagers.

### Motivation point

In March-April at the beginning of this year's competition establishment, Jilin Province, where our team is located,
suffered from a more serious COVID-19 epidemic. The quarantine control policy made students trapped in the dormitory,
and the scheduled experimental plan could not be carried out normally, and all the study work was forced to be online
or terminated. During the days in the dormitory, we found that: college students studying in other countries around us
became more and more anxious and dull due to the multiple effects of the epidemic, their studies, and their lives. We
began to think: Can we use our required knowledge of synthetic biology combined with emotions to soothe and relax the
depression and misery in everyone's mind?

### Source of inspiration

In a group discussion in March, the fragrance of gardenias in full bloom outside the window came with the wind,
bringing new inspiration to everyone. This light floral scent soothed the other students who were present. We then got
inspired: can we follow the main line of scent and see what kind of scent we can synthesize? Try something that could
serve to calm a sad mood or relieve the stress of anxiety.

### Preliminary design

After preliminary literature research, questionnaire survey and market research, we found that:

1. Smell and emotion are closely related: we found that the neural connection between smell and emotion is very
strong,
and 75% of human emotions are produced
by smell. The main site of interaction with the olfactory center is the amygdala. No intuitive system other than smell
can affect the amygdala, the brain region that controls human emotions, in such a unique and direct way. All of our
sensory organs react after thinking, except for smell, where the brain reacts first and then enters into thinking.

2. After literature research, at the same time we issued questionnaires to our friends around and online, analysis of
survey data found that currently aromatic alcohols and phenylethyl alcohol are very commonly used as monomeric
fragrance raw materials, and their rose scent and lily of the valley scent are currently the two most popular scents
in the survey.

3. We found that aromatic substances have a good effect on regulating bad emotions, and there are such practical means
of
aromatherapy in psychotherapy. Therefore, we consider that we can use aromatic substances to stimulate the body to
respond positively to bad emotions when they appear and regulate them in time.

4. Many aromatherapy and essential oil products already exist on the market, but all these products can only release a
single odor substance, so we want to develop an odor releasing product with recognition function, which can release
different odor substances according to the user's emotion.

Combining synthetic biology knowledge and Artificial Intelligence technology, we came up with the idea that we could
quantitatively identify different emotions and use light promoters to stimulate the synthesis of engineered bacteria
to release different odor substances. To complete our idea, we have carried out the following design:

##### (1) AI Emotion Recognition

After understanding the face recognition system, we decided to use facial expression and bracelet heart rate to
achieve emotion recognition and designed a set of related input and output devices. The camera and the bracelet are
used as input devices, and the main function is to capture the facial expressions of the face. The main function of
the software system as the output device is to output the current user's emotion by comparing these expressions with
the expression data stored in the database.

##### (2) Signal Transformation

We use light to link emotions and engineered bacteria together, and connect the recognition system to the expression
system through a circuit. In our project, we chose sad emotion as the object of study. When the result of AI emotion
recognition is sad, the system will turn on the blue light and irradiate the engineered bacteria.

##### (3) Plasmid Expression

We designed a complete blue light expression system. When sadness is output as blue light through signal conversion,
the light source irradiates the engineered bacteria to activate EL222 protein and initiate the expression of
downstream dual enzyme genes KdcA and Adh1, which synthesize phenylethyl alcohol that can relieve anxiety.

##### (4) Release of Incense System.

In order to effectively release odor substances, we designed a set of odor-releasing incense devices, and through a
mode similar to a leafless fan, achieve smooth and directional release. At the same time, we extended the music,
lighting and other multi-sensory approaches to a later stage to assist the odor for emotional regulation.

As shown in the picture, this is our mascot, who will take you through the mystery of the engineering

![](https://static.igem.wiki/teams/4427/wiki/engin-233.png)

# Target 1

How to recognize emotions effectively? ----------AI facial recognition
and
bracelet heart rate

### First Circle

##### First Design

The first engineering goal facing our project is: how to effectively identify the expression interest. Because only
on
the basis of the correct recognition of emotions, we can achieve an effective emotion regulation process.

In this regard, Luo Zijin, who is responsible for the software in our group, decided to use python to call the
camera
to implement expression capture. In the way of using convolutional neural network contains the picture of the face
will be transformed into a 48\*48 matrix as the input of convolutional neural network, using the algorithm
convolutional neural network to compare the different expressions in the database material in the open source. The
screenshots of expressions in the dataset are shown below:

![](https://static.igem.wiki/teams/4427/wiki/engine-2.png)

##### First Design

We build systems that capture facial convolutional neural networks and perform emotion judgment through software
analysis and database comparison. For the input device, we dynamically capture the user's facial information through
a
camera and collect the data required for emotion analysis. For the output device, we wrote a program that can
analyze
the convolutional neural network and compare the analysis results, and output the input information and data
analysis
and comparison as the corresponding real-time emotion of the user. The preliminary schematic-construction is shown
in
the following figure:

![](https://static.igem.wiki/teams/4427/wiki/engine-3.jpeg)

As shown above, the image input is mainly processed by the convolutional layer, and then the fully-connected layer
performs the expression score classification, and finally outputs the corresponding result

##### First Test

As shown in the picture, our classmate in the software group, Zijin Luo, tested the facial recognition program we
wrote to call the camera to judge its own expression using convolutional neural network. We call the camera that
comes
with the laptop, and the program will contain the picture of the face will be transformed into a 48\*48 matrix as a
convolutional neural network input. The algorithm Convolutional Neural Network is used to compare the combined
scores
of different expressions of the database material, and finally the most similar emotion results are inferred
according
to the relevant scores. This is the accuracy figure of our first round of recognition results.

![](https://static.igem.wiki/teams/4427/wiki/engine-4.png)

##### First Learn:

As can be seen by analyzing the data in the above figure, the accuracy of our first round of model testing rose
rapidly at the beginning of training, and began to slow down after the tenth round, and basically increased to a
halt
after twenty rounds, maintaining it in the 60% judgment accuracy range. In the first round of iterations, we
achieved
an effective expression recognition process. However, the overall accuracy is effective, and the final accuracy of
68%
does not provide effective recognition and judgment guidance.

### Second Circle

##### Second Design

In the first iteration, we camera to achieve expression capture, the use of algorithms Convolutional Neural Network
to
compare different expressions of database material in open source although the method can achieve process closure.
However, the overall recognition effect was average, and the 62% accuracy did not satisfy the actual usage effect.

In the group meeting of the software group, Wang Zhixin proposed that: based on the traditional machine learning
process, it is necessary to have better data sets as samples for giving machine learning. Our initial recognition
selection was open source of someone else's black and white processed expression data, but it was obviously a bit
harsh to let the machine learn to determine the change of expressions of different students in real time.

After discussion, we decided to use a combination of the user's own facial features capture and database assisted
reference, using the user's own features as the key input reference to retest the improved process.

##### Second Build

(1) First, we call the laptop's own camera to capture the user's expressions (after user authorization) while
capturing the user's own key facial features, taking into account the positioning office scenario and the user's
hardware burden.

(2) Then, the image containing the face will be transformed into a 48\*48 matrix as the input to the Convolutional
Neural Network

(3) The algorithm Convolutional Neural Network is used to compare its own feature values with the database samples
and
calculate the combined scores of different expressions

The expression with the highest score will be displayed

The combination of our user's own facial feature capture, and database assisted reference, uses the user's own
features as a key input reference to retest the improved process.

##### Second Test

We improved the user's own facial feature extraction by adding a pre-processing module for user feature extraction
to
the above model calculation, and the final recognition accuracy obtained is shown in the following figure:

![](https://static.igem.wiki/teams/4427/wiki/engine-5.png)

##### Second Learn

We found that after adding the pre-processing of the user's own features, the recognition accuracy of the model was
greatly improved, adding up to an increase of about 11 percentage points from the original 68% accuracy. Although
the
second modified model has one more step of the pre-processing process and the effect converges more slowly in the
first ten training rounds, the recognition accuracy is significantly increased afterwards.

### Third Circle

##### Third Design

After the second iteration, we developed the recognition program as software and combined it with the light source
system afterwards. After a period of practical application, we found that: in the test of the emotion recognition
system, our system is basically accurate for the recognition of expressions. But sometimes people's complex emotions
may be subjectively or objectively passively disguised. For example, they may pretend to be calm when they are sad,
or
consciously and deliberately make expressions that are different from their actual emotions. This all brings new
challenges to the effective recognition of our system.

To address this issue, we have explored alternative approaches to emotion recognition through literature research.
Although emotions such as expressions are subjectively influenced, they are easy to capture and quick to respond,
but
there is an inherent defect in recognizing specific situations. To address this situation, I started to think about
whether there is a more accurate emotional response signal that does not "lie" and is easy to capture. This would
complement the failure of facial recognition methods in specific situations.

##### Third Build:

In response, Zhixin Wang from our group thought that we could use a common device, the smart bracelet, to assist us
in
judgmental recognition. Using the bracelet to achieve the directional capture of heart rate, especially for the
special peak change to match the situation.

We finally designed a judgment method combining facial recognition and heart rate, so as to improve the accuracy of
judgment and avoid the possibility of certain mis-touch judgment. So we designed a bracelet user's heart rate for
detecting auxiliary input with reference to sports bracelets and other related devices.Eventually we constructed a
new
comprehensive judgment system combining heart rate recognition of a bracelet and facial recognition as shown in the
following figure:

![](https://static.igem.wiki/teams/4427/wiki/engine-6.png)

##### Third Test:

We built a bracelet system to capture heart rate, using PPG (photoplethysmographic, photo volumetric pulse wave
tracing method) to record the pulsation state of blood vessels and measure pulse waves by illuminating human blood
vessels with LED light sources and using sensors to measure the attenuated light reflected from human blood vessels
and tissues.

Firstly, Wang Zhixin of our group chose a sports bracelet for heart rate detection. BLE (Bluetooth Low Energy)
communication is used to transmit between the bracelet and the computer. Compared with the classic Bluetooth method,
BLE also has the characteristics of fast transmission speed, long transmission distance and large broadcasting
capacity. The bracelet measures the user's heart rate and then broadcasts the user's current heart rate outward via
BLE. The phone receives the heart rate and uploads it to the cloud for analysis.

![](https://static.igem.wiki/teams/4427/wiki/engine-7.png)

Then, Zijin Luo from our group combined the previous facial recognition algorithm with the heart rate condition
measured by the bracelet. In order to calculate the combination of both data, we developed the relevant
comprehensive
judgment software. We also combined gender, age and other key information input to achieve the correction of the
heart
rate model of the bracelet, and added the protection of user privacy instructions. The overall software
implementation
process is as follows:

(1)Enter the program and a help window will pop up, prompting the user to enter the range of parameters to be
obtained:

![](https://static.igem.wiki/teams/4427/wiki/engine-8.png)

(2) When the user enters the parameters correctly, clicking the Start button will bring up the Privacy Statement
button, asking the user to confirm permission to use the camera and stating that the information entered will be
encrypted and will not be saved:

![](https://static.igem.wiki/teams/4427/wiki/engine-9.png)

(3) After the user accepts, the recognition program will start running, the main window will display the heart rate
(the program currently uses random numbers for heart rate), while the camera window and the window displaying
real-time MAL will pop up, and the MAL window will display the recognized emotions

![](https://static.igem.wiki/teams/4427/wiki/engine-10.png)
![](https://static.igem.wiki/teams/4427/wiki/engine-11.png)

(4) Overall recognition accuracy effect comparison: we can find that with the addition of the bracelet heart rate
sampling as an auxiliary set, the overall effect of the model achieves an effective increase, and the effect is more
obvious after 20 rounds of iterations.

![](https://static.igem.wiki/teams/4427/wiki/engine-12.png)

##### Third Learn:

After several rounds of iterations, we added pre-processing extraction of user expressions in the pre-recognition
stage of facial expression recognition, which led to an overall increase of 11 percentage points in model
recognition.After that, we added the hand ring as a supplement to physiological signal acquisition, which
effectively
avoids the shortage of facial emotion recognition in some specific situations. We combined the two multiple signals
to
corroborate each other, so that the model as a whole achieved high convergence accuracy.

It can be said that it is also this iterative engineering upgrade that effectively improved the overall recognition
model, and also laid the pilot foundation for later transformation into light signal and emotion regulation, and
also
let me experience the charm of engineering.

# Target 2

How to identify emotional outcomes to odor regulation --------
Light-Responding Switch

### First Circle

##### First Design

The first problem we have to solve in order to construct a light-initiated expression system is how to get a working
light source promoter. Only if the promoter is working properly can we achieve our goal of using the light source to
initiate gene pathway expression.

In response, we designed a gene expression pathway that uses the blue light photosensitive protein
EL222([BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003))to regulate the red fluorescent protein
mRFP. E. coli BL21 can synthesize the blue light
photosensitive protein EL222 upon induction of L-arabinose, and blue light irradiation can alter its LOV structural
domain and initiate the expression of the downstream red fluorescent protein mRFP1（ [BBa\_K4427006](http://parts.igem.org/Part%3ABBa_K4427006)）. By examining the
final product, red fluorescent protein, we can determine whether the blue light initiation system is working
properly.

![](https://static.igem.wiki/teams/4427/wiki/engine-13.png)

##### First Build

To achieve the above functions, we constructed the pSB1C3-stufffer-pro ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)) plasmid using pSB1C3:

![](https://static.igem.wiki/teams/4427/wiki/engine-14-new.png)

*①Ara operon([BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003)):UCL iGEM 2017 employed a
constitutive promoter ahead of EL222 to construct the light
control system. However, by literature retrieval, we found there was a risk of expression leakage for constitutively
expressed EL222, making it difficult to demonstrate the efficacy of blue light induction intuitively (compared to
quantified characterization). Thus, ara operon was involved in the pathway in control of the expression
EL222.*
*②EL222([BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003)):EL222 is a blue light photosensitive
protein. Upon irradiation with blue light at a wavelength
of 465 nm, the LOV structural domain of EL222 undergoes metatable activation and the metatable EL222 binds to the
PBlind promoter to initiate the expression of downstream genes.*
*③mRFP1( [BBa\_K4427006](http://parts.igem.org/Part%3ABBa_K4427006)):mRFP1 is a red fluorescent protein. The
excitation wavelength is 584.9nm and the emission
wavelength is 612.*

##### First Test

Firstly, we tested whether our pSB1C3-stufffer-pro plasmid transformed properly in BL21 by CmR resistance screening,
plasmid miniprep, XbaI and SpeI double digestion nucleic acid electrophoresis experiments, and the experimental
results proved that we obtained the engineered bacterium BL21-Pro.

We then used arabinose to induce the synthesis of EL222 protein by BL21-Pro under light-proof conditions, followed
by
cyclic experiments with alternating blue light irradiation and dark incubation of the engineered bacteria, and
finally
UV irradiation, bacterial confocal characterisation and fluorescence spectroscopy to detect the red fluorescent
protein mRFP1.

The experimental results (available on the RESULTS page[https://2022.igem.wiki/jlu-china/results)](https://2022.igem.wiki/jlu-china/results) that
BL21-Pro synthesizes the red fluorescent protein mRFP.

![](https://static.igem.wiki/teams/4427/wiki/engine-15.png)

(A1 and A2: Expression control results of blue light-initiated red fluorescent protein mRFP1, A1 is the experimental
group with red fluorescence produced under UV, right is the blank control; A2 is the experimental and control PCR
tubes without UV irradiation; B: Fluorescence spectroscopy results of red fluorescent protein mRFP1. The excitation
wavelength is 612 and the emission wavelength is 589, which is consistent with the characteristics of mRFP1; Right:
Confocal characterization of BL21-Pro microstructure, 1000x )

*(top left: blue light-initiated expression control results for red fluorescent protein mRFP1; bottom left: red
fluorescent protein mRFP1 fluorescence spectroscopy results; right: BL21-Pro Microstructure confocal
characterisation,1000x.)*

##### First Learn

After research and analysis of the experimental results within the group, our blue light starter system is able to
effectively control the expression of gene pathways. Compared to other light-controlled components, it can be better
adapted to our needs. We can replace the red fluorescent protein gene with a gene related to the synthesis of 2-PE
on
the basis of this pathway.

### Second Circle

##### Second Design

After proving that the blue light promoter works, the second problem we had to solve was how to use blue light to
regulate 2-PE expression. We found the biosynthetic pathway of 2-PE.

![](https://static.igem.wiki/teams/4427/wiki/engine-16.png)

Based on this pathway, we have improved on the blue-red fluorescent protein expression pathway and designed a
heterologous blue-light expression system for overexpression of.

![](https://static.igem.wiki/teams/4427/wiki/engine-17.jpg)

This system allows E. coli to initiate the expression of 3-deoxy-D-arabinoheptulose-7-phosphate synthase and the
bifunctional enzymes branching acid metatase (CM)-prephenate dehydratase (PT), KdcA and Adh1, which catalyse the
progressive synthesis of 2-PE from glucose via a cascade reaction, and this pathway enhances 2-PE production through
overexpression of the enzyme, under the modulation of blue light.

①DS:DS (3-dcoxy-7-phosphoheplulonate synthas) is an important protease
that controls the
synthesis of aromatic amino
acids by regulating enzyme activity through an allosteric mechanism. The synthesis of PEP (phosphoenolpyruvate) to
DAHP (3-deoxy-D-arabino-hept-2-ulosonate 7-phosphate) can be catalysed.

②CM-PT:CM-PT (chorismate mutase/prephenate dehydratase) is a key
enzyme
in the L-Phe
anabolic branching pathway,
catalyzing the decarboxylation, aromatization and deacylation of prephenic acid (PRE) to produce phenylpyruvate,
which
is then subjected to a pyridoxal phosphate (PLP)-dependent transamination reaction to produce L-Phe. This is
followed
by a pyridoxal phosphate (PLP)-dependent transamination reaction to produce L-Phe.

③KdcA([KdcA
BBa\_K4427005](http://parts.igem.org/Part%3ABBa_K4427005)):KdcA (branched chain alpha-keto acid
decarboxylase)
is a keto acid
decarboxylase that converts
phenylalanine PPA to PAD, which in turn is converted to 2-PE.

④Adh1( [BBa\_K4427004](http://parts.igem.org/Part%3ABBa_K4427004)):Adh1 (alcohol dehydrogenase) converts the
above-mentioned PAD catalyzed
by KdcA into the final
product 2-PE, a geotactic process that uses the genetically catalyzed product PAD of the former enzyme, which is
then
converted to the final product 2-PE by Adh1.

##### Second Build

To achieve the desired function, we integrate this synthesis pathway and constructed the
pSB1C3-stuffer-all（[BBa\_K4427012](http://parts.igem.org/Part%3ABBa_K4427012)
） plasmid. The map is as follows:

![](https://static.igem.wiki/teams/4427/wiki/engine-18.jpg)

##### Second Test

First of all, the transformation of pSB1C3-stuffer-all into BL21 was examined before the functional validation.
Theoretically, bacteria with successfully transformed plasmids should be chloramphenicol-resistant due to the
function of CmR. However, only a small number of colonies were actually observed on the culture media with
chloramphenicol after several repeated experiments. Amplification and plasmid extraction was performed on the
"fortunate" minority, but only to find the result disappointing—no qualified plasmids were obtained.

##### Second Learn

A group discussion was held to look for possible explanations for the frequent failure of plasmid transformation. We
speculated the main reason to be the extra size of our plasmid, which contained a whole lot of genes to be
expressed, making it difficult for the engineered bacteria to absorb or process them.

To find a solution, we reviewed a large amount of literature and found that E. coli itself contains the gene pathway
to synthesize PPA, the precursor of 2-PE. So, all we needed to do was to introduce plasmids merely containing two
key enzyme genes, KdcA and Adh1 to enable the synthesisis of 2-PE in the engineered bacteria by shortening the
overall length of the exogenous genes.

### Third Circle

##### Third Design

Based on the assumptions above, we designed a new blue-light expression system, the EL222-KdcA-Adh1 expression
pathway.

![](https://static.igem.wiki/teams/4427/wiki/engine-19-1.jpg)

In this system, BL21 synthesized protein EL222 in response to L-arabinose induction. Upon blue light irradiation,
EL222 initiated the expression of downstream genes KdcA and Adh1 to synthesize 2-PE from its precursor PPA
previously produced by E. coli itself.

##### Third Build

The shortened plasmid pSB1C3-stuffer-LP (BBa\_K4427002) was
then constructed by omitting the aroF gene for DS
synthesis and the pheA (fbr) gene for CM-PT synthesis. The map of the updated plasmid is as follows:

![](https://static.igem.wiki/teams/4427/wiki/engine-20-new.png)

##### Third Test

In order to test whether the newly designed plasmid could be transformed properly, the same screening process was
carried out by culturing the post-transformation BL21 on the medium with chloramphenicol. As expected, the bacteria
managed to survive, demonstrating our engineering success.

Next, to verify the synthesis 2-PE, we performed a series of experiments on the engineered bacteria, which included
1) plasmid extraction with electrophoresis to validate the transformation completion, 2) double-digestion with
electrophoresis to validate the correct insertion of the three genes of interest, and 3) SDS-PAGE and measurement of
enzyme activity to validate the capability of the engineered bacteria to synthesize active KdcA and Adh1.

![](https://static.igem.wiki/teams/4427/wiki/engine-33.png)

![](https://static.igem.wiki/teams/4427/wiki/engine-21.png)

(top left: UV absorption spectrum of coenzyme NADH in the KdcA cascade reaction; top right: concentration-dependent
enzymatic activity of Adh1 enzyme; bottom left: absorption spectrum of Adh1 coenzyme NADH; bottom right: cascade
catalytic activity of a blue light-initiated bacterial solution containing a dual enzyme plasmid of KdcA and Adh1)

Results above (see details on the RESULTS page
<https://2022.igem.wiki/jlu-china/results>) demonstrated that
the
engineered bacteria could produce 2-PE. Quantitative assays were further conducted on our samples and gas
chromatography was performed with standard 2-PE. The results confirmed that our samples were 2-PE with yields of
90-100 mg/L.

![](https://static.igem.wiki/teams/4427/wiki/engine-22.png)

(top: mass spectra of extracted 2-PE samples in bacterial broth; bottom: gas chromatograms of extracted 2-PE samples
in bacterial broth)

##### Third Learn

On reflection and reviewing, we learnt that the BUCT team were also synthesizing 2-PE. We reached out to team BUCT
and found that our designs differed in the expression pathway. The synthetic strategies of the two teams are shown
respectively as follows.

1.JLU\_China:

![](https://static.igem.wiki/teams/4427/wiki/engine-23-1.jpg)

2.BUCT:

![](https://static.igem.wiki/teams/4427/wiki/engine-24.png)

![](https://static.igem.wiki/teams/4427/wiki/engine-25.png)

Our engineered bacteria are capable of synthesising 2-PE in yields of 90-100 mg/L, compared to 20-60 mg/L for BUCT's
method of synthesis.

In order to raise our yields, we held several discussions with BUCT. Combined with additional literature research,
we learned that tyrB encodes an aromatic amino acid transaminase that plays a key role in the synthesis of aromatic
amino acids and 2-PE. Lack of the tyrB gene might restrain the intracellular movement of phenylethylaldehyde,
leading to its massive accumulation inside the cell, thus inhibiting the cell growth.

We therefore assumed the addition of an exogenous tyrB gene to the engineered bacteria could possibly reduce this
effect and might increase the expression of 2-PE.

### Fouth Circle

##### Forth Design

In order to obtain relatively higher 2-PE expression, we designed a fourth-generation pathway of the blue light
expression system.

![](https://static.igem.wiki/teams/4427/wiki/engine-26.jpg)

TryB(tyrosine aminotransferase):Deletion of the tyrB gene resulted in unbalanced flux, large amounts ofaccumulated
phenylacetaldehyde and repressed cell growth.

In comparison to the original blue-light expression system, the expression of 2-PE was increased by reducing the
inhibition of cell growth by aldehyde.

##### Forth Build

We modified the pSB1C3-stuffer-LP ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) plasmid by
adding the TyrB gene to construct the pSB1C3-stuffer-LP2
( [BBa\_K4427021](http://parts.igem.org/Part%3ABBa_K4427021)) plasmid. The improved plasmid was ligated as
follows:

![](https://static.igem.wiki/teams/4427/wiki/engine-27.jpg)

Constrained by the time limit, we did not perform a complete test of the fourth-generation blue-light expression
system. If possible, we'll validate this system and explore more strategies to boost the production of phenylethanol
in the future.

# Target 3

How to ensure biosafety when using products - Red and blue light suicide
systems

### First Circle

##### First Design

In order to ensure the safety of our device in real-life application, we designed the red- and blue-light suicide
system together with Hainan University on the basis of the lethal gene (
 [BBa\_K3739028](http://parts.igem.org/Part%3ABBa_K3739028)) provided by Xiamen University.
When irradiated with both red and blue light, the engineered bacteria would initiate the expression of toxic
proteins.

![](https://static.igem.wiki/teams/4427/wiki/engine-34.png)

Considering the experimental time, we only designed the blue light-induced suicide system by replacing the mRFP1
gene in the pSB1C3-stuffer-pro plasmid with a virulence protein gene, and then performed the verification
experiment.

Due to the specificity of the pSB1C3-stufffer-pro plasmid, the experiments could not be carried out with suitable
enzymatic sites found on both sides of the fluorescent protein, so the experimental steps were adjusted and
modified, and four methods were found for the extraction and ligation of the plasmid and the toxic protein sequence.

##### First Build

We constructed the pSB1C3-stuffer-BDH（[BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)）plasmid by
replacing the red fluorescent protein gene in the
p1C3-stufffer-pro ([BBa\_K4427015](http://parts.igem.org/Part%3ABBa_K4427015)) plasmid with a virulence
protein gene.

![](https://static.igem.wiki/teams/4427/wiki/engine-28-new.png)

After thorough communication, the agreed experimental procedure was to amplify the pro plasmid-containing punctures
and extract the pro plasmid, followed by enzymatic ligation and verification of the ligation results, followed by
blue
light induction of expression after transformation to obtain the experimental results.

Due to the specificity of the pro plasmid, it was not possible to use the appropriate enzymatic sites on both sides
of
the fluorescent protein for the experiments, so the experimental procedure was modified and four methods were found
for the extraction and ligation of the plasmid and the virulent protein sequence.

Option 1: The pro plasmid vector is linearised by reverse pcr, with primers for both NdeⅠ and PacⅠ at both ends for
pcr. The virulent gene fragment is amplified by pcr and then double ligated and transformed. The disadvantage is
that
the reverse pcr length of the pro plasmid is too long, over 4000bp.

Option 2: The vector is linearised in such a way that the pro plasmid fluorescent protein sequence is disrupted and
some fluorescent protein sequence remains. Blra toxicity gene fragment was amplified by the PCR method. The target
plasmid was then obtained by double digestion ligation method.

Option 3: The vector is linearised in such a way that the fluorescent protein sequence is destroyed and some of the
fluorescent protein sequence remains. and enzymatic sites are not common, amplification efficiency needs to be
considered. blra toxicity gene fragment amplified by the PCR method. A homologous recombination kit is then used and
a
one-step cloning is performed.

Option 4: The pro plasmid vector is linearised by reverse pcr without enzymatic sites and the virulence gene
fragment
is amplified by pcr and then cloned in one step using a homologous recombination kit. The disadvantage is that the
reverse pcr length of the pro plasmid is too long, over 4000bp.

![](https://static.igem.wiki/teams/4427/wiki/engine-29.png)

(top left: amplification of puncturing bacteria; top right: pro plasmid attempted pcr pre-amplification , 1 and 5
are
fragments obtained from method one and method four respectively, 2 and 6 are dilutions of the template 10-fold, 3
and
7 are dilutions of 20-fold, 4 and 9 are dilutions of 100-fold, 8 is added incorrectly and 9 is a make-up of 8.
(Below:
amplification of the blrA toxicity gene fragment)

![](https://static.igem.wiki/teams/4427/wiki/engine-30.png)

(From left to right, top to bottom, Blra gum recovery concentrations for scenarios I, II, III and IV)

##### First Test

After obtaining the recombinant plasmid using the above method, we carried out transformation
experiments
using BL21 to obtain BL21-BDH and carried out control experiments with blue light irradiation and dark culture for
BL21-BDH. The experimental results showed that no colonies grew in either the control or experimental groups, and
the
pro plasmid was difficult to reconstitute by conventional methods.

![](https://static.igem.wiki/teams/4427/wiki/engine-31.png)

##### First Learn

After conducting several experiments, none of the expected results were achieved. After communication with the
Hainan
University team, an analysis of possible causes was carried out.

1. Not completely protected from light and toxic genes leaked into natural light.
2. Transformation failure due to inaccurate control of heating temperature and heating duration.
3. The plasmid base number was too large and spontaneous spitting occurred, so we had to keep changing the sensory
   state to try.
4. Due to reverse pcr, the pro plasmid is too long and there are various factors that affect the trailing band and
   stray band.
5. Enzyme are not common.

### Second Circle

##### Second Design

The design has been continuously improved so that all problems except for the difficulty of constructing the pro
plasmid can be effectively solved. As the pro plasmid is difficult to reconstitute by conventional methods and we
have previously validated the function of the pro plasmid. Given the limited time available, we decided to validate
the toxin alone first. If the virulence protein works properly, then the lethal task can be accomplished by ligating
the blue light initiation system ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)) to the
virulence protein.

##### Second Build

In order to verify whether the virulence gene protein expressed normally, we designed a control experiment together
with HainanU\_China, expecting bacteria that expressed the virulence protein not to grow on the plate, while the
bacteria without this part to grow as the control group to better prove the detrimental function of the virulence
gene that put our bacteria to death.

##### Second Test

HainanU\_China added 1 μL of the virulence gene plasmid to 50 μL of competent E. Coli cells, spread the plates after
transformation, added 50 μL of the solution to each plate, and had them incubated overnight at 37°C. The results are
shown below, which proved their engineering success.

![](https://static.igem.wiki/teams/4427/wiki/engine-32.png)

(Left: engineered bacterium introducing a virulent protein pellet; right: engineered bacterium introducing a plasmid
but without a virulent protein gene.)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Our experiment consists of the functional validation of two systems, the blue-light system and the
red-light system. The main body of our experiment is divided into 3 aspects: the blue-light system to release 2-PE
(phenylethanol), the red-light system to release 3-oxo-C12-HSL, and the simultaneous activation of both systems to kill
the engineered *E. coli.*{% endblock %}
{% block page\_left\_content %}

* [Overview](#1)
* [1. Introduction](#2)
* [2. Blue light system](#3)
* [3. Red light system](#4)
* [4. Suicide system](#5)
* [5. References](#6)

{% endblock %}
{% block page\_content %}

# Overview

Our experiment consists of the functional validation of two systems, the
blue-light system and the red-light system. The main body of our experiment is divided into 3 aspects: the blue-light
system to release 2-PE (phenylethanol), the red-light system to release 3-oxo-C12-HSL, and the simultaneous activation
of both systems to kill the engineered *E. coli*.

For the blue-light pathway, we selected the EL222 protein, which could
dimerize to bind to the PBLind promoter, initiating the expression of the downstream gene expression under blue
light[1]. In contrast, for the red-light pathway, we used BphP1 and PpsR2 as a photosensitive system, which could be
activated by red light to initiate downstream gene expression[2][3].

For the selection of target gene, we focused on 2-PE. As a terpenoid,
2-PE has been shown to be beneficial in relieving sadness with its distinctive floral scent, and this mood-relieving
effect can be scientifically confirmed by the detection of brain waves at sleep[4]. Past researches have reported the
successful expression of 2-PE in E.coli . We need to use its natural production and transfer the genes downstream to
build an Ehrlich pathway in E.coli to achieve efficient expression of 2-PE[5]. This ensures that the selected target
gene, 2-PE, can be expressed properly in E.coli. In addition, we associate the target gene with EL222, so that we can
use blue light to control the expression of the target gene.

In consideration of the biosafety of the project, we proposed to use both
red and blue light together to kill the engineered bacteria in case of bio-contamination. This idea was achieved by
producing an inducer 3-oxo-C12-HSL under red light, and opening AND Gate channels with the inducible substance when
irradiated with blue light to induce the expression of downstream virulent protein genes[1]. Having the product
expressed inside the cell was not the end of the story, instead, they should be released. Fortunately, as small
molecules, 2-PE had innate properties that allowed it to cross the cell membrane along the concentration gradient.
Moreover, as an alcohol, 2-PE could diffuse out of E. coli more easily due to its high fat-solubility[5].

# 1. introduction

### ① Plasmid construction

We plan to realize the function of producing phenylethanol under the control of blue light[6], so we designed the
plasmid pSB1C3-stuffer-all ([BBa\_K4427012](http://parts.igem.org/Part%3ABBa_K4427012)).

![](https://static.igem.wiki/teams/4427/wiki/wet-1.jpg)

Fig.1 Complete path of Blue Light system

![](https://static.igem.wiki/teams/4427/wiki/wet-2.jpg)

Fig.2 the plasmid pSB1C3-stuffer-all ([BBa\_K4427012](http://parts.igem.org/Part%3ABBa_K4427012))

However, we found that the construction of such a huge plasmid was not only difficult, but also the actual
transformation and expression effect was not satisfactory. Therefore, we properly simplified the plasmid, removed some
non critical enzymes, and temporarily removed the toxic protein gene blra from the whole plasmid. Therefore, our
simplified plasmid pSB1C3-stuffer-LP ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) came out.

![](https://static.igem.wiki/teams/4427/wiki/wet-3.jpg)

Fig.3 Simplified path of Blue Light system

![](https://static.igem.wiki/teams/4427/wiki/wet-4.jpg)

Fig.4 the plasmid pSB1C3-stuffer-LP ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002))

In order to better explore whether the function of each component can be realized normally, we built several sets of
plasmids separately for several components in the plasmid pSB1C3-stuffer-LP ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) to verify its function.

![](https://static.igem.wiki/teams/4427/wiki/wet-5.jpg)

Fig.5 the plasmid pSB1C3-stuffer-pro ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009))

We constructed a plasmid pSB1C3-stuffer-pro ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)) to
independently verify the function of photosensitive
protein EL222. By adding the fluorescent protein gene mrfp1, we can directly judge whether the function is normal by
the presence or absence of fluorescence.

![](https://static.igem.wiki/teams/4427/wiki/wet-6.jpg)

Fig.6 the plasmid pET-22b-Adh1 ([BBa\_K4427007](http://parts.igem.org/Part%3ABBa_K4427007))

![](https://static.igem.wiki/teams/4427/wiki/wet-7.jpg)

Fig.7 the plasmid pET-30a-KdcA ([BBa\_K4427008](http://parts.igem.org/Part%3ABBa_K4427008))

In order to investigate whether the two key enzymes Adh1 and KdcA related to phenylethanol production are normal, we
constructed plasmids pET-22b-Adh1 ([BBa\_K4427007](http://parts.igem.org/Part%3ABBa_K4427007)) and
pET-30a-KdcA ([BBa\_K4427008](http://parts.igem.org/Part%3ABBa_K4427008)) respectively. These two plasmids can
express Adh1 and KdcA separately, so that we can better study the properties of these two enzymes in vitro.

Similar to the blue light system, we also independently verified each part of the red light system.

![](https://static.igem.wiki/teams/4427/wiki/wet-8.jpg)

Fig.8 Path of Red Light system

First, we designed the plasmid pET-22b-RLP ([BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016)) to
independently verify the function of the red light
starting system. By adding the fluorescent protein sfGFP gene, we can directly judge whether the function is normal by
the presence or absence of fluorescence.

![](https://static.igem.wiki/teams/4427/wiki/wet-9.jpg)

Fig.9 the plasmid pET-22b-RLP ([BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016))

We originally planned to carry out red and blue light co adjustment experiments to verify whether the function of the
AND gated channel was normal. Unfortunately, due to time problems, we could only save this verification for a later
time.

### ② E.coli selection

In order to balance the accuracy of the experimental results and the stability of the experimental process, we decided
to choose E.coli BL21 as the experimental strain after comparing the normal curves and product yield of E.coli DH5α
and BL21.

# 2. Blue Light System

To ensure the accuracy and reliability of the blue light system, we used a separate validation format, transforming
plasmids of each key protein in the system (EL222, Adh1, KdcA) into E.coli separately and verifying their expression
as well as their function.

### ① EL222（[BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003)）

We first transformed pSB1C3-stuffer-pro ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009))into
E.coli and first confirmed the successful transformation of
the plasmid into E.coli by gel electrolysis, (note that thereafter all experimental manipulations were performed under
shade) adding the EL222 expression inducer to the E.coli culture media to get the system started.

![](https://static.igem.wiki/teams/4427/wiki/wet-10.jpg)

Fig.10 Ehrlich pathway in E.coli[5]

### ② KdcA（ [BBa\_K4427008](http://parts.igem.org/Part%3ABBa_K4427008) ）

KdcA (branched-chain alpha-ketoacid decarboxylase) is a ketoacid decarboxylase that converts phenylalanine PPA to PAD,
which in turn converts PAD to 2-PE.

### ③ Adh1（ [BBa\_K4427007](http://parts.igem.org/Part%3ABBa_K4427007)）

Adh1 (alcohol dehydrogenase) converts the PAD mentioned above catalyzed by KdcA into the final product 2-PE. This
geotactic process uses the genetically catalyzed product PAD of the former enzyme, which is then converted to the
final product 2-PE by Adh1.

# 3. Red Light system

To ensure the accurate reliability of the red light system, we used separate validation, i.e. transforming plasmids
of each key protein in the system into E. coli separately to characterize their expression as well as their
function.

### BphP1 ([BBa\_K4427000](http://parts.igem.org/Part%3ABBa_K4427000)), PpsR2 ([BBa\_K4427001](http://parts.igem.org/Part%3ABBa_K4427001)) and Ho1 ([BBa\_K4427010](http://parts.igem.org/Part%3ABBa_K4427010))

![](https://static.igem.wiki/teams/4427/wiki/wet-11.jpg)

Fig.11 Principle of red light system[3]

We exploited this system of reversible light-induced binding between the bacterial phytochrome BphP1 and its natural
partner PpsR2. In this system, Ho1 first catalyzes heme to BV, BV binds to BphP1 and acquires light-sensitive ability,
and can bind to PpsR2 under 760nm NIR light irradiation to inhibit the suppression of PpsR2, highly expresses
downstream genes. While under 680nm red light irradiation, the suppression effect is quickly disabled and suppressed
the expression of the downstream gene again[3]. For the red light part, we also used a block validation approach to
demonstrate the proper functioning of the red light system.

# 4. Suicide system

In order to avoid unnecessary nutrient depletion of other flora caused by E.coli entering the decay phase, and also so
that the process of gas synthesis and release can be effectively controlled to achieve an immediate on/off effect, we
cleverly used the existing blue and red light pathways and added the E. coli virulence protein BlrA used by the iGEM
team at XMU in 2021 downstream of the pathway intersection, and made a logical breakthrough to verify its function of
causing the death of E. coli.

Our in-depth partner HNU\_China helped us to complete the functional verification of the suicide system. As for the
hardware design, we still adopted the mode of blue light intermittent irradiation to activate EL222 expression. In
other words, the alternate treatment of 2 hours of irradiation followed by 2 hours of darkness to the engineered
bacteria (to reduce the toxic effect of blue light irradiation on the bacteria, and at the same time to shift the
chemical balance) continued for 8 hours in total. Subsequently, the bacteria lysed and died due to the large
accumulation of BlrA toxic protein in E.Coli.

### ① blrA （[BBa\_K4427028](http://parts.igem.org/Part%3ABBa_K4427028)）

Firstly, while the blue light system was normally expressed, we have added a "small tail" at the end of the
pathway—the LasI promoter—which, while not activated, prevented the normal production of the virulence protein BlrA
and kept the bacteria active to synthesize and release odour molecules. Once the AND gate opens, the LasI promoter
will be activated and will transcribe a large amount of BlrA toxin, which causes the bacterium to autolysis and die,
thus terminating the synthesis and release of odour molecules.

### ② HSL

As mentioned above for logic gap, when would the AND logic gate turn on? We designed the normal irradiation of red
light to produce 3-oxo-C12-HSL, which can open the AND gate channel thereby initiating the expression of the
downstream toxic protein blrA.

# 5. References

[1] Jayaraman P, Devarajan K, Chua T K, etal. Blue light-mediated transcriptional activation and repression of gene
expression in bacteria[J]. Nucleic Acids Research, 2016, 44(14):6994-7005.

[2] Baumschlager A, Khammash M. Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional
Light-Control in Bacteria[J]. Advanced Biology, 2021, 5(5).

[3] Ong N.T, Olson E.J, Tabor J.J. Engineering an E. coli Near-Infrared Light Sensor[J]. ACS Synthetic Biology,
2019(7):240-248.

[4] Herz RS. Aromatherapy facts and fictions: a scientific analysis of olfactory effects on mood, physiology and
behavior. Int J Neurosci. 2009;119(2):263-90.

[5] Liu, C., Zhang, K., Cao, W. et al. Genome mining of 2-phenylethanol biosynthetic genes from Enterobacter sp. CGMCC
5087 and heterologous overproduction in Escherichia coli.[J] Biotechnol Biofuels.

[6] Schwaiger K N, Voit A, Hana Dobiašová, etal. Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes
Whole-Cell Production of Cellobiose[J]. Biotechnology Journal, 2020, 15(11).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}We successfully proved our engineered bacteria could synthesize 2-PE, which could regulate mood, by
verifying the blue light-responding switch, the red light-responding switch, the blue light-controlled system. We also
separately verified the switch and suicide functions of the red and blue suicide system. And we will verify more
light-responding switches and light-controlled systems in the future.{% endblock %}
{% block page\_left\_content %}

* [Overview](#1)
* [1. Blue Light-Responding Switch](#2)
* [2. The Red Light-Responding Switch](#3)
* [3. Blue Light-Controlled System](#4)
* [4. The Suicide system](#5)

{% endblock %}
{% block page\_content %}

# Overview

The wet experiment was designed to demonstrate the engineering feasibility of our blue light-controlled expression
system to synthesize the odour substance phenylethanol (2-PE), which can modulate sadness. In this section we
present the results of experiments in four aspects: the blue light-responding switch, the blue light-controlled
system, the red light-responding switch, and the suicide system. The success of the blue light-responding switch
showed that we could control the expression of downstream genes through a blue light switch, and the validation of
the blue light-controlled system illustrated that our project had the ability to synthesize 2-PE. Together with the
successful validation of the red light-responding switch, the suicide system could be constructed under the control
of red and blue light, lying in the foundation for further expansion of other light sources corresponding to the
regulation of more emotions.

# 1. Blue Light-Responding Switch

In the blue light-responding switch, we constructed the pSB1C3-stuffer-pro plasmid ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)), which includes
the blue light sensitive protein EL222 gene and the red fluorescent protein gene (mRFP1). It expresses EL222 protein
with the induction of L-arabinose, and under blue light, EL222 dimerize to bind to the PBlind promoter,
initiating
the expression of the downstream mRFP1. By detecting the presence of mRFP1, we could verify that the blue
light-responding switch was working properly.

### 1.1 Agarose Gel Electrophoresis and Double Digestion

To ensure that the EL222 protein gene ([BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003)) and mRFP1
([BBa\_K4427006](http://parts.igem.org/Part%3ABBa_K4427006)) gene were inserted in the correct
position, and to verify that the plasmids were correctly transformed, we performed plasmid extraction, double digestion
(XbaI and SpeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pSB1C3-stuffer-pro
([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)). The results of the electrophoresis after plasmid
extraction showed that our plasmid was successfully
transformed into BL21, and the results of the XbaI and SpeI double digestion experiments showed that our target gene was
inserted into the target site correctly.

![](https://static.igem.wiki/teams/4427/wiki/result-1.png)

Figure 1. The map of
pSB1C3-stuffer-pro plasmid.

![](https://static.igem.wiki/teams/4427/wiki/result-2.png)

Figure 2. The result of agarose
gel electrophoresis after double digestion.
Lane 1: pSB1C3-stuffer-pro (Pro) plasmid;
Lane 2: DNA
segments
after digestion.

### 1.2 The Blue Light-Responding Switch Control the Expression of the Red Fluorescent Protein mRFP1

After demonsrating that the plasmid was correctly constructed and successfully transformed, we verified the
light-sensitive function of the pSB1C3-stuffer-pro plasmid ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)) in a dark room. With reference to the
literature, we performed periodic experiments on engineered bacteria BL21 in a dark room with blue light irradiation
for 2 hours and dark treatment for 2 hours alternatively, for a total of 8 hours, and a control experiment was
conducted in complete darkness. The experimental results showed that the engineered bacteria containing the
pSB1C3-stuffer-pro plasmid ([BBa\_K4427009](http://parts.igem.org/Part%3ABBa_K4427009)) successfully
expressed mRFP1, indicating the efficacy of our blue
light-responding switch.

##### 1.2.1 SDS-PAGE of mRFP1

We performed SDS-PAGE on both sets of bacteria and obtained the following result. mRFP1 has a molecular weight of
about 25 kDa, which is comparable to the molecular weight of the marker corresponding to the band in the
electrophoresis graph (Fig. 3), tentatively proving the successful expression of the target protein in the
engineered bacteria.

![](https://static.igem.wiki/teams/4427/wiki/result-3.png)

Figure 3. SDS-PAGE of mRFP1.

##### 1.2.2 The expression levels of red fluorescent protein

We used a UV lamp to irradiate the two groups of bacteria solution and the results showed (Fig. 4) that a clear
fluorescence appeared in the experimental group while no fluorescence appeared in the control group. We observed the
microstructure of the bacterial solution for the experimental group under a laser confocal microscope (Fig. 5).
These results showed the engineered bacteria synthesized red fluorescent protein under the control of the blue
light-responding switch.

![](https://static.igem.wiki/teams/4427/wiki/result-4.png)

Figure 4. A: comparison of
fluorescent proteins under UV irradiation, blue light irradiated group A1 appears clearly fluorescent,
control group A2 does not.
Figure 4. B: comparison under the naked eye, blue light irradiated group B1 appears light pink, control
group B2 is colourless.

![](https://static.igem.wiki/teams/4427/wiki/result-5.png)

Figure 5. Laser confocal.

##### 1.2.3 Determination of Fluorescent Red Fluorescent Protein mRFP1 by Fluorescence Spectrometry

In order to determine whether the protein expressed by the engineered bacteria was the mRFP1 we expected, we
performed fluorescence spectroscopy on the bacteria solution. The results (Fig. 6) showed the protein had an
excitation wavelength of 584 nm and an emission wavelength of 612 nm, consistent with the standard mRFP1. It
determined that our engineered bacteria successfully synthesized mRFP1 under the control of the blue
light-responding switch.

![](https://static.igem.wiki/teams/4427/wiki/result-6.png)

Figure 6. Fluorescence spectrum
of the bacterial solution after blue light irradiation.

In summary, we successfully verified the validity of the blue light-responding switch. Also, we found the control
group (Fig. 7) also had a lower emission peak. Such a leakage expression phenomenon is of concern. But for time
reasons, we did not explore the leakage expression further.

![](https://static.igem.wiki/teams/4427/wiki/result-7.png)

Figure 7. Fluorescence spectra
of the control solute without blue light exposure.

# 2. The Red Light-Responding Switch

In the red light-responding switch, we constructed pET-22b-RLP ( [BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016)), which includes Ho1 ([BBa\_K4427010](http://parts.igem.org/Part%3ABBa_K4427010)),
the red light sensitive protein BphP1 ([BBa\_K4427000](http://parts.igem.org/Part%3ABBa_K4427000)) and the
deterrent protein PpsR2 ([BBa\_K4427001](http://parts.igem.org/Part%3ABBa_K4427001)). It expresses
Ho1, BphP1 and PpsR2 under the induction of IPTG. Ho1 catalyzes heme to BV, BV binds to BphP1 and acquires
light-sensitive ability, and can bind to PpsR2 under 760nm NIR light irradiation to inhibit the suppression of
PpsR2, highly expresses downstream genes. While under 680nm red light irradiation, the suppression effect is quickly
disabled and suppressed the expression of the downstream gene again. To validate the above function of our red light
initiation system pET-22b-RLP ([BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016)), we performed the
following validation:

### 2.1 Agarose Gel Electrophoresis and Double Digestion

To ensure the BphP1 and PpsR2 genes ([BBa\_K4427018](http://parts.igem.org/Part%3ABBa_K4427018)) and the
mutant green fluorescent protein (sfGFP) gene
([BBa\_K4427011](http://parts.igem.org/Part%3ABBa_K4427011)) were inserted in the correct positions, we
performed plasmid extraction, double digestion (XhoI and
NdeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pET-22b-RLP
plasmid([BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016)). The results showed (Fig. 9) that our
target gene was transformed successfully and inserted
into the target site correctly .

![](https://static.igem.wiki/teams/4427/wiki/result-8.png)

Figure 8. The map of
pET-22b-RLP plasmid.

![](https://static.igem.wiki/teams/4427/wiki/result-9.png)

Figure 9. The result of agarose
gel electrophoresis after double digestion.Lane 1: DNA segments after digestion; Lane 2: pET-22b-RLP
(Redlight promotor) plasmid.

### 2.2 The Red Light-Responding Switch Control the Expression of Green Fluorescent Protein Mutant sfGFP

After demonsrating that the plasmid was correctly constructed and successfully transformed, we verified the
light-sensitive function of pET-22b-RLP ([BBa\_K4427016](http://parts.igem.org/Part%3ABBa_K4427016)) in a
dark room. With reference to the literature, we
performed irradiation experiments experiments on engineered bacteria BL21 with far-red light irradiation for 4 hours
and a control experiment with complete light-proofing. After the irradiation, the following observations were made
on the bacterial solution.

##### 2.2.1 The expression levels of green fluorescent protein

We used a UV lamp to illuminate the two groups of bacterial fluids and the results (Fig. 10) showed that clear
fluorescence appeared in the experimental group, while no fluorescence appeared in the control group.

![](https://static.igem.wiki/teams/4427/wiki/result-10.png)

Figure 10. Comparative graph of
fluorescent protein under UV irradiation, control A1 did not fluoresce and red light irradiated group A2
showed significant fluorescence.

![](https://static.igem.wiki/teams/4427/wiki/result-11.png)

Figure 11. Comparison graph
under the naked eye, control group A1 is colourless, red light irradiated group A2 appears light green.

##### 2.2.2 Fluorescence spectra of sfGFP

To determine whether the protein expressed by the engineered bacteria was the green fluorescent protein (sfGFP) that
we expected, we performed fluorescence spectroscopy on the bacterial solution. The results (Fig.13) showed the
protein had an excitation wavelength of 488 nm and an emission wavelength of 510 nm, which is consistent with the
standard sfGFP. It determined that our engineered bacteria synthesized sfGFP successfully. However, the results
(Fig.12) of the fluorescence spectra of the bacterial broth in the light-avoidance group also showed that sfGFP
was synthesized and that leakage of gene expression may have occurred.

![](https://static.igem.wiki/teams/4427/wiki/result-12.png)

Figure.12: Fluorescence
spectrum of the control solute without red light irradiation, protected from light.

![](https://static.igem.wiki/teams/4427/wiki/result-13.png)

Figure 13. Fluorescence spectra
of the experimental group with red light irradiation.

For our project, the red light repression function of the BphP1-PpsR2 system was not indispensable. Due to time
constraints, we only performed experimental validation of the activated expression function and did not complete
functional validation of the repressed expression function.

# 3. The Blue Light-Controlled System

In the blue light-controlled system, we constructed the pSB1C3-stuffer-Lp plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)). It expresses EL222
protein with the induction of L-arabinose, and under blue light, EL222 dimerize to bind to the PBlind
promoter,
initiating the expression of the downstream ethanol dehydrogenase (Adh1) and alpha-keto acid decarboxylase (
KdcA),and catalyzes the synthesis of the substrate phenylpyruvate into phenylethanol (2-PE). To validate the above
functions of our blue light expression system pSB1C3-stuffer-Lp plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)), we performed the following
validation.

### 3.1 Agarose Gel Electrophoresis and Double Digestion

To ensure that the EL222 protein gene ([BBa\_K4427003](http://parts.igem.org/Part%3ABBa_K4427003)), the Adh1
gene ( [BBa\_K4427004](http://parts.igem.org/Part%3ABBa_K4427004)) and the KdcA gene ([KdcA BBa\_K4427005](http://parts.igem.org/Part%3ABBa_K4427005))
were inserted in the correct positions and the plasmids were correctly transformed, we performed plasmid extraction,
double digestion (XbaI and SpeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with
the pSB1C3-stuffer-Lp([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)). The results of the
electrophoresis after plasmid extraction showed that our
plasmid was successfully transformed into BL21, and the results of the XbaI and SpeI double digestion experiments
showed that our target gene was inserted into the target site correctly.

![](https://static.igem.wiki/teams/4427/wiki/result-14.png)

Figure 14. The map of
pSB1C3-stuffer-Lp plasmid.

![](https://static.igem.wiki/teams/4427/wiki/result-15.png)

Figure 15. The result of
agarose gel electrophoresis after double digestion. Lane 1: pSB1C3-stuffer-Lp (lp) plasmid; Lane 2: DNA
segments after digestion.

### 3.2 Functional validation of the complete blue light-controlled system

After determining that the plasmid was correctly constructed and successfully transformed, we verified the
light-sensitive function of the pSB1C3-stuffer-Lp plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) in a dark room. With reference to the
literature, we performed periodic experiments on engineered bacteria BL21 in a dark room with blue light irradiation
for 2 hours and duck treatment for 2 hours alternatively, for a total of 8 hours, and a control experiment was
conducted in complete darkness. The experimental results showed that the engineered bacteria containing the
pSB1C3-stuffer-Lp plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) successfully
expressed 2-PE, which validated the effectiveness of our blue
light-controlled system.

##### 3.2.1 SDS-PAGE of crude enzyme solution

We performed SDS-PAGE experiments on the two groups of bacterial solution, obtained the following figure (Fig. 16).
The molecular weight of Adh1 is 37 kDa and KdcA is 62 kDa, which is comparable to the molecular weight of the Marker
corresponding to the bands in the electropherogram, which can tentatively prove that the pSB1C3-stuffer-Lp plasmid
([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) successfully expresses the target protein we
need.

![](https://static.igem.wiki/teams/4427/wiki/result-16.png)

Figure 16. SDS-PAGE of crude
enzyme solution.

##### 3.2.2 chromatograph characterisation of the end product phenylethanol (2-PE)

The above experiments demonstrated that E. coli BL21 transfected with pSB1C3-stuffer-Lp plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002)) could
catalyze the synthesis of the substrate phenylpyruvate into 2-PE. We then performed the final product expression
experiments, and compared the samples with the standard 2-PE. The following results were obtained, identifying our
samples as 2-PE.

The results of the experiment were as follows:

(1)Gas chromatogram of Standard 2-PE sample

![](https://static.igem.wiki/teams/4427/wiki/result-30.png)

(2)Gas chromatogram of the bacterial extract sample

![](https://static.igem.wiki/teams/4427/wiki/result-31.png)

2-PE cleavage mechanisms in electron bxombardment ion sources:

![](https://static.igem.wiki/teams/4427/wiki/result-32.png)

(3) Mass spectra of standard 2-PE samples

![](https://static.igem.wiki/teams/4427/wiki/result-33.png)

(4) Mass spectra of extracted 2-PE samples in bacterial broth

![](https://static.igem.wiki/teams/4427/wiki/result-34.png)

The results of gas chromatography and mass spectrometry showed that the bacteriophage extract samples contained
2-PE, indicating that the overall blue light expression system was successful. We also used liquid chromatography to
roughly determine our 2-PE yields as follows:

![](https://static.igem.wiki/teams/4427/wiki/result-table-1.png)

### 3.3 Separate individual validation of KdcA and Adh1

To describe our process in more detail, we constructed two plasmids, pET-22b-Adh1 ( BBa\_K4427007) and pET-30a-KdcA
([BBa\_K4427008](http://parts.igem.org/Part%3ABBa_K4427008)), for two key enzymes in phenylethanol
synthesis, ethanol dehydrogenase (Adh1) and α-keto acid
decarboxylase (KdcA). And validated them individually as follows.

##### 3.3.1 Nucleic acid electrophoresis, double digestion assay

To ensure that the Adh1 gene ( [BBa\_K4427004](http://parts.igem.org/Part%3ABBa_K4427004)) and KdcA gene ([KdcA BBa\_K4427005](http://parts.igem.org/Part%3ABBa_K4427005)) were inserted in the correct position and
to verify that the plasmids were correctly transformed, we performed plasmid extraction, double digestion (XhoI and
NdeI) and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pET-22b-Adh1
( [BBa\_K4427007](http://parts.igem.org/Part%3ABBa_K4427007)) and pET-30a-KdcA ([BBa\_K4427008](http://parts.igem.org/Part%3ABBa_K4427008)). The results showed that our plasmid was
successfully transformed
into DH5α successfully and our target gene was inserted into the target site correctly

![](https://static.igem.wiki/teams/4427/wiki/result-17.jpg)

Figure 17. The map of
pET-22b-Adh1 plasmid.

![](https://static.igem.wiki/teams/4427/wiki/result-18.png)

Figure 18. The map of
pET-30a-KdcA plasmid.

![](https://static.igem.wiki/teams/4427/wiki/result-19.png)

Figure 19. The result of
agarose gel electrophoresis after double digestion. Lane 1: pET-22b-Adh1 (ADH1) plasmid. Lane 2: DNA
segments after digestion. Lane 3: pET-30a-KdcA (KDCA) plasmid. Lane 4: DNA segments after digestion.

##### 3.3.2 SDS-PAGE of Adh1 and KdcA

(1)Adh1:

To initially determine whether the engineered bacteria expressed Adh1, we respectively performed SDS-PAGE of the
proteins in BL21 before and after IPTG induction, and the supernatant and precipitate after induction of the crude
extract. The results (Fig. 20)showed that the target protein Adh1 was present in all four samples at the same time,
although initially most of the protein was expressed in the inclusion bodies, but after subsequent adjustment of the
induction expression conditions,we obtained the desired protein in the supernatant finally.

![](https://static.igem.wiki/teams/4427/wiki/result-20.png)

Figure 20. SDS-PAGE of Adh1.

(2) KdcA:

To initially determine whether the engineered bacteria expressed KdcA, we respectively performed SDS-PAGE of the
proteins in BL21 before and after IPTG induction, and the supernatant and precipitate after induction of the crude
extract. The results (Fig. 21) showed that the target protein KdcA was present in all four samples at the same time,
and a high level of heteroproteins appeared. After mapping the conditions of Adh1 expression, we succeeded in
obtaining a large amount of the desired target protein in the supernatant in the expression of KdcA.

![](https://static.igem.wiki/teams/4427/wiki/result-21.png)

Figure 21. SDS-PAGE of KdcA.

##### 3.3.3 Enzyme activity assay

Our aim was to ensure the enzymes synthesised by the engineered bacteria would complete the catalysis of the
substrate and ultimately synthesise the odour substance we wanted. Therefore, we performed in vitro enzyme activity
assays for the two key enzymes involved in the reaction, Adh1 and KdcA, respectively. However, considering the
toxicity of phenylacetaldehyde, we decided to switch the substrates for the in vitro experiments to safer pyruvate
and acetaldehyde for safety reasons.

(1) NADH absorption spectroscopy of Adh1

Adh1 from Saccharomyces cerevisiae reduces aldehyde groups to hydroxyl groups and converts NADH to NAD+ in order to
maintain glycolysis. Both NADH and NAD+ have UV absorption peaks at 260 nm, but only NADH has a UV absorption peak
at 340 nm. We used this property to check the absorbance of the reaction system at 340 nm to indirectly verify
whether the reaction is taking place.

We used acetaldehyde and NADH as reaction substrates and Tris-HCl at pH = 7 as a buffer. The absorbance of the
solution was measured using a UV spectrophotometer at reaction times of 0, 3, 6, 9, 12, 18 min to verify the
catalytic effect of Adh1 on this reaction. The results (Fig. 22) showed that the absorbance of the system decreased
with increasing reaction time when irradiated with UV light at a wavelength of 340nm, which proved that our enzyme
was active.

![](https://static.igem.wiki/teams/4427/wiki/result-23.png)

Figure 22. NADH absorption spectroscopy of Adh1.

(2) Enzyme concentration-dependent enzymatic activity assay for Adh1

After spectroscopic determination, we continued with the enzyme concentration-dependent enzymatic activity assay of
Adh1 under 340 nm UV light. We carried out five sets of Adh1 gradient experiments at 3, 4, 4.5, 5 and 6 μL
respectively. The experimental results showed that the enzyme solution volume at 3-6 μL showed an accelerated rate
of substrate reduction with increasing enzyme concentration and a higher reaction rate.

![](https://static.igem.wiki/teams/4427/wiki/result-24.png)

Figure 23. Enzyme concentration-dependent enzymatic activity assay for Adh1.

(3) Absorption spectroscopic determination of NADH in the KdcA cascade reaction

KdcA from Saccharomyces cerevisiae catalyzes the conversion of alpha-keto acids to aldehydes, and the resulting
aldehydes can participate in subsequent reactions catalyzed by Adh1. Therefore, we can verify the effectiveness of
KdcA by showing that the entire cascade reaction is proceeding normally by the reaction of Adh1 with known enzyme
activity.

![](https://static.igem.wiki/teams/4427/wiki/result-25.jpg)

We used pyruvic acid (simplest α-keto acid) as the reaction substrate, Tris-HCl at pH = 7 as a buffer. KdcA can
catalyse the reaction of pyruvic acid to form acetaldehyde, and then we used Adh1 to catalyse the reaction of
acetaldehyde to ethanol to determine the activity of KdcA. We used a UV spectrophotometer to measure the absorbance
of the solution at reaction times of 1, 2, 3, 4 and 5 min. The results showed that NADH had a high absorbance when
irradiated with UV light at a wavelength of 340nm and its content decreased with increasing reaction time, proving
that our enzyme was active.

![](https://static.igem.wiki/teams/4427/wiki/result-26.png)

Figure 24. Absorption spectra of NADH in the KdcA cascade reaction.

(4) Enzyme concentration-dependent enzymatic activity assay of KdcA

After spectroscopic measurements, we continued with the enzyme concentration-dependent enzymatic activity assay of
KdcA under UV light at 340 nm. We added 10, 15, 20, 30, 35 and 40 μL of KdcA to the substrate and performed six sets
of control experiments. The results showed that the rate of substrate reduction became increasingly faster with
increasing enzyme concentration at enzyme solution volumes of 10-30 μL, the higher the enzyme activity. Anomalously,
the reaction rate at 35 μL of enzyme solution slowed down compared to 30 μL, which was outside the confidence
interval for the overall data trend, so we decided to discard this set of unreasonable data, which was tested by the
Grubbs method as a suspect value that should be discarded.

![](https://static.igem.wiki/teams/4427/wiki/result-27.png)

Figure 25. Enzyme concentration-dependent enzymatic activity assay of KdcA.

##### 3.3.4 Cascade catalytic activity of bacterial fluids.

We measured the activity of KdcA and Adh1 using pyruvate as the reaction substrate and a citric acid/sodium
hydroxide solution at pH=6 as a buffer to verify the functionality of the pSB1C3-stuffer-LP plasmid ([BBa\_K4427002](http://parts.igem.org/Part%3ABBa_K4427002))
by using a cascade reaction in which KdcA catalyzes the reaction of pyruvate to produce acetaldehyde and Adh1 then
acetaldehyde catalyzes ethanol. We added 10, 15, 20, 30, 35 and 40 μL of the dual enzyme enzyme solution to the
substrate and performed six sets of control experiments. The results (Fig. 25) showed the rate of substrate
reduction was increasingly rapid with increasing enzyme concentration at 10-40 μl of enzyme solution. This is
consistent with the results of the cascade reaction between KdcA and Adh1, which suggests that our expression system
is effective.

![](https://static.igem.wiki/teams/4427/wiki/result-28.png)

Figure 26. Cascade catalytic activity of bacterial fluids.

# 4. The Suicide system

The suicide system consists of an initiation system and a virulence protein (blrA). The switch system consists of a
red light-responding switch and a blue light-responding switch in parallel, and it is the simultaneous irradiation
of red and blue light that initiates the expression of downstream virulence proteins. Due to time issues, the red
light-responding switch, the blue light-responding switch and the toxin function of the suicidel system were
verified separately.

### 4.1 Light switch for the suicide system

The initiation switch of the suicide system consists of a red light-responding switch and a blue light-responding
switch in parallel, and it is the simultaneous irradiation of red and blue light that initiates the expression of
downstream toxic proteins. Due to time issues, we only verified the blue light-responding switch and the red
light-responding switch separately as described above, and the results showed that both switches were effective.

### 4.2 Functional validation of toxic proteins

We conducted a collaborative experiment with HainanU\_China to validate the virulence proteins. They added 1 μL
virulence gene plasmid to 50 μL competent cell, coated the plates after transformation according to the
transformation process, added 50 μL solution to each plate and incubated them overnight at 37°C. The final result is
shown here. The plate coated with the bacteria that have transformed the blrA has no colony, while the control group
grows normally.

![](https://static.igem.wiki/teams/4427/wiki/result-29.png)

Figure 27. Toxic protein validation assay. A1 engineered bacteria with introduced toxin protein pellet; A2
engineered bacteria with introduced plasmid but no toxin gene.

{% endblock %}