iGEM 2022 — Team njmu-china

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{% block title %}Notebook{% endblock %}
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{% endblock %}
{% block name %}Notebook{% endblock %}
{% block overview %}

On this page, we record our project progress briefly. Notably, this web page is a summary at the end of
iGEM. During the iGEM, we actually used an online team calendar to schedule and record our team's
regular meetings and big events. In addition, detailed records of our daily wet lab outcomes can be
found on the
[Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records) page.

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{% block page\_content %}

Nootbook

March
April
May
June
July
August
September
October

March

Week 1
Week 2
Week 3
Week 4

April

Week 5
Week 6
Week 7
Week 8

May

Week 9
Week 10
Week 11
Week 12
Week 13

June

Week 14
Week 15
Week 16
Week 17

July

Week 18
Week 19
Week 20
Week 21

August

Week 22
Week 23
Week 24
Week 25
Week 26

September

Week 27
Week 28
Week 29
Week 30

October

Week 31
Week 32

## Week of March 06th to 12th

### Project

On March 6, the members of the 2022 NJMU-China
finally assembled and began this trip full of opportunities and challengeschallenges. It
is the third year that NJMU-China has participated in iGEM, and we aim for better.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-01-1.jpg)

## Week of March 13th to 19th

### Project

Brainstorming. Generally, we plan to design engineering bacterias for autism treatment this year.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-02-1.jpg)

### Human Practices

We revisited Star of the Sea, the institution for autistic children, and got our inspiration from our conversation with parents of autistic children there.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-02-2.jpg)

## Week of March 20th to 26th

### Project

Brainstorming. Through literature research, we noticed the intestinal disorders, heavy metal poisoning symptoms, and mitochondrial dysfunction in autistic children.

### Human Practices

Overall planning. We held online meetings and formed a plan for future HP activities.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-03-1-jpg.jpg)

## Week of March 27th to Week of April 02nd

### Project

Brainstorming. We found a study in 2021 that allowed us to integrate what we had previously discovered and we hope to design based on this. Read the inspiration part on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page for more information.

### Human Practices

We mainly focused on education and partnership this week and attended a seminar on mitochondria as well. Since World Autism Awareness Day took place this week, we also organized a series of activities related to this special day.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-04-1.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-04-2.jpg)

## Week of Arpil 03rd to 09th

### Project

Brainstorming. We planned to design three engineering bacteria for autistic children: One for the treatment of mitochondrial dysfunction, one for the prevention of heavy metal poisoning, and one for the detection of mitochondrial dysfunction.

### Human Practices

We handed out a questionnaire on the mental health of parents with autistic children.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-05-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-05-2.jpg)

## Week of April 10th to 16th

### Project

Engineering Bacteria a: Consider lactic acid as a response substance; Consider carnitine, coenzyme Q, and Vbs as the target products of a cocktail therapy.

Engineering Bacteria b: Consider the MerR family as a sensor; Consider metallothionein as the target product.

Engineering Bacteria c: Look for urinary biomarkers of mitochondrial dysfunction, and look for how to develop color reactions and keep the flora stable on the test strip.

### Human Practices

We mainly prepared for the online educational program on ASD for schoolchildren this week and carried out the program on the weekend and it continued into the next week.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-06-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-06-2.jpg)

## Week of April 17th to 23th

### Project

By reviewing the literature and the previous work of gut-related teams in iGEM, we found that the environment of the digestive tract is so complex that a drug delivery system is essential. Hence, we expand our project to four systems:

**System 1:**aim to improve mitochondrial function

**System 2:**aim to prevent the possible accumulation of heavy metals

**System 3:**aim to build a drug delivery system

**System 4:**aim to build a test strip to diagnose mitochondrial dysfunction in autistic children.

### Human Practices

We continued with the educational program this week.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-07-1.jpg)

## Week of April 24th to 30th

### Project

**System 1:**The concentration of lactic acid is affected by the secretion of lactic acid by lactic acid-secreting bacteria in the intestinal tract, another sensor for mitochondrial dysfunction is needed; Consider a sensor that senses NAD+ reduction; NAD + may be used as the drug.

**System 2:**Found the sequence of human or animal metallothionein; Considered using human metallothionein due to biosafety; Consider fusing metallothionein with Lpp-OmpA.

**System 3:**Consider encapsulating engineering bacteria with sodium alginate.

**System 4:** Look for the lactic acid sensor.

### Human Practices

Since we have come up with an initial idea for our project, we held an online meetup with iGEM NAU-China and discussed our design.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-08-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-08-2.jpg)

## Week of May 01st to 07th

### Project

**System 1:**Looks for NAD + or its precursor's synthetic pathway.

**System 2:**Look for the sequence of the MerR family.

**System 3:**Consider microencapsulation.

**System 4:**Look for the lactic acid sensor.

### Human Practices

We interviewed Professor Yichao Wang on the feasibility of our project.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-9-1-jpg.jpg)

## Week of May 08th to 14th

### Project

Genetic circuit construction and mathematical simulation learning. More information can be found on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-10-1.jpg)

### Human Practices

We gave an educational lecture on ASD and mitochondrial dysfunction for university students.

## Week of May 15th to 21th

### Project

Genetic circuit construction and mathematical simulation learning. More information can be found on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

### Human Practices

We organized another meetup with iGEM WHU-China and updated our project designs.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-11-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-11-2.jpg)

## Week of May 22th to 28th

### Project

Genetic circuit construction and mathematical simulation learning. More information can be found on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

### Human Practices

We uploaded educational videos about ASD and synthetic biology on Bilibili and wrote educational articles as well.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-12-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-12-2.jpg)

## Week of May 29th to 04th

### Project

Genetic circuit construction and mathematical simulation. More information can be found in the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) and [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

### Human Practices

We mainly focused on the design of our team merch and prepared for the art exhibition on gut microbiota. Additionally, we attended the NIA meetup online on June 4th.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-13-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-13-2.png)

## Week of June 05th to 11th

### Project

We held a lab sefety lecture.

### Human Practices

We held an art exhibition on gut health and gut microbiota in two buildings at our university.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-14-1.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-14-2.jpg)

## Week of June 12th to 18th

This period is the exam month of our university, and the iGEM work has been paused until July.

## Week of June 19th to 25th

This period is the exam month of our university, and the iGEM work has been paused until July.

## Week of June 26th to 02nd

This period is the exam month of our university, and the iGEM work has been paused until July.

## Week of July 03rd to 09th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Human Practices

We interviewed Professor Zhinuan Su and had a better understanding of the feasibility of the project.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-18-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-18-2.jpg)

## Week of July 10th to 16th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

We did Pathway Enrichment Analysis and Further
explained the intimate connection between autism and energy metabolism. More information can be
found on the [Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) page.

### Human Practices

We mainly designed and handed out a questionnaire on the acceptance of synthetic biological products among parents of autistic children.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-19-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-19-2.png)

## Week of July 17th to 23th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Human Practices

We made a brochure on synthetic biology and designed a set of puzzles that educate children on microorganisms.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-20-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-20-2.jpg)

## Week of July 24th to 30th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Human Practices

We revisited Star of the Sea and did voluntary work there.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-21-1-jpg.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-21-2.jpg)

## Week of July 31th to 06th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Game development. More information can be found on the [Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D) page.

### Human Practices

We continued with our community service at Star of the Sea and held an online interview with a family member of an autistic adult.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-22-1.jpeg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-22-2.jpg)

## Week of August 07th to 13th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Hardware design. More information can be found on the [Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) page.

### Human Practices

We continued with our community service at Star of the Sea.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-23-1.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-23-2.png)

## Week of August 14th to 20th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Hardware design. More information can be found on the [Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-24-1.jpg)

## Week of August 21th to 27th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Hardware 3D print. More information can be found on the [Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) page.

### Human Practices

We interviewed Professor Xiaoyan Ke for the further demonstration of our project.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-25-1.jpg)

## Week of August 28th to 03th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Human Practices

We organized a summer program on synthetic biology for both schoolchildren and high school students, attended several meetups with our fellow iGEMers, and educated the public on the application of synthetic biology.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-26-1.jpg)

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-26-2.jpg)

## Week of September 04th to 10th

### Wet Lab

Plasmid transformation and functional verification. More information
can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Human Practices

We interviewed Xbiome and wrote the initial version of our business plan.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-27-1.jpg)

## Week of September 11th to 17th

### Wet Lab

Functional verification and data collection. More information can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Data analysis and model improvement. More information can be found on the [Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

### Human Practices

We had a discussion on biosafety with team NFLS\_Nanjing.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-28-1.png)

## Week of September 18th to 24th

### Wet Lab

Functional verification and data collection. More information can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Data analysis and model improvement. More information can be found on the [Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

### Human Practices

We discussed our entrepreneurship approaches with iGEM WHU-China online.

## Week of September 25th to October 1st

### Wet Lab

Functional verification and data collection. More information can be found in the [experiment record](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#experiments-records).

### Dry Lab

Data analysis and model improvement. More information can be found on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/notebook/week-30-1.jpg)

## Week of October 2nd to 8th

Final improvement and wiki documentation.

## Week of October 9th to 15h

Final improvement and wiki documentation.

{% endblock %}

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{% block title %}Engineering Success{% endblock %}
{% block name %}Engineering{% endblock %}
{% block style %}

{% endblock %}
{% block overview %}

This page shows the engineering iterations of our four systems and proves our engineering success. For DESIGN,
through a process of questioning, we developed our design progressively. For BUILD, AGE and SDS-PAGE were used to
verify the successful construction and transformation of our plasmids. For TEST, we performed different experiments
for each system and successfully verify their function. For LEARN, we provided conclusions of our successes, failures,
and future plans for each system after iGEM.

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

System 1

· Design

· Build

· Test

· Learn

System 2

· Design

· Build

· Test

· Learn

System 3

· Design

· Build

· Test

· Learn

System 4

· Design

· Build

· Test

· Learn

System 1

Design

### Wet Lab Design

|  |
| --- |
| What substance will we choose to enhance mitochondrial function? Why? |
| What role does it play in enhancing mitochondrial function? |
| Which genes will we select to directly or indirectly produce the substance? Why? How does it work? |
| Which strategy will we choose to assure security? Why? |
| What chassis bacteria will we use? Why? |

To answer the above question, we did literature reviews and brainstorming sessions to complete the design of
system one. You can find more information on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/system-1-map.png)
The map of system 1

Build

### Wet Lab Build

We successfully constructed the LDHLH673-PncA plasmid and verified it by agarose gel electrophoresis. You can find
more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

Test

### Wet Lab Test

You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

In our pre-experiments, we knocked out the PncA gene in
Lactobacillus Plantarum L168 and found it would produce fewer NAD+, which provides indirect evidence to the
test stage. You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

### In Silico Test

For the purpose of understanding the absorption and distribution of NAD+ in the human body, we developed a 3D
visualization and two videos. The organ model files are from [bodyparts 3D](http://lifesciencedb.jp/bp3d/?lng=en). The model was created using Blender, Python
scripts, and Meshlab, and
the video was rendered using Blender.

In the video, the transparent pink contour represents the human body, the red and pink organs represent the
liver and intestine, the fluorescent green spheres represent NAD+, and the blue blood vessels represent veins.

Video 1 provides an overview of our 3D visualization, in which we
can observe the distribution of NAD+ in the small intestine, liver, mesenteric vein, and hepatic portal vein.

Video 2 is the focused view of our three-dimensional
visualization, in which we can observe NAD+ being absorbed in the small intestine, entering the mesenteric
vein, following the blood into the hepatic portal vein, and then entering the liver.

This 3D representation allows us to intuitively comprehend the
distribution and absorption of NAD+ in the body. Originally, it was part of our pharmacokinetic model,
however, without sufficient experimental data or literature support, it was not able to solve and develop this
model. But we still kept the video and hope to complete it after iGEM.

Learn

### Successes

1. Construct the pLDHLH673-PncA plasmid

### Failures

1. Amplify the pMG36e and pLDHLMCS plasmid
2. Transform the pLDHLH673-PncA into Lactobacillus Plantarum L168

Possible reasons: First, the plasmid extraction kit we used was
ineffective. In the lysing bacteria step, the color of the solution should have become clear after adding the
reagent, but it remained unchanged. Second, the chassis bacteria we choose, Lactobacillus Plantarum, are
gram-positive bacteria that have different physiological characteristics from the gram-negative bacteria
commonly used in plasmid extraction.

### Future Plans

1. Verify the function of PncA directly
2. Verify the function of the Nisin system.

System 2

Design

### Wet Lab Design

|  |
| --- |
| What substance will we choose to prevent the accumulation of heavy metals in children with autism? Why? |
| How does it work and what role does it play in preventing the accumulation of heavy metals in children with autism? |
| What genes will we select to synthesize the substance? Need some improvement? |
| Which strategy will we choose to assure security? Why? |
| What chassis bacteria will we use? Why? |

To answer the above question, we did literature reviews and
brainstorming sessions to complete the design of
system two. You can find more information on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/system-2-map.png)
The map of system 2

Build

### Wet Lab Build

At the DNA level, we performed enzyme digestion/plasmid PCR and
ran agarose gel to prove the successful construction or transformation of the plasmid.

At the protein level, we use the heavy metal solution of different
concentrations to induce the protein expression and ran SDS-PAGE gal to prove the successful expression of our
target gene.

You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

### In Silico Build

We established an ordinary differential (ODE) model to simulate
the induction-expression process in this system. You can find more information on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

Test

### Wet Lab Test

We drew the bacterial growth curve. We and verified their heavy
metal elimination function and as well as kill switch function.

### In Silico Test

Combined with experimental data, the induction-expression process
in this system was visualized. You can find more information on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Model%27%29%20%7D%7D) page.

Learn

### Successes

1. Transform the pET28a-MT plasmid into Escherichia coli Nissle 1917
2. Verify the protein expression
3. Draw the growth curve
4. Verified the heavy metal elimination function
5. Verify the kill switch function

### Failures

1. Determine whether the target protein is anchored to the bacterial outer membrane
2. Determine The elimination capacity of our engineering bacteria to other heavy metal ions

### Future plans

In the future, we hope to complete experiments on the sense and
elimination capacity of engineered bacteria to different heavy metal ions (lead, mercury, copper, zinc,
lithium), we hope to perform immunofluorescence staining that can determine the localization of the target
protein in the engineered bacteria.

System 3

Design

### Wet Lab Design

|  |
| --- |
| Why do we consider drug delivery? |
| What drug delivery methods are available and what are they? |
| What is the definition of microencapsulation and what is its function? |
| How can we achieve microencapsulation? |
| How can we validate the microencapsulation results? |

To answer the above question, we did literature reviews and
brainstorming sessions to complete the design of
system three. You can find more information on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

Build

#### Web Lab Build

To confirm the establishment of our microencapsulation, electron microscopic images were taken. By counting
the number of bacteria before and after microencapsulation, we also measured the microencapsulation yield. You
can find more information on th [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/part1.png)
Illustration of microencapsulation

Test

#### Wet Lab Test

We let our engineered bacteria with or without microencapsulation pass the simulating gastrointestinal fluid
and then evaluate their survival rates to test if microencapsulation could help engineered bacteria survive
the harsh environment of the human digestive tract. You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/part2.png)
![](https://static.igem.wiki/teams/4205/wiki/wet-lab/part3.jpg)
Illustration of microencapsulation verification

Learn

During the process of adding the suspension to the calcium
chloride solution using a sterile nozzle syringe to form bacterial microcapsules, it was found that
controlling the syringe at a certain height and injecting at a uniform speed resulted in more homogeneous
microcapsules. No attention was paid to controlling the height and speed of injection during the first
experiment and this was improved during the second experiment.

### Successes

We have successfully produced microencapsulated engineered bacteria and confirmed its activity with functional validation.

### Failures

We tried using yogurt as a vehicle for drug delivery, but the smear plate results showed that the Lactobacillus plantarum colony count was much higher than expected. This may have been caused by the bacteria continuing to proliferate during multiple failed vacuum drying attempts.

### Future plans

In the future, we hope to complete the functional validation of microencapsulated engineered bacteria with adsorbed heavy metal ions.

System 4

Design

#### Wet Lab Design

|  |
| --- |
| What substances do we use as markers of mitochondrial dysfunction? Why? |
| What system do we choose to sense lactic acid? Why? How does it sense lactic acid? |
| What substances do we choose to report? Why? |
| Which strategy will we choose to assure security? Why? |
| What chassis bacteria will we use? Why? |

To answer the above question, we did literature reviews and
brainstorming sessions to complete the design of
system four. You can find more information on the [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/system-4-map.png)
The map of system 4

Build

#### Wet Lab Build

At the DNA level, we performed enzyme colony/plasmid PCR and ran
agarose gel to prove the successful construction or transformation of the plasmid.

At the protein level, we use the lactate of different
concentrations to induce the protein expression and ran SDS-PAGE gal to prove the successful expression of our
target gene.

You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

### In Silico Build

We established an ordinary differential (ODE) model to simulate
the induction-expression process in this system. You can find more information on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

Test

### Wet Lab Test

We verified the lactate test function

You can find more information on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

### In Silico Test

Combined with experimental data, the induction-expression process
in this system was visualized. You can find more information on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Model%27%29%20%7D%7D) page.

Learn

### Successes

1. Transform ALPaGA-lacZ into Escherichia coli DH5α-T1
2. Verify that the engineered bacteria could produce more Beta-galactosidase under the increasing of lactate
   concentration.
3. Prediction curve of lactate-protein expression
4. On the test paper, the engineered bacteria produced color reaction with x-gal.

### Failures

According to the conclusion that engineering bacteria can produce
more Beta-galactosidase by a high concentration of lactic acid, we propose to distinguish different
concentrations of lactate according to the degree of color reaction, so as to diagnose autism, however, only a
little bit of difference was found in our functional test, which may be related to the material
of filter paper, the lower activity of bacteria after fixing on the testing strip, and the unsuitable reaction
environment.

### Future plans

Further improve the sensitivity of the functional
verification experiment.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block name %}Experiments{% endblock %}
{% block style %}

{% endblock %}
{% block overview %}

This page includes the experiment protocols and records.

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

Experiment protocols

· Microbiological Experiment

· Molecular Experiment

· Function Verification

Experiment records

Experiment protocols

Microbiological Experiment

### Medium

##### LB Medium

* Yeast Extract 5 g / L
* Sodium Chloride 10 g /L
* Tryptone 10 g / L
* Agar 20g/L(solid)

▲ Dissolve in demineralized water, stir and autoclave.

##### MRS Medium

* Peptone 10 g / L
* beef extract 10 g / L
* yeast extract 5 g / L
* glucose 5 g / L
* sodium acetate trihydrate 5 g / L
* polysorbate 80 (also known as Tween 80) 1 g / L
* dipotassium hydrogen phosphate 2 g / L
* triammonium citrate 2 g / L
* magnesium sulfate heptahydrate 0.2 g / L
* manganese sulfate tetrahydrate 0.05 g / L
* agar 15g / L
* calcium carbonate 20g / L

▲ Dissolve in demineralized water, stir and autoclave.

### Antibiotics

##### Kanamyicin

Working concentration: 100 μg/ml (E. coli), storage concentration: 100 mg/ml, pro-tected from
light, stored at -20°C. Sterilize with filter membrane before use.

##### Erythromycin

working concentration: 300 μg/ml, storage concentration: 50 mg/ml erythromy-cin-ethanol solution,
protected from light, stored at -20°C. Sterilize with a filter membrane before use.

### Bacteria Culture

##### Escherichia coli culture

Solid: the preserved strains/cultured strains were coated/strewed onto the pre-prepared LB solid
medium and placed in a 37℃ constant temperature incubator for aerobic static culture for 18h.

Liquid: Inoculate the preserved strain/culture liquid into a shaker tube containing 5 ml
pre-prepared LB liquid medium, placed on a constant temperature shaker, and aerobically
cultured at 185rpm at 37℃ for 18h.

##### Lactobacillus plantarum culture

Solid: the preserved strains/cultured strains were coated/strewed onto the pre-prepared MRS solid
medium and placed in a 37℃ constant temperature incubator for aerobic static culture for 12h.

Liquid: Inoculate the preserved strain/culture liquid into a shaker tube containing 5 ml
pre-prepared MRS liquid medium, placed on a constant temperature shaker, and aerobically
cultured at 185rpm at 37℃ for 12h.

### Bacteria Inoculation

Liquid to Liquid: Pipette 50μL of culture medium into a shaking tube containing 5 ml of culture
medium (diluted at a ratio of 1:100), rinse the tip of the pipette, and discard it.

### Bacteria Preservation

Liquid to Solid: 80% glycerol solution and cultured bacterial liquid are added to the same
preservation tube at a volume of 1:4, pipetted evenly, and stored in a refrigerator at -80℃.

### Bacteria Recovery

Solid to Liquid: Use a pipette tip to pick a single colony into a shaking tube containing 5 ml of
culture medium, rinse the pipette tip, and discard it.

### Chemical Transformation of Escherichia Coli DH5A

##### Preparation of competent cells

1. The bacteria to be transformed are cultured in advance to the plateau or late
   loga-rithmic growth stage. Then the bacterial suspension is inoculated in a 5 ml LB
   liquid medi-um at a ratio of 1:100-1:50 and cultured with shaking at 37°C 3 hours to
   OD600 = 0.5-0.7.
2. Transfer 1 ml of culture medium into a 1.5 ml centrifuge tube, place it on ice for 5
   minutes and then centrifuge at 4°C and 8000 rpm for 2 minutes.
3. Discard the supernatant, gently suspend the cells in 200 μL of pre-chilled 0.05 mol/L
   CaCl2 solution, and then place on ice for 10 min.
4. Centrifuge for 2 minutes at 4°C and 8000 rpm. (If there is no need to preserve the
   seeds, this step can be omitted. )
5. Discard the supernatant, add 200 μL of pre-cooled 0.05 mol/L CaCl2 solution con-taining
   15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a
   competent cell suspension. (If there is no need to preserve the seeds, this step can
   be omitted) Competent cells can be used for a transformation immediately or stored at
   -80°C for half a year.

##### Transformation

1. Take 200μL of competent cell suspension from -80℃ refrigerator and thaw on ice. (If
   there is no need to preserve the seeds, this step can be omitted.)
2. Add plasmid DNA solution (the volume should not exceed 5 μL, depending on the
   concentration), shake gently, and place on ice for 20 min.
3. Heat in a 42℃ water bath for 60 seconds, and quickly place on ice to cool for 3 minutes
   after heat shock.
4. Add 800 μL of LB liquid medium (without antibiotics) to the tube, place it in a shak-er
   at 37°C, and incubate for 10-30 minutes.
5. Centrifuge at 3000 rpm for 2 minutes, discard part of the supernatant (100-200 μL of
   the supernatant is best), gently pipette the cells to resuspend them in the
   supernatant solu-tion.
6. Spread the above-mentioned bacterial liquid on a screening plate containing
   antibi-otics (place it face up for half an hour, and invert the culture dish after
   the medium absorbs the bacterial liquid completely ). Then incubate at 37°C for 16-24
   hours.

### Electric Transformation of Escherichia Coli Nissle 1917

##### Preparation of competent cells

1. Day 1: Remove the preserved strains from the refrigerator at -80℃, thaw them on ice,
   streak them on the GM17 solid medium, and cultivate them overnight at 30℃.
2. Day 2: Take a single colony from the plate on Day 1, place it in 100 ml G-SGM17 medium,
   and cultivate it anaerobic at 30 ℃ until the OD600 is 0.6-0.8.
3. Aliquot into 50 ml centrifuge tubes, pre-cool on ice for 20-30 minutes, and centrifuge
   at 4°C and 4000 rpm for 10 minutes.
4. Discard the supernatant, add 3 ml of pre-chilled ddH2O, gently blow to suspend the
   bacterial pellet, dispense it in a 1.5 ml centrifuge tube, and centrifuge at 4°C and
   4000 rpm for 10 min.
5. Repeat step (4)
6. Change ddH2O to 10% glycerol and repeat step (4)
7. Repeat step (6)
8. Finally, add 500 μL of 10% glycerol to each centrifuge tube, which can be used for a
   transformation immediately or stored at -80°C.

##### Electric Transformation

1. Set the electro-transfer parameters as follows: voltage 2000 V, capacitance 25 μF,
   re-sistance 200 Ω.
2. After electroporation, immediately add 1 ml of resuscitation medium SGM17MC, mix well
   and place on ice for 10 minutes.
3. Transfer the solution from the electroshock cup to a 1.5 ml centrifuge tube and
   incu-bate at 30°C for 2 h anaerobic.
4. Centrifuge at 4000 rpm for 10 minutes, discard part of the supernatant，and spread the
   plate. Place it on the front side for half an hour.
5. After the bacterial solution is entirely absorbed by the culture medium, place the
   petri dish upside down. Anaerobic culture at 30°C for 2-3 days.

### Electric Transformation of Lactobacillus Plantarum L168

##### Preparation of competent cells

1. Day 1: Remove the preserved strains from the refrigerator at -80℃, thaw them on ice,
   streak them on the GM17 solid medium, and cultivate them overnight at 30℃.
2. Day 2: Take a single colony from the plate on Day 1, place it in 100 ml G-SGM17 medium,
   and cultivate it anaerobic at 30 ℃ until the OD600 is 0.6-0.8.
3. Aliquot into 50 ml centrifuge tubes, pre-cool on ice for 20-30 minutes, and centrifuge
   at 4°C and 4000 rpm for 10 minutes.
4. Discard the supernatant, add 3 ml of pre-chilled ddH2O, gently blow to suspend the
   bacterial pellet, dispense it in a 1.5 ml centrifuge tube, and centrifuge at 4°C and
   4000 rpm for 10 min.
5. Repeat step (4)
6. Change ddH2O to 10% glycerol and repeat step (4)
7. Repeat step (6)
8. Finally, add 500 μL of 10% glycerol to each centrifuge tube, which can be used for a
   transformation immediately or stored at -80°C.

##### Electric Transformation

experimental group: Put 100 μL of competent cells into a pre-cooled 2 mm electric shock cup
containing 10 μL of plasmid DNA solution, and place them on ice for 10 min.

control group: Put 100 μL of competent cells into a pre-cooled 2 mm electric shock cup containing
10 μL ddH2O, and place them on ice for 10 minutes.

1. Set the electro-transfer parameters as follows: voltage 2000 V, capacitance 25 μF,
   re-sistance 200 Ω.
2. After electroporation, immediately add 1 ml of resuscitation medium SGM17MC, mix well
   and place on ice for 10 minutes.
3. Transfer the solution from the electroshock cup to a 1.5 ml centrifuge tube and
   incu-bate at 30°C for 2 h anaerobic.
4. Centrifuge at 4000 rpm for 10 minutes, discard part of the supernatant，and spread the
   plate. Place it on the front side for half an hour.
5. After the bacterial solution is entirely absorbed by the culture medium, place the
   petri dish upside down. Anaerobic culture at 30°C for 2-3 days.

Molecular Experiment

### PCR

System (20μL in total):

|  |  |
| --- | --- |
| r Taq Buffer（10x | 2 μL |
| dNTPs | 1.6μl |
| Pre primer | 0.5μL |
| Back primer | 0.5μl |
| rTap | 0.5μl |
| Template sequence | 1.0μl |
| ddH2O | 14.8μl |

Reaction procedure:

|  |  |
| --- | --- |
| 94℃ | 5min |
| 90℃ | 30 s |
| 55℃ | 30 s |
| 72℃ | x min |
| 72℃ | 8 min |
| 16℃ | ∞ min |

Step2 to Step4 30cycles

### Plasmid Amplification

##### Materials:

* Sterile pipet tips.
* Sterile 1.5 ml Eppendorf tubes.
* Plasmids to amplify.
* DH5alpha, transformation competent. Stored at -80oC. Thaw rapidly immediately
  before use and keep on ice.
* Agar plates, with appropriate antibiotics. Leave on room temperature bench for
  1-2days to get a semi-dry surface. Protect from light.
* LB medium
* SOC medium

##### Procedure

1. Mix 0.5 - 1 ul plasmids with 0.1 ml competent E. coli in a sterile Eppendorf tube
   on ice. Mix gently by tapping. Also prepare a no DNA control tube.
2. Incubate on ice for 30 min.
3. Incubate at 42oC for exactly 45 seconds. Use floater in a circulating water bath.
4. Incubate on ice for 2 minutes.
5. Add 900 ul SOC medium, mix by gentle tumbling (optional, for high efficiency
   transformation to increase the number of transformants).
6. Incubate for 30-60 min at 37oC.
7. Pipet 10 - 300 ul onto an agar plate. For larger volumes, break them into multiple
   small puddles. Tilt the plate around to make even coverage.
8. Smear the E. coli gently with sterile glass rod (ethanol soaked and flamed) or
   pipets across the agar surface. Hold the lid over the surface with one hand and
   rotate the plate to smear in multiple directions.
9. Incubate the plate overnight at 37oC, place upside down to avoid condensation
   prob-lem.
10. Add 3 ml of LB with antibiotics to a 15 ml conical tube.
11. Select a well-formed, smooth-edged, isolated colony on the agar plate. Do not use
    colonies with tiny dots around (these may be non-transformed E. coli growing in
    regions of depleted antibiotics).
12. Pick E. coli from the colony with a sterile toothpick. Dip the tip into the LB
    medi-um.
13. Place on a shaking incubator at 37o C, 200-250 rpm for 12 hours. The medium should
    turn cloudy. Store the tube overnight at 4o C if necessary.
14. Plasmid extraction

### Plasmid Extraction

Small extraction: See TIANGEN plasmid small extraction kit.

Large-scale extraction based on TIANGEN plasmid large-scale extraction
kit.

1. Take 100 mL (choose the appropriate amount according to the concentration of the
   cultured cells, 200 mL is recommended for a soft copy) overnight cultured
   bacteria liquid into the centrifuge tube, room temperature 8000 rpm (~8228×g)
   centrifugation for 3 minutes to collect the bacteria liquid, try to suck Except
   the supernatant. Note When there is much bacterial liquid, the bacterial pellet
   can be collected in a centrifuge tube by centrifugation. The amount of bacterial
   liquid should be sufficiently lysed. Too much bacterial liquid will lead to
   insufficient lysis and reduce the efficiency of plasmid extraction.
2. Try to absorb the supernatant as much as possible. To ensure that the supernatant
   is completely absorbed, please use clean absorbent paper to absorb the water
   droplets on the bottle wall. 3. Add 8 mL of solution P1 (please check whether
   RNaseA has been added) to the centrifuge tube with bacterial pellet, and use a
   pipette or vortex to lyse the suspended bacterial cell pellet completely.

### Note

Be sure to
suspend the bacterial sediment thoroughly. If some bacteria are clumps that
are not thoroughly mixed, it will affect the lysis effect, resulting in low
extraction vol-ume and purity. For low-copy plasmids, increase the number
of bacteria while increasing the proportion of P1, P2. The amount of P4.

3. Add 8 mL of solution P2 to the centrifuge tube, turn it upside down gently 6-8
   times, and leave it at room temperature for 5 minutes.

### Note

Mix gently, do
not shake vigorously to avoid contamination of genomic DNA. At this time,
the bacterial liquid should become transparent and viscous. If it does not
become apparent, it may be due to too much bacteria and incomplete lysis,
so fewer bacteria should be reduced.

4. Add 8 mL of solution P4 to the centrifuge tube, and immediately turn it upside
   down gently 6-8 times, mix well, until the solution appears white dispersed
   flocculent precipitate. Then leave it at room temperature for about 10 minutes.
   Centrifuge at 8000 rpm (~8228×g) for 5-10 min to allow the white precipitate to
   separate to the bottom of the tube (the centrif-ugation time can be increased
   appropriately), and carefully pour all the solution into the fil-ter CS1 (please
   avoid pouring a large amount of precipitate to block the filter ), slowly push
   the push handle to filter, and collect the filtrate in a clean 50 mL tube
   (provided by yourself).

### Note

Mix the solution
immediately after adding solution P4 to avoid local precipita-tion. If the
solution poured into the filter CS1 after centrifugation has white
precipitation, it would not affect the filtration. If there are too many
bacteria (>100 mL), it is recommended to extend the centrifugation time to
20-30min.

5. Add isopropanol equal to the filtrate volume to the filtrate, mix upside down, and
   centrifuge at 8000 rpm (~8228×g) for 5 min.
6. Pour and discard the supernatant. Take care to prevent the precipitate from being
   poured out. Add 6 mL of 70% ethanol to wash the precipitate. Centrifuge at 8000
   rpm (~8228×g) at room temperature for 5 min. Carefully discard the ethanol.
   Repeat the opera-tion once.
7. Leave the lid open in the air for 5-10 minutes, and dissolve the precipitate with
   an appropriate volume of ddH2O as needed.

### Enzyme Digestion

##### Materials:

* Plasmid DNA: x μL (according to the actual concentration)
* Restriction enzyme 1: 1μL
* Restriction enzyme 2: 1μL
* 10×buffer　: 2μL
* ddH₂O: 20-x μL

##### Protocol:

1. The total system is 20 μL, add samples and mix well, 37℃ water bath for 1h-3h,
   electrophoresis verification.

### Agarose Gel Electrophoresis

##### Materials:

* An electrophoresis chamber and power supply.
* Gel casting trays, which are available in a variety of sizes and composed of
  UV-transparent plastic.
* Sample combs, around which molten agarose is poured to form sample wells in the gel.
* 1X TAE solution

##### Protocol:

1. Dissolve 0.3 g of agarose in 30 mL of 1X TAE solution, heat it until the bubbles stop,
   and the solution becomes transparent, cool slightly and add 3 μL Goldview (10000x)
2. Pour the gel into the gel membrane tool inserted with the comb (the sample amount
   determines the specific size), and wait for it to set.
3. Add 1/2/3 μL 6× loading buffer to the 5/10/15μL sample and mix by pipetting.
4. Put the gel into the electrophoresis tank and add 1X TAE buffer to completely cover the
   gel and squeeze out the bubbles in the gel hole.
5. Add the mixed sample and marker to the gel hole. Specific experiments deter-mine the
   sample loading volume. The marker loading volume is based on the following standards，
   11 wells gel corresponds to 2 μL, 8 wells correspond to 5 μL, and 6 wells corresponds
   to 10 μL.
6. Run electrophoresis under 180V voltage until the colour band of loading buffer is
   electrophoresed to the middle or two-thirds of the gel, stop electrophoresis.
7. Use ultraviolet light to image the gel. If necessary, cut rubber for recycling.

Function Verification

### SDS-Page

##### Materials:

* Gels
* Electrophoresis Chambers
* Protein Samples
* Running Buffer
* Staining and Destaining Buffer
* Protein Ladder

##### Protocol

1. When the gel is ready to be put, add TEMED.
2. The gel used to separate is poured in the casting chamber.
3. You need to put butanol before polymerization to remove the unwanted air.
4. bubbles present.
5. Between the glass plate, the comb is inserted.
6. The “gel cassette” is the polymerized gel.
7. Sample Preparation
8. Boil some water in a beaker.
9. Add 2-mercaptoethanol to the sample buffer.
10. Put the buffer solution in microcentrifuge tubes and add protein samples to it.
11. Take MW markers in separate tubes.
12. Bring the samples to boil for less than 5 minutes to completely denature the proteins.
13. The gel cassette is put off from the casting stand and placed separately in the
    electrode assembly.
14. The clamp stand is fixed to the electrode assembly.
15. a 1X electrophoresis buffer is added to the opening of the casting frame to fill the
    wells of the gel.
16. Fit 30ml of the denatured sample in the well.
17. The tank is covered with a lid and the unit is attached to a power supply.
18. The sample is made to run at 30mA for about 1 hour.
19. UV light is used to observe the light.

### Tips

Avoid overloading gel.

### Test of The Death of Bacteria

##### Materials:

* Engineering bacteria
* LB broth
* Incubator of 37 °C
* LB agar plates containing 50 μg/mL antibiotics of choice
* Shaking incubator of 37 °C
* Three concentrations of Arabinose LB solution: 0umol/L, 1.25umol/L, 2.5umol/L

##### Protocol

1. Distribute EcN 1917-pet28a(control) & EcN 1917-pet28a-mi126a into three parts
   respectively, each 10 ml.
2. Check the OD600 value. Adjust it to 0.3.
3. Centrifuge at 1500 rpm at 4℃ for 10 minutes.
4. Resuspend the precipitation with three concentrations of Arabinose LB solution
5. Each concentration is distributed into 10 eppendorfs, each 1 ml.
6. Cultured in all the bacteria in a shaker at 37 ℃.

To the three concentrations of the Arabinose LB solution cultured bacteria, every round
we pick out a eppendorf to coat the plate. Take one of the treatment of the example:

7. After a thorough mixing, take 100ul to 900ul ddH2O. Mix thoroughly.
8. Repeat step 1 for 8 times, that is dilute original sample for 10－9.
9. Add 200ul to solid medium, use a sterile coater for coating, to ensure the full coating
   plate.
10. For each concertation, repeat the same procedure 2 times.

At the end of the round, a total of 9 coating plates is done. Place them in a 37 ℃ incubator, and
counting CFU after 24h.

### Test of Heavy Metal Eliminate Effection

##### Equipment

* Atomic absorption photometer

##### Protocol

1. Prepare EcN 1917 bacterial solution without plasmid and 1917-pEt28a-MT bacterial
   solution for overnight culture in advance.
2. Prepare eight 1.5ml EP tubes. Dilute the bacterial solution in each EP tube with LB at
   a ratio of 1:9, add corresponding amount of Pb2+, and culture it in a shaker at 37 ℃.
   The composition formula and test time of each EP tube are as follows:

Tube 1: The concentration of divalent lead ion is 112mg/L, add
1917-pEt28a-MT bacterial
solution, culture for 4h, and take it out

Tube 2: The concentration of divalent lead ion is 112mg/L, add
1917 bacterial solution not
transferred into plasmid, culture for 4h, and take it out

Tube 3: The concentration of divalent lead ion is 112mg/L, add
1917-pEt28a-MT bacterial
solution, culture for 4h, and take it out

Tube 4: The concentration of divalent lead ion is 112mg/L, add
1917 bacterial solution not
transferred into plasmid, culture for 4h, and take it out

Tube 5: The concentration of divalent lead ion is 112mg/L, add
1917-pEt28a-MT bacterial
solution, and take it out after 2h of culture

Tube 6: The concentration of divalent lead ion is 112mg/L, add
1917 bacterial solution not
transferred into plasmid, culture for 2h and take it out

Tube 7: The concentration of divalent lead ion is 112mg/L, add
1917-pEt28a-MT bacterial
solution, and take it out after 2h of culture

Tube 8: The concentration of divalent lead ion is 112mg/L, add
1917 bacterial solution not
transferred into plasmid, culture for 2h and take it out

3. Centrifuge the above 8 EP pipes at 12000 rpm
4. Take 40uL of supernatant from each of the above ep tubes and add it into the
   corresponding glass tube, then add 1ml of 65% concentrated nitric acid for overnight
   digestion, and add 9ml of deionized water the next day to adjust the acidity to about
   5% - 7%.
5. Finally, each sample is 10ml, and a total of 18 samples are packed in a glass tube. The
   maximum concentration of divalent lead ion does not exceed 448ug/L
6. Use graphite furnace atomic absorption spectrometry to determine the content of Pb2+in
   the sample. The results are as follows:
7. According to the initial concentration of each tube, the data can be analyzed to
   calculate the absorption ratio

### Microencapsulation

#### Culture of Lactobacillus plantarum

Lactobacillus plantarum strain 200μl was mixed with 15ml MRS broth and incubated at 37°C for 14
hours. Then, the tubes containing MRS broth and Lactobacillus plantarum were centrifuged at
4000 rpm/min for 15 min, the supernatant was removed, washed with PBS, centrifuged, and
repeated three times.

#### Microencapsulation and encapsulation of Lactobacillus plantarum

In the present study, Lactobacillus plantarum was microencapsulated in sodium alginate and coated
with whey protein.

1. First, solutions of sodium alginate (1% w/v), whey protein (5.5% w/v) and calcium
   chloride (0.1 M) were prepared separately and sterilized at 121°C for 15 min.
2. Microencapsulation of Lactobacillus acidophilus was carried out by the extrusion
   method. One milliliter of Lactobacillus acidophilus suspension (containing 6.21 × 108
   CFU/mL bacteria) was added to 5 mL of sodium alginate solution and gently stirred at
   500 RPM for 30 minutes.
3. The suspension obtained was then added to a calcium chloride solution (0.1 M) using a
   sterile nozzle syringe to form bacterial microcapsules. The obtained microcapsules
   were isolated by centrifugation (3,700 g, 5 min) and washed twice with distilled
   water.
4. Afterwards, the microcapsules were immersed in 5 ml of coating solution (i.e. whey
   protein, stirred at 500 RPM for 30 min, transferred to a sterile glass container and
   placed in a freezer (-80°C, 2 h).
5. Finally, dry in a vacuum freeze dryer at -47°C for 48 hours. Collect the coated
   microcapsule powder and store in the refrigerator (4°C).

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/1-ed.jpg)

##### Microencapsulation yield calculation

To determine the encapsulation rate, the number of Lactobacillus acidophilus in the encapsulated
microcapsules and bacterial suspension was calculated.

1. First, 0.1 g of microencapsulated and/or coated Lactobacillus acidophilus was added to
   0.9 ml of sterile sodium citrate (2% w/v, pH 7) and stirred for 5 min until the
   capsule was completely dissolved and the bacteria were released.
2. Next, bacterial serial dilutions were prepared and incubated by decantation on plates
   containing MRS agar medium, which were subsequently placed in an incubator at 37°C
   for 48 hours and the bacterial counts were counted.
3. Then, the microencapsulation yield was calculated as follows: microencapsulation rate
   (%) = (N / N$\_0$ ) × 100%.

N is the number of bacteria released after microencapsulation (log CFU/g) and N$\_0$ is the
number of bacteria in the suspension before microencapsulation and coating (log CFU/g).

##### Preparation of simulated gastric and intestinal fluids

Simulated gastric juice (SGI) was prepared by adding 0.3 g of pepsin to 100 ml of NaCl solution
(0.2% w/v) and adjusting the pH to 2.0 using 0.1 N HCl). Simulated intestinal fluid was
prepared by suspending trypsin (0.1% w/v) and bile salts (0.45% w/v) in sodium citrate (2%) and
adjusting the pH to 7.4 using 0.1 N NaOH (Fazilah). both SGJ and SIJ were sterilized by
membrane filtration.

##### Viability of Lactobacillus plantarum lyophilized powder after exposure to simulated gastrointestinal conditions

1. First, a sample of Lactobacillus plantarum lyophilized powder 0.1 g stored in the
   refrigerator was placed in a tube containing 0.9 ml SGJ and incubated at 37°C for 120
   min.
2. Next, 1 ml of SIJ was added and then incubated at 37°C for 300 min. Finally, the number
   of Lactobacillus acidophilus was counted as described above.

E. coli operates in the same way as Lactobacillus plantarum

Experiment records

#### PDF preview

You can download the PDF by clicking "Download" in the upper right corner after it
opened.

#### PDF: Experiment records of system 1👈

[Large screen preview on new page](https://static.igem.wiki/teams/4205/wiki/pdf/experiment-records-system-1-2022njmu-china.pdf)

#### PDF: Experiment records of system 2👈

[Large screen preview on new page](https://static.igem.wiki/teams/4205/wiki/pdf/experiment-records-system-2-2022njmu-china.pdf)

#### PDF: Experiment records of system 3👈

[Large screen preview on new page](https://static.igem.wiki/teams/4205/wiki/pdf/experiment-records-system-3-2022njmu-china.pdf)

#### PDF: Experiment records of system 4👈

[Large screen preview on new page](https://static.igem.wiki/teams/4205/wiki/pdf/experiment-records-system-4.pdf)

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On this page, we document our wet lab results for each system. This year, we successfully constructed three plasmid
expression systems, the pLDHLH673-PncA, pET28a-MT, and the ALPaGA-lacZ. Their name, plasmid skeleton, and main
components are as below.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/overview.png)

At the DNA level, we performed enzyme digestion/plasmid PCR/colony PCR and ran agarose gel to prove the successful
construction or transformation of the plasmid. At the protein level, we use inducers of different concentrations to
induce the protein expression and run SDS-PAGE gel to prove the successful expression of our target gene. As to the
function analysis, we performed different experiments for each system and successfully verified their function.
Experiment protocols and records can be found on the [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

During the whole iGEM season, we tried our best to design, build and test our systems, however, we did experience
more failures than expected. Although we were a little disheartened and discouraged with the outcome, we did not
give up until the last minute, and that makes the results you see on the page. Our experiences, reflections, and
future plans can be found in the learn section on the [Engineering success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page. We hope
our studies on this project, regardless of failures or successes, will be the cornerstone of our subsequent
research, and the inspiration for future iGEM teams.

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Wet Lab Results

· Results of System 1

· Results of System 2

· Results of System 3

· Results of System 4

## Wet Lab Results

### Results of System 1

In system one, we aim at boosting NAD+ levels for its critical role in mitochondrial function. We choose
Lactobacillus Plantarum L168 as the chassis and pLDHLH673 as the vector. The gene circuit begins with the
promoter ldhl, followed by the PncA gene, whose overexpression enables our bacteria to produce additional NAD+
for the host.

### The DNA Level

$Lactobacillus Plantarum$ L168, a strain we obtained from our PI, Xingyin Liu's lab, has been proven to be
able
to alleviate social behavior deficits in animal models of autism. It carries the PncA gene itself and our aim
is to amplify the gene, insert it into the vector, and transform the vector back to the L168 which enables the
engineering bacteria to overexpress the PncA encoding protein. Thus, we designed primers, performed PCR, and
ran gel to verify the successful amplification of the PncA gene.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/1-pncapcr.png)
Figure 1. Proof of the successful amplification of the PncA gene

As shown in the figure above, after PCR, the bands we obtained are
near 600 bp, which was consistent with the target band (621 bp). This further indicated that we have
successfully amplified the PncA gene.

We then cut bands out of the agarose gel and purify the DNA
samples. Using pLDHLH673 as the vector, we constructed the pLDHLH673-PncA plasmid expression system, the main
components of which is the PncA gene.

To prove our construction is successful, we performed enzyme
digestion verification on the plasmid, adding restriction endonuclease NdeI and XbaI for double enzyme
digestion, and ran the agarose gel to verify.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/1-ed.png)
Figure 2. Proof of the successful construction of the pLDHLH673-PncA plasmid

As shown in the figure above, after enzyme digestion, the lower
bands we obtained are near 600 bp, which was consistent with the target band (632 bp). The upper bands we
obtained were higher than the marker and were near 5500bp, which was as we expected. This further indicated
that have successfully constructed the pLDHLH673-PncA plasmid.

Although the construction was quite successful, we encountered
difficulty in the electric transformation step. In fact, our original design used pMG36e as the vector. To
amplify the plasmid, we need to transform it into *Escherichia coli* DH5a. However, every time we performed the
transformation, extracted the plasmid, and ran the gel, we found no expected band on the gel. Considering that
pMG36e was a low-copy plasmid, we switched our plasmid extraction kit from FastPure Plasmid Mini Kit (Vazyme)
to TIANprep Midi Plasmid Kit (TIANGEN). However, the result was the same. Then, we changed the condition
during the heat shock of the plasmid but the result showed no change. In this case, we had to change our
vector to pLDHLMCS then to pLDHLH673, and finally constructed the target plasmid successfully. By this time,
however, with few days left to finish the project, we were trapped in the electric transformation. After
careful thinking and consulting our PI, we came to several explanations. First, the plasmid extraction kit we
used was ineffective. In the lysing bacteria step, the color of the solution should have become clear after
adding the reagent, but it remained unchanged. Second, the chassis bacteria we chose, *Lactobacillus plantarum*,
are gram-positive bacteria that have different physiological characteristics from the gram-negative bacteria
commonly used in plasmid extraction.

In fact, in the design stage, we discussed about using the chassis
bacteria that are more common in synthetic biology. Due to its capacity to ameliorate social behavior
deficiencies in animal models of autism, however, we elected to continue using it. Although we have not fully
verified the functionality of this system, we believe that in the future, when given enough time, we can
demonstrate its functionality and viability.

### Function Verification

In our pre-experiments, we knocked out the pncA gene in
*Lactobacillus plantarum* L168 to determine whether it would produce fewer NAD+.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/nad-level-and-pnca-knocked-out.png)
Figure 3. NAD+ level and pncA knocked out

As shown in the figure above, the bacteria with pncA knocked out
produce fewer NAD+ compared with that of Wildtype of LP-L168 supernatant through mass spectrometry assay,
which implicated that our experimental design is reasonable. Despite the fact that this result does not
directly demonstrate the NAD+-boosting ability of our engineering bacteria, it does provide indirect evidence.

### Results of System 2

In system two, we aim at eliminating heavy metal ions such as Hg2+ and Pb2+ in the intestine of autistic
children. We choose *Escherichia coli* 1917 as the chassis and pET-28a(+) as the vector. The gene circuit begins
with two sensors which can be induced by Hg2+ and Pb2+ respectively and follows the Lpp-OmpA-MT gene, which
encodes the protein that can anchor to the bacterial outer membrane and chelate the heavy metal ions. For
biosafety, a kill switch that can be induced by arabinose is assembled downstream.

### The DNA Level

We constructed the pET28a-MT plasmid, transformed it into
*Escherichia coli* Nissle 1917, and extracted the plasmid to prove the success of our transformation. First, we
performed enzyme digestion verification by adding restriction endonucleases SphI and Xhol for double enzyme
digestion and running an agarose gel. Second, we performed the plasmid PCR verification, by designing primers
to amplify the target segment and running an agarose gel.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-pcr.jpg)
Figure 4. Proof of the successful transformation
(A) Enzyme digestion verification. Lanes 1: digestion sample of the extracted plasmid.
(B) plasmid PCR verification. Lanes 1: PCR sample from the extracted plasmid.

As shown in the figure above, after enzyme digestion, the sample
from the extracted plasmid ran out bands near 2000 bp, which was consistent with the target band (2399 bp).
After plasmid PCR, the sample from the extracted plasmid also ran out bands near 2000 bp, which was consistent
with the target band (2399 bp). This further indicated that our plasmid transformation was successful.

### The Protein Level

To verify the expression of our functional genes Lpp-OmpA-MT, we
induced the bacteria at different concentrations of lead ion salt solution. After lysing the bacteria and
performing the centrifugation, we took the supernatant or precipitation at the first or third hour. Then we
ran the SDS-PAGE to check MT protein level for all samples

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-sds.jpg)
Figure 5. SDS-PAGE of Lpp-OmpA-MT expression in different induction conditions
Lane 1-6: 1h. Lane 7-12: 3h. Lane 1,3,5,7,11: Supernatant sample. Lane
2,4,6,8,10,12: Precipitation sample.
Lane 13: negative control of the supernatant sample. Lane 14: negative control of
the precipitation sample.

The pre-stained protein ladder is from 10 to 180 kDa. We induced
the protein expression at different concentrations of lead ion salt solution (112 mg/L, 61mol/L, and 30.5
mol/L) and used double distilled water as the negative control. As shown in the figure above, the bands we
obtained were nearly 40 kDa, which was almost twice the molecular weight we expected. After careful
consultation with our PI and searching literature, we found it has been reported that MT easily form a dimer
in vivo. Hence, we believed this indicated that Lpp-OmpA-MT proteins would combine and form a dimer in the
bacteria, which confirmed the expression of our target protein.

Then, using ImageJ, we quantitatively analyzed the band brightness
and obtained the following results:
(ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare
it with the marker to get the protein expression of each lane)

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-pe.png)
Figure 6. Pb$^{2+}$ concentration and the corresponding protein expression.
(A) Supernatant. (B) Precipitation.

As shown in the figure above, the protein expression was greater
in the precipitation than in the supernatant and was greater after three hours than after one, which was
consistent with our expectations. Using the above data, we solved the model of inducer-protein expression.
More information can be found on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

### Function Verification

To determine whether our engineering bacteria can grow in the
presence of heavy metals, we measured the growth curve. The most frequent model for describing the growth
kinetics of bacteria is the logistic equation, which is frequently used to fit growth curves based on optical
density (OD) in order to comprehend the dynamics of biological systems. Its differential expression is **$\dfrac{1}{N}\dfrac{\partial N\_t}{\partial t} = r(1-\dfrac{N\_t}{K})$**, by variation, we can obtain**$N\_t = \dfrac{K}{1 + \frac{K - N\_0}{N\_0} \times e^{-rt}}$**

We use MATLAB to fit the data and obtain the figure and formulas
shown below.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-logistic.png)
Figure 7. growth curve

Table 1. fit results of the growth curve

| Variable | Formula | R$^2$ |
| --- | --- | --- |
| Wild Type in LB | $N\_t = \dfrac{1.4240}{1 + 28.0422 \times e^{1.5709t}}$ | 0.9979 |
| Wild Type in Pb(NO$\_3$)$\_2$ solution | $N\_t = \dfrac{1.3153}{1 + 18.8537 \times e^{1.7823t}}$ | 0.9964 |
| pET28a-MT in LB | $N\_t = \dfrac{1.5713}{1 + 11.5395 \times e^{1.1985t}}$ | 0.9798 |
| pET28a-MT in Pb(NO$\_3$)$\_2$ solution | $N\_t = \dfrac{1.4928}{1 + 16.9268 \times e^{1.5685t}}$ | 0.9956 |

We used untransformed *Escherichia coli* Nissle 1917 (wild type) as
the control. The concentration of Pb(NO$\_3$)$\_2$ solution is 112 mg/L. As shown in the figure above, the
growth of
our engineered bacteria will not be affected by heavy metals. Also, at the logarithmic phase, when the
bacteria are most sensitive to changes in the environment, our engineering bacteria grew faster than the wild
type, which indirectly proved that our engineering bacteria have the ability to eliminate heavy metals.

As to prove that our engineering bacteria can eliminate heavy
metals directly, we put the engineering bacteria into the metal salt solution Pb(NO$\_3$)$\_2$ and cultured them
for 2
hours. Then, we took the supernatant and measured the concentration of the metal salt solution using atomic
absorption photometer.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-e.png)
Figure 8. proof of the heavy metal elimination ability

We used untransformed *Escherichia coli* Nissle 1917 (wild type) as
the negative control. As shown in the figure above, the transformed bacteria eliminated more heavy metals than
the wild type, and the difference was significant($P$$<$0.05). This indicated that our engineered bacteria
were able to eliminate heavy metals. In the future, we will adjust the reaction time with the metal salt
solution to further optimize the experiment condition. Kill Switch Verification

To validate the function of our arabinose-induced kill switch,
we inoculated 3% fresh bacterial solution in the culture medium and added 0 mM and 10 mM arabinose
respectively as the control and experimental groups. Every 1 hour, 2 hours, and 4 hours, we draw 100 ul of
bacteria to the smear plate and cultured at 37 ℃ overnight. The colony counts are shown below.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-k.png)
Figure 9. colony count in the kill switch verification after dilution (the dilution multiple is
10$^9$)
(A) 1h. (B) 2h. (C).3h

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/2-kf.png)
Figure 10. kill switch verification

As shown in the figure above, in one, two, and four hours, the
number of colonies with arabinose induction are all lower than that without arabinose induction. This
indicated the effectiveness of our kill switch.

### Results of System 3

In system three, we aim at increasing the bioavailability of our
engineering probiotics. We wrap them in alginate, coat them with whey protein and perform freeze-drying.
Microencapsulation enables our engineering bacteria to go through the human digestive tract environment with
less damage and fatalities. The experiments are conducted using the engineered probiotics in systems one and
two, respectively. For convenience, our results are referred to by their chassis names.

### Morphology

To observe the morphology of the microencapsulated bacteria, we
took electron microscope photos. Since the morphology observation was considered to be the same for different
chassis, we used *Escherichia coli* as an example.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/3-dj.png)
Figure 11. Electron microscopy $(*Escherichia coli*)$

As shown in the figure above, the black structure is the bacteria
and the greyish mist surrounding the bacteria is the coating of sodium alginate.

Then, we investigated the microencapsulation yield and effect of
Lactobacillus Plantarum and *Escherichia coli*, respectively.

* $Lactobacillus$ $Plantarum$
* $Escherichia$ $coli$

### Microencapsulation Yield

To determine whether sufficient bacteria remain after
microencapsulation, we counted the number of our engineering bacteria before and after
microencapsulation and calculated the microencapsulation yield.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/3-l1.png)
Figure 12. Microencapsulation Yield of $Lactobacillus Plantarum.$
(A) Bacterial count before microencapsulation. (B) Bacterial count after
microencapsulation (Dissolved in Sodium Citrate).

Table 2 Microencapsulation yield of $*Lactobacillus plantarum*&. (with dilution multiples
calculated)

| Num | Bacterial count before microencapsulation $N\_{L0}$ | Bacterial count after microencapsulation (Dissolved in Sodium Citrate) $N\_{L1}$ |
| --- | --- | --- |
| 1 | $7.2 \times 10^9$ | $6.1 \times 10^{10}$ |
| 2 | $4.7 \times 10^9$ | $7.1 \times 10^{10}$ |
| 3 | $3.4 \times 10^9$ | $7.1 \times 10^{10}$ |
| Mean | $7.1 \times 10^{10}$ | $6.5 \times 10^{10}$ |

Microencapsulation yield of $Lactobacillus Plantarum$ **$=\dfrac{lgN\_{L1}}{lgN\_{L0}}
\times 100\% = 111.38\%$**

The yield of microencapsulation was as expected, which
indicated that sufficient engineering bacteria remained after microencapsulation. During freeze-drying,
the engineering bacteria may continue to grow, which is normal and may lead to the yield exceeding 100%.

### Microencapsulation Effect

To verify the effect of microencapsulation, we prepared
simulated gastric juice and intestinal juice. Then we used them to process our engineering bacteria. The
procedure was called simu-digest for convenience.

We defined survival rate as the ratio of the bacteria count
after digestion to the bacteria count before digestion, which is

Survival rate = $\dfrac{\text{bacteria count after digestion}}
{\text{ bacteria count before digestion}} \times 100\%$

By comparing the mean survival rates of Microencapsulated
and Unmicroencapsulation bacteria, we can verify the effect of microencapsulation. Noted, the count of
bacteria unmicroencapsulated and microencapsulated before digestion is identical to the count of
bacteria before and after microencapsulation in the Microencapsulation Yield section, so we reused the
data of them.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/3-l2.png)
Figure 13. Microencapsulation Effect of $*Lactobacillus plantarum*$.
(A) Unmicroencapsulated bacteria. (B) Microencapsulated bacteria.

Table 3 Microencapsulation Effect of $Lactobacillus Plantarum$.

| Num.  Unmicroencapsulated bacteria  Microencapsulated bacteria | | | | |
| --- | --- | --- | --- | --- |
| Before digestion | After digestion | Before digestion | After digestion |
| 1 | $7.2 \times 10^9$ | $9.2 \times 10^8$ | $6.1 \times 10^{10}$ | $7.0 \times 10^{10}$ |
| 2 | $4.7 \times 10^9$ | $8.6 \times 10^8$ | $6.3 \times 10^{10}$ | $5.6 \times 10^{10}$ |
| 3 | $3.4 \times 10^9$ | $6.2 \times 10^8$ | $7.1 \times 10^{10}$ | $4.7 \times 10^{10}$ |
| Mean | $5.1 \times 10^9$ | $8.1 \times 10^8$ | $6.5 \times 10^{10}$ | $5.8 \times 10^{10}$ |

According to the data in the table above, we can know that
the mean survival rate of the unmicroencapsulated bacteria was 15.69% whereas the mean survival rate of
the microencapsulated bacteria was 89.23%. This indicated that the microencapsulation of Lactobacillus
Plantarum is effective.

### Microencapsulation Yield

To determine whether sufficient bacteria remain after
microencapsulation, we counted the number of our engineering bacteria before and after microencapsulation
and calculated the microencapsulation yield.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/3-e1.png)
Figure 14. Microencapsulation Yield of $*Escherichia coli*$.
(A) Bacterial count before microencapsulation. (B) Bacterial count after
microencapsulation (Dissolved in Sodium Citrate).

| Num | Bacterial count before microencapsulation &N\_{E0}& | Bacterial count after microencapsulation (Dissolved in Sodium Citrate) &N\_{E1}$ |
| --- | --- | --- |
| 1 | $5.7 \times 10^{11}$ | $5.9 \times 10^{10}$ |
| 2 | $1.1 \times 10^{12}$ | $4.7 \times 10^{10}$ |
| 3 | $1.0 \times 10^{12}$ | $5.2 \times 10^{10}$ |
| Mean | $8.9 \times 10^{11}$ | $5.3 \times 10^{10}$ |

Microencapsulation yield of $Lactobacillus Plantarum$ **$=\dfrac{lgN\_{E1}}{lgN\_{E0}}
\times 100\% = 89.73\%$**

The yield of microencapsulation was as expected, which
indicated that sufficient engineering bacteria remained after microencapsulation.

### Microencapsulation Effect

To verify the effect of microencapsulation, we prepared
simulated gastric juice and intestinal juice. Then we used them to process our engineering bacteria. The
procedure was called simu-digest for convenience.

We defined survival rate as the ratio of the bacteria count
after digestion to the bacteria count before digestion, which is

Survival rate = $\dfrac{\text{bacteria count after digestion}}
{\text{ bacteria count before digestion}} \times 100\%$

By comparing the mean survival rates of Microencapsulated and
Unmicroencapsulation bacteria, we can verify the effect of microencapsulation. Noted, the count of
bacteria unmicroencapsulated and microencapsulated before digestion is identical to the count of bacteria
before and after microencapsulation in the Microencapsulation Yield section, so we reused the data of
them.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/3-e2.png)
Figure 15. Microencapsulation Effect of $*Escherichia coli*.$.
(A) Unmicroencapsulated bacteria. (B) Microencapsulated bacteria.

Table 2 Microencapsulation yield of $*Lactobacillus plantarum*&. (with dilution multiples
calculated)

| Num.  Unmicroencapsulated bacteria  Microencapsulated bacteria | | | | |
| --- | --- | --- | --- | --- |
| Before digestion | After digestion | Before digestion | After digestion |
| 1 | $5.7 \times 10^{11}$ | $6.0 \times 10^{10}$ | $5.2 \times 10^{10}$ | $3.1 \times 10^{10}$ |
| 2 | $1.1 \times 10^{12}$ | $4.2 \times 10^{10}$ | $5.9 \times 10^{10}$ | $3.2 \times 10^{10}$ |
| 3 | $1.0 \times 10^{12}$ | $1.5 \times 10^{11}$ | $4.7 \times 10^{10}$ | $3.2 \times 10^{10}$ |
| Mean | $8.9 \times 10^{11}$ | $8.4 \times 10^{10}$ | $5.27 \times 10^{10}$ | $3.17 \times 10^{10}$ |

According to the data in the table above, we can know that
the mean survival rate of the unmicroencapsulated bacteria was 9.44% whereas the mean survival rate of
the microencapsulated bacteria was 60.12%. This indicated that the microencapsulation of Lactobacillus
Plantarum is effective.

### Results of System 4

In system four, we aim at sensing the elevated concentration of
lactate to detect the potential mitochondrial dysfunction in autistic children. We choose *Escherichia coli* DH5α
as the chassis and pSB4K5 as the vector. The gene circuit begins with a sensor that can be induced by lactate
and follows the lacZ gene, which encodes the protein β-Galactosidases that can have a color reaction with X-gal.

### The DNA Level

We constructed the ALPaGA-lacZ plasmid and transformed it into
*Escherichia coli* DH5α. To prove the success of our transformation, we designed primers, performed plasmid PCR
and colony PCR to amplify the LldR gene, and ran the agarose gal.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/4-lldrpcr.png)
Figure 16. Proof of the successful transformation.
(A) plasmid PCR. Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample
form untransformed DH5α.
(B) colony PCR. Lanes 1-4: PCR sample form transformed DH5α. Lanes 5-8: PCR sample
form untransformed DH5α.

As shown in the figure above, after plasmid PCR and colony PCR, the
sample from the transformed DH5α both ran out bands near 750 bp, which was consistent with the target band (777
bp). This further indicated that our plasmid transformation was successful.

For our reporter gene lacZ, in order to determine whether our
chassis bacteria *Escherichia coli* DH5α themselves carry this gene, which may interfere with the results of our
functional analysis. We designed primers, performed colony PCR to amplify the lacZ gene, and ran agarose gel.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/4-laczpcr.png)
Figure 17. PCR amplification of lacZ reporter gene.
Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample form
untransformed DH5α.

As shown in the figure above, after colony PCR, the sample from the
transformed DH5α ran out bands near 3000 bp, which was consistent with the target band (3075bp). The sample from
the untransformed DH5α can also run out bands near 3000 bp but with much weaker light. This indicated that our
chassis bacteria *Escherichia coli* DH5α themselves carried the lacZ gene and could express them in micro amounts.

### The protein Level

To verify the expression of our reporter genes lacZ, we induced the
bacteria at different lactate concentrations. After lysing the bacteria and performing the centrifugation, we
took the supernatant and ran SDS-PAGE.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/4-sds.png)
Figure 18. SDS-PAGE of β-galactosidases expression in different induction conditions.

The pre-stained protein ladder is from 10 to 180 kDa. We induced the
protein expression at different lactate concentrations (1 mol/L, 10-3 mol/L, and 10-6 mol/L) and used double
distilled water as the negative control. As shown in the figure above, the bands we obtained were close to the
target band (113 kDa). This confirmed the expression of our target protein.

Then, using ImageJ, we quantitatively analyzed the band brightness
and obtained the following results:
(ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare it
with the marker to get the protein expression of each lane)

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/4-pe.png)
Figure 19. Lactate concentration and the corresponding protein expression

As shown in the figure above, as the lactate concentration
increased, the protein expression also increased gradually, which was consistent with our expectations. Using
the above data, we solved the model of inducer-protein expression. More information can be found on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) page.

### Function Verification

The function of this system is to sense the elevated concentration
of lactate and then detect potential mitochondrial dysfunction in autistic children. To verify this, we used
filter paper, a common supply in the laboratory, to perform the experiment. First, we dropped the bacteria
solution onto the filter paper. After 30 minutes, our engineering bacteria were firmly attached to the filter
paper. Then we added lactate and x-gal onto the filter paper and observed the color changes.

![](https://static.igem.wiki/teams/4205/wiki/wet-lab/4-test.png)
Figure 20. color of the filter paper under different lactate concentrations

We performed the function analysis at different lactate
concentrations (1 mol/L, 10$^{-3}$ mol/L, and 10$^{-6}$ mol/L) and used double distilled water as the negative
control.

As shown in the figure above, without the induction of lactate, the
lacZ protein produced by the chassis bacteria was insufficient to color the filter paper. When adding 10$^{-6}$-1
mol/L lactate, the filter paper turned blue, and as the concentration of lactate increased, the blue became
darker. This confirmed that our lactate test strip was functional.

However, the color change was not very significant to the naked eye,
which may be related to our plasmid being a low copy, the material of filter paper, the lower activity of
bacteria after fixing on the test paper, and the unsuitable reaction environment.

{% endblock %}