iGEM 2022 — Team lzu-hs-china-a

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

## Catalog

[Part 1. Pre-iGEM course](#_h1_0)
[Part 2. Deciding our research](#_h2_0)
[Part 3. Doing experiments](#_h3_0)

# NoteBook

## Part 1. Pre-iGEM course

In order to be familiar with iGEM , we attended various course about different aspects of iGEM. We watched
some great market surveys, mathematical modelling and Wiki designing to give us a general idea of what we
need to do for our own research. In addition, we learned requirements and timing of iGEM to arrange our
schedule.

### 3.5

#### 1. Definition

DNA is the genetic information that stores almost all the proteins and RNA molecules that determine the
traits of a species
In addition to chromosomal DNA, very little structurally different DNA exists in mitochondria and chloroplasts of eukaryotic cells
The genetic material of viruses is also DNA, very rarely RNA, and in very special cases proteins (prions)

#### 2.Chemical component

 A. Preliminary hydrolysis:
deoxynucleotide
B. Complete hydrolysis: Deoxyribose, nitrogenous base, phosphoric acid.
C. Basic unit: deoxynucleotide

#### 3.Basic group

A=adenine
G=guanine
C=cytosine
T=thymine

### 4.10 RNA

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/1.png)

#### 1. Definition

RNA is a class of nucleic acids, is a ribonucleotide through the 3', 5' phosphodiester bond through a
series of condensation formed by long chain molecules.
A. In many viruses, RNA is the only carrier of genetic information;
B. In some RNA viruses, RNA is the genetic material;
C. In all mammals, RNA is essential for protein synthesis.

#### 2.Classify& function

A. rRNA: Classified ribosomal RNA (rRNA): Components of ribosomes
B. tRNA: Transfer RNA (tRNA): Transport amino acids
C. mRNA: Messenger RNA (mRNA): template for protein synthesis
D. hnRNA: Heterogeneous nuclear RNA (hnRNA) a precursor of mature mRNA
E. snRNA: Small nuclear RNA (snRNA): Participates in the splicing and transport of hnRNA
F. snoRNA: Small nucleolar RNA (snoRNA): Processing and modification of rRNA
G. scRNA: Small cytoplasmic RNA (scRNA/ 7S-RNA): component of the signal recognition body synthesized by protein er localization

#### 3. Codon

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/2.png)

A codon is a set of three adjacent nucleotides in a messenger RNA molecule that represents a pattern of
amino acids in protein synthesis.

### 5.7 Basic concepts of synthetic biology

Central dogma
Regulatory element
Logic gate genetic circuitry
Synthetic biological systems are hierarchical
Example of movement circuit

### 7.5 iGEM& Synthetic biology

1. iGEM
International Genetic Engineering Machine Competition
·Synthesis biology
·Mode
·Human practice
·Wiki
·Safety
·Giant jamboree

2. Synthetic biology
Depending on multiple disciplines, business and industry Integration. Working to standardize genetic components, The ultimate goal of reliability performance research is to Design and code to meet the needs of human society Design biology.
Typical composition biological plasmids include RBS, structural genes, Terminator and BioBrick, the sequence.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/3.png)

#### 3. Case studies in synthetic biology

A. Four constituent promoters involved in the central glycolysis pathway were selected to maximize P8
production.
B. The concentration of P8 secreted by PP\* P8 was quantified by ELISA.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/4.png)

C.
· Transcription circuits are layered by using promoters to transmit signals between circuits.
· The sequence of connection points between the input promoter and the circuit can affect the input of the circuit.
· Regardless of the identity of the input promoter, they all produce the same number of transfer functions.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/5.png)

D. Similar to the lactose operon, the negative feedback operon contains repressor proteins and manipulation
sequences.
E. Theory By combining LuxI quorum sensing system with φX174E lysis gene, we constructed bacteria capable of periodic colony death.
F. In E. coli, the starting codon of 14% of genes is GUG and 3% is UUG.
G. By combining LuxI quorum sensing system with φX174E lysis gene, we constructed bacteria capable of periodic colony death.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/6.png)

H. DSB is repaired by non-homologous terminal linking (NHEJ) or homologous directed repair (HDR).  In the
NHEJ pathway, small nucleotide insertions or deletions (INdels) often occur at DSB sites because.
I.  This results in the deletion, insertion or displacement of a gene fragment.  By providing the HDR pathway, the donor DNA with the cosource arm can be used for accurate gene editing by HRD.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/7.png)

## Part 2. Deciding our research

By searching online, we found that chitosan and chito-oligosaccharides have excellent biological
activities. Chitin is the second most abundant polymer in nature. Discarded shrimp shells Crab shells
contain a lot of chitin. Based on these studies, our topic was determined. Chitin can be extracted from
shrimp and crab shells and chitosan can be obtained by deacetylation. Chitosan can be further hydrolyzed to
obtain chitosan oligosaccharides with better water solubility, easier utilization and better biological
activity.

### 7.12

We discuss the IGEM competition system
There are 4 sub-units
We know the background and the requirement of joining the competition
The stages of competition and missions

Our team studied the basic synthetic biology
We first know about the history of the synthetic biology
Second, we studied how the synthetic biology theory was put forward.
Then, we understand what methods and tools do we used in synthetic biology research.
Last, we ask the teacher what is the orientation of synthetic biology.

Our team learn the basics of molecular biology,
The past of the molecular biology
We learned what we should do when we are using the molecular biology
Then we learn about the gene engineering
Last, we learn the trend of the future development of gene engineering.

### 7.13

Our team studied the knowledge about cells, including the form of cells and development, structures, and
message transport between cells. Then, we studied the method that we might use in studying cytobiology.
This week we also studied microbiology, to gain the fundamental cognition about microorganisms.
First, we join the microorganisms by studying the history of the microscope
Second, we learned about autogenetic and Koch's postulates.
Last, we studied the movements of bacteria.

### 7.14

we first studied the previous teams’ wiki, and poster, to make sure we know the basic elements that we need
to contain in our own product.
Second, we get a deeper understanding of our topic and know the background of the experiment and the benefits that it can bring to society.

### 7.15

Starting with learning the rules in the lab and the operation that we need to know and mastered before we do
the experiment.
Therefore, to make sure we can have the ability to measure our data, we learn some basic mathematical modeling.

### 7.16

Still, our skills in doing experiments are not tough enough, so we make review the rules and methods that we
are going to use in our experiment.
Second, there are many essays waiting for us to complete, so we give a specific time to improve our English writing skills.

## Part 3. Doing experiments

We extracted the RFP and insert plasmids from E. Coli DH5α, and synthesized them to form a new plasmid.
Then we put this new plasmid into E. Coli BL21 and observed the green fluorescence in it.

### Week1

#### Process:

Preparing consumable materials
1. Filling large (1 mL), regular (200 μL), small (20 μL), 5mL pipette tips two boxes for each. Pasting tapes
on each box.
2. Distributing 40 test tubes and filling bottle necks with plugs into 7 tubes per group. Wrapping each group with a piece of newspaper.
3. Preparing dd H2O (500ml), 0.1 M CaCl2 (50ml).
Configurating Amp antibiotic solution
Accurately weighing 1g Amp powder and add 10mL dH2O to dissolve it. Filtering it into a sterile 2 mL EP tube and storing it at -20℃.
Preparing antibiotic plate
Cooling LB solid medium until 60℃ and adding 250μL 100mg/mL Amp antibiotic. Shaking it completely and pouring it into single-used petri dish.

#### Topic & Keywords:

1. Activate E. coli DH5α/pSB1A3-mRFP, E. coli DH5α/\_pMD18T-insert
2. Amplification
3. Get specific plasmid

#### Team cooperation:

Due to the epidemic, some of our students could not come to the offline laboratory. There are altogether 14
students in the offline laboratory. The specific division of labor of the laboratory is as follows:
Zongyue Zhang, Yaxin Jiao, Xiaomi Ai, and Lohan Lu were responsible for controlling the ultra-clean table to complete the solution preparation.
Huijia Zhang, Yiwei Yang, Jinqi Li, Sifei Sheng, Wenjia Qu and Fangning Yu assist in disinfection, preparation, and waste disposal. Normally, a super clean station has two assistants in addition to the main operator.
Mingze Wang and Haozhe Feng took pictures and showed them to the students online.
The online students were mainly responsible for some of our paperwork.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/8.png)

### Week2

#### Process:

 1. plasmid extraction:
According to the instruction of plasmid extraction kit, we extracted four tubes of pSB1A3-mRFP and one tube of pMD18T-insert plasmid.
2. Configure nucleic acid glue (lab instructors helped us to complete this step)
3. PCR amplification:
We used high-fidelity PrimeStar@HS to amplify the target fragment from PSB1A3-insert.
4. PCR amplification procedure
We stored PCR products at 4℃ environment.
5. Double digestion:
We used EcoRl and Xhol for double digestion of extracted plasmid pSB1A3-mRFP and the amplified target fragment.
6. Running nucleic acid glue:
We added pSB1A3-mRFP, target gene enzyme digestion products, 5μl DL 2000, and DL 5000 DNA marker to agarose gel. Then electrophoresis was performed. After electrophoresis, we used gel image system for observation and the lab instructor helped us to cut and place the target DNA gel in a 2mL centrifuge tube for labeling.

#### Topic & Keywords:

1. Get specific plasmid
2. PCR amplification
3. Chut specific gene we need and differ from other genes

#### Team cooperation:

After electrophoresis, another iGEM team recover the DNA gel according to the instruction of the glue
recovery kit. Then preserve it at -20℃.
Lab instructors helped us to configure nucleic acid glue and cut and place the target DNA gel in a 2mL
centrifuge tube for labeling.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/9.png)
![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/10.png)

### Week3

#### Process:

We complete four goals of the experiment:
-Preparing competent cell
-Connect two DNA fragments (RFP plasmid and insert)
-Insert new plasmid into competent cells (DH5α)
-And Cultivate the E. coli in order to prepare for the next day’s experiment.

#### Topic & Keywords:

1. Competence Preparation
2. Inserting gene of interest into plasmid

#### Team cooperation:

Due to the epidemic, some of our students could not come to the offline laboratory. There are altogether
13 students in the offline laboratory. The specific division of labor of the laboratory is as follows:
Zongyue Zhang, Yaxin Jiao and Xiaomi Ai were responsible for preparing competent cells.
Huijia Zhang, Yiwei Yang, Jinqi Li, Sifei Sheng, Wenjia Qu and Fangning Yu insert the target gene is inserted into the plasmid.
Haozhe Feng and Lohan Lu plant the strain on the medium.
Zongyue Zhang took pictures and showed them to the students online.
The online students were mainly responsible for other work.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/11.png)
![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/12.png)
![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/13.png)

### Week4

#### Process:

Today our team has been through several processes to gain positive clones.
First, we use PCR to the extent insert gene, the part that we want.
Then we use gel electrophoresis to gain the target gene.

#### Topic & keywords:

1. Linked plasmids selected (single colony)
2. Method: Colony PCR
Right colony->colonial expansion (LB + Amp antibiotic + right colony)
Polymerase chain reaction, （abbreviation: PCR）is also known as polymorphic chain reaction. PCR is a nucleic acid synthesis technique that uses the principle of double-stranded DNA replication to replicate specific DNA fragments in vitro. The in vitro amplification of DNA is based on the principle of semi-conserved DNA replication and the principle of base complementary pairing, i.e., the double-stranded DNA is unstranded during the replication process, from double-stranded to single-stranded, usually at 95°C. After the temperature drops, the primers bind to the single-stranded DNA. This process can be summarized as the three basic steps of 'denaturation-annealing-extension'.

#### Team cooperation:

A total of 13 people attended offline.
1. The lab instructor helped us to prepare and measure 2x mix, F enzyme, R enzyme and distilled water in advance.
2. Zongyue Zhang, Huijia Zhang, Xiaomi Ai, Lehan Lu, Fangning Yu, Sifei Sheng, Shu Zheng, Sheng Zheng, Wenjia Qu, Yiwei Yang, Yaxin Jiao, Haozhe Feng, Mingze Wang was involved in the selection of target colonies (mixed with distilled water)
3. Zongyue Zhang, Huijia Zhang, Xiaomi Ai, Lehan Lu, Fangning Yu, Sifei Sheng, Shu Zheng, Sheng Zheng, Wenjia Qu, Yiwei Yang, Yaxin Jiao, Haozhe Feng, Mingze Wang was involved in the preparation of 2x Taq colony PCR 15ul reaction system and centrifugation.
4. Zongyue Zhang, Yaxin Jiao, Sheng Zheng, Huijia Zhang, Lehan Lu, Mingze Wang participated in the expansion of the colonies
5. The lab supervisor helped us to perform the colony PCR
6. The lab instructor helped us to perform nucleic acid electrophoresis

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/14.png)

### Week5

#### Process:

First, perform column equilibration. We put the adsorption column in the collection tube, and added 500 μL
of equilibration solution BL to it, and centrifuged in a centrifuge at 12000 rpm for one minute. Then we
discarded the waste liquid in the collection tube and put the adsorption column back into the collection
tube.
Next, we took 1-5mL of bacterial liquid into a centrifuge tube and centrifuged for one minute, and aspirated the supernatant. Then we added 250 μL of solution P1 to the bacterial pellet to precipitate the bacteria using a pipetting point, and added 250 μL of solution P2 to the centrifuge tube, gently turned the tube up and down to fully lyse the cells, and sent them into a centrifuge for centrifugation.

Third, we added 350 acetonitrile solution P3 to the high core tube, and immediately turned it up and down gently for 6-8 times and mixed well. At this time, a white precipitate appeared and centrifuged for 10 min.

Fourth, we transferred the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), centrifuge for 30-60 sec, and poured out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube. Then, each of us added 500 mL of deproteinized solution PD to the attachment column CP3, centrifuged for 30-60 sec, poured off the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube.

Fifth, we added 600 µL of rinsing solution PW to the adsorption column CP3, centrifuged at 12,000 rpm for 30-60 sec, poured off the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube. Then we repeated the previous steps. We put the adsorption column CP3 into the collection tube and centrifuged at 12,000 rpm (~13,400xg) for 2 min to remove the residual rinsing solution in the adsorption column.

Next, we put the adsorption column CP3 in a clean centrifuge tube, added 50-100 μL of elution buffer EB to the middle part of the adsorption membrane, placed it at room temperature for 2 min, and centrifuged at 12,000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

Finally, in order to increase the recovery rate of the plasmid, the obtained solution can be re-added to the adsorption column, placed at room temperature for 2 minutes, centrifuged for 2 minutes, and the plasmid solution is collected into a centrifuge tube.

#### Topic & keywords:

Plasmid extraction

#### Team cooperation:

Each team member completed the whole process of plasmid extraction by themselves.
Yaxin Jiao, Yiwei Yang, Xiaomi Ai, and Haozhe Feng got the reagents from the lab instructors.
Lehan Lu, Zongyue Zhang, Yaxin Jiao, Huijia Zhang, and Haozhe Feng set up the centrifuge.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/15.png)

### Week6

#### Process:

First, we added 500 P1 equilibrium BL to the adsorption column placed in the collection tube, and then
centrifuged for 1min. After centrifugation, we poured out the waste liquid in the collection tube and put
back the adsorption column into the collection tube.
Second, we cut a piece of agarose gel and placed it in a clean centrifuge tube and weighed it.
Then, the same volume of solution PN was added to the glue block and placed in a 50°C water bath. We gently turned up and down the centrifugal tube to ensure that the glue block was fully dissolved.
We added the solution obtained in the previous step to an adsorption column, placed it at room temperature for 2min, and centrifuged it for 30-60 SEC. we drained the waste liquid from the collection tube and placed adsorption column CA2 into the collection tube.
Then 600ul of bleaching solution PW was added to the adsorption column and 30-60 SEC centrifugation was performed to drain the skin solution in the collection tube and put adsorption column CA2 into the collection tube.
Then we repeated the rinsing procedure above.
The adsorption column was put back into the collection tube and centrifuged for 2min to remove as much rinse liquid as possible.
Let adsorbed cinnamyl CA2 sit at room temperature for several minutes and dry thoroughly.
We placed the adsorption column CA2 in a thousand net centrifuge tube, and suspended an appropriate amount of elution rinse EB in the middle of the adsorption film, placed it at a constant temperature for 2 min, and then centrifuged it for 2 min to collect DNA solution.
Finally, the resulting solution was placed under a microscope to observe the fluorescent protein.

#### Topic & keywords:

Gel recycle
Observing green fluorescence of E. coli under the microscope

#### Team cooperation:

Each team member completed the whole process of observing green fluorescence of E. coli under the
microscope.
Zongyue Zhang, Huijia Zhang, Xiaomi Ai, Lehan Lu, Fangning Yu, Sifei Sheng, Shu Zheng, Sheng Zheng, Wenjia Qu, Yiwei Yang, Yaxin Jiao, Haozhe Feng, Mingze Wang was involved in the gel recycle.
Zongyue Zhang, Yaxin Jiao, Sheng Zheng, Huijia Zhang, Lehan Lu, Mingze Wang set up the centrifuge.
The lab supervisor helped us to analyze issues.

![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/16.png)
![](https://static.igem.wiki/teams/4330/wiki/wiki/notebook/17.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

## Catalog

[Design 1](#_h1_0)
[Build 1](#_h2_0)
[Test 1](#_h3_0)
[Learn 1 Learning and Optimization](#_h4_0)
[Design 2](#_h5_0)
[Build 2](#_h6_0)
[Test 2](#_h7_0)
[Learn 2](#_h8_0)

# Engineering

## Design 1

After studying the existing technology, we found that the technology used in the market today has problems
such as pollution and low efficiency. We eventually found that using transgenic technology could be a
possible way. Basically, chitosanase genes bind to the genes of E. coli to form a new plasmid and are
expressed inside E. coli cells.

![](https://static.igem.wiki/teams/4330/wiki/wiki/engineering/5.png)

## Build 1

In the first attempt to use transgenic technology. After we obtained a plasmid with a chitosanase gene, we
put it into E. coli Dh5alpha and multiplied it, and finally successfully demonstrated chitosanase in cells.

## Test 1

Although we were able to successfully express chitosanase. In terms of the yield of chitosan
oligosaccharides, we found that chitosan oligosaccharide preparation efficiency was extremely inefficient.

## Learn 1 Learning and Optimization

Based on the results of the first test of our design, we realized that there could be a more productive way
to produce chitosan oligosaccharides. In the first round of experiments, we transferred the chitosanase gene
into E. coli and found that the protein expressed by it could only be in the cell, and this makes the
transmembrane transport of chitosan necessary to break down chitosan, which greatly reduced the production
efficiency of the enzyme. We realized that the problem was with the transmembrane transport of chitosan, and
perhaps we could find a way to break down chitosan without crossing the cell membrane. In the second cycle
of the experiment, following the professor's suggestion, we introduced the N-terminal gene of the ice
crystal nucleoprotein, and improved our experiment to achieve this purpose.

## Design 2

After receiving expert advice, we conducted a second trial. The chitosanase gene was extracted, but the
difference is that we fused the chitosanase gene with the N-terminal gene of the INP (Ice Nucleation
Protein) in advance to become our target gene. We synthesize the gene of interest with the gene of E. coli
to form a new plasmid and re-insert it into E. coli Dh5alpha, at which time the protein expressed by the
N-terminal gene of the INP will be anchored in the cell membrane for fixation. In addition, chitosanase is
expressed on the cell surface, which successfully eliminates the tedious step of chitosan transmembrane into
the cell.

![](https://static.igem.wiki/teams/4330/wiki/wiki/engineering/6.png)

## Build 2

In order to make chitosanase work better, we display chitosanase on the cell surface through cell surface
display technology. The INP gene fragment in the newly synthesized plasmid after successful expression
allows chitosanase to express and function outside the cell membrane. Furthermore, breaking down the exposed
chitosan into chitosan oligosaccharides.

## Test 2

First, we compared the crude chitosanase and detected the effect of cell surface presentation technology on
maintaining the stability of the enzyme, as shown in Figure A, after 40 days of placement, the engineered
bacteria still maintained 80% of the initial enzyme activity, while the crude chitosan only maintained 31%
of the initial enzyme activity after 40 days at room temperature. Therefore, it can be seen that the cell
surface display technology can maintain and enhance the activity of chitosanase.

Furthermore, we further compare the amount of chitosan produced by engineered bacteria and crude chitosan
enzymes with substrate hydrolysis. As shown in Figure B, it can be seen that within day 1-7, the hydrolysis
capacity of engineered bacteria maintains a good stability, but the crude enzyme solution is almost
inactivated after a day of interaction with the substrate and cannot continue to hydrolyze the substrate. It
can be seen that engineering bacteria can work more stably.

## Learn 2

Using cell surface presentation technology, the efficiency of our preparation of chitosan oligosaccharides
has been greatly improved. This contributes to our next goal of industrializing the project. While the
technology has been improved, we are also thinking about how to further improve the efficiency of production
and control the yield constant. For example, we may make modifications to existing engineered bacteria to
make them more tolerant of different conditions and make them more usable.

{% endblock %}