iGEM 2024 — Team jiashu-china

![](https://static.igem.wiki/teams/5480/head-banner/2.png)

- Week 1 (5.20 Mon-5.26 Sun) -

The handshake experiment tests how many functional bacteria can be transferred through
handshakes: pipet some functional bacteria to 1st person’s hand, handshake with the 2nd person,
and the 2nd person goes on to handshake with the 3rd person, until the 5th person, press their
hands on the Petri dishes that contain kanamycin respectively in the end, cultivate these
bacteria to the next day to see the results.

Notes: Academic pieces of literature prove E.coli safety:

1. Reference: Centers for Disease Control and Prevention (CDC) & National Institutes of
   Health
   (NIH). (2009). Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th
   Edition.
   Retrieved from CDC’s official website.
2. Reference: FDA. (2020). Bacteriological Analytical Manual (BAM). Chapter 4:
   Enumeration of
   *Escherichia coli* and the Coliform Bacteria. Retrieved from FDA’s official
   website.
3. Reference: FDA. (2009). Guidance for Industry: Bottled Water: Microbiological
   Quality and
   Testing. Retrieved from FDA’s official website.
4. Reference: FDA. (2020). Cosmetic Microbiological Safety Issues. Retrieved from FDA’s
   official
   website.

- Week 2 (5.27 Mon-6.2 Sun) -

· 1. PCR of gene knockout

![notebook3](https://static.igem.wiki/teams/5480/notebook/notebook-3.png)

*Design of gene knockout*

![notebook4](https://static.igem.wiki/teams/5480/notebook/notebook-4.png)

*Principle of gene knockout*

![notebook5](https://static.igem.wiki/teams/5480/notebook/notebook-5.png)

*Construction of the gene knockout*

![notebook6](https://static.igem.wiki/teams/5480/notebook/notebook-6.png)

· 2. Electrophoretic PCR products

![notebook7](https://static.igem.wiki/teams/5480/notebook/notebook-8.png)

· 3. PCR products recycling

![notebook7](https://static.igem.wiki/teams/5480/notebook/notebook-9qian.png)

· 4. Product concentration test

- Week 3 (6.3 Mon-6.9 Sun) -

1. PCR of Decaf pathway

![notebook9](https://static.igem.wiki/teams/5480/notebook/notebook-9.png)

*Design of Decaf pathway*

![notebook10](https://static.igem.wiki/teams/5480/notebook/notebook-10.png)

*Construction of the Decaf Pathway*

2. Electrophoretic PCR products

![notebook10](https://static.igem.wiki/teams/5480/notebook/notebook-10hou.png)

3. PCR products recycling

4. Product concentration test

- Week 4 (6.10 Mon-6.16 Sun) -

1. The construction of plasmid of Decaffeination pathway and gene knockout through Golden gate
and Gibson methods respectively as directed.

![notebook11](https://static.igem.wiki/teams/5480/notebook/notebook-11.png)

2. PCR of decaffeination pathway constructed plasmid through Golden Gate.

3. Electrophoretic PCR products

4. PCR products recycling

5. Product concentration test

6. Bacterial conversion of “Male” product(Decaffeination pathway), the bacteria were cultured to
the next day for growth curve and phenotype verification, took one night for the colonies to
appear.

![notebook12](https://static.igem.wiki/teams/5480/notebook/notebook-12.png)

7. PCR of gene knockout constructed plasmid through Gibson

8. Electrophoretic PCR products

9. PCR products recycling

10. Product concentration test

11. Bacterial conversion of “Female” product(Gene knockout), the bacteria were cultured to the
next day for growth curve and phenotype verification, took one night for the colonies to appear.

- Week 5 (6.17 Mon-6.23 Sun) -

1. Bacteria colonies were appeared

![notebook13](https://static.igem.wiki/teams/5480/notebook/notebook-13.png)

*Three Petri dishes are on the way. The upper left Petri dish, the upper right Petri dish, and
the middle and lower Petri dish are Decaf Pathway, Decaf Pathway, and up-KanR-down
respectively.*

2. Perform colony PCR

![notebook14](https://static.igem.wiki/teams/5480/notebook/notebook-14.png)

*PCR result of KanR fragment*

![notebook15](https://static.igem.wiki/teams/5480/notebook/notebook-15.png)

 *Decaf on the left and Up - KanR - Down on the right.*

- Week 6 (6.24 Mon-6.30 Sun) -

1. Plasmid extraction followed the instruction after colony PCR.

![notebook16](https://static.igem.wiki/teams/5480/notebook/notebook-16.png)

2. Enzyme digestion verification/ Enzyme-cut vertification, includes Electrophoretic/ run-glue
verification. (KpnI and *DpnI*)

3. Electrophoresis of different endonuclease(Enzyme-cut vertification)

· results:

The first two lanes have two bands, which are around 2500 and 2000-2500 base pairs. In the
Snapgene simulation, the desired lengths are 2654 and 2103 bp respectively. The electrophoresis
result corroborates the restriction enzyme digestion. Lanes 3 and 4 are under Dpnl digestion,
which can be visualized in 2 bands around 1000 and 750 bp, as well as smaller bands around 250.
This is a reasonable output, since various cutting sites of *DpnI* could be detected on the
plasmid of pYB1s, the genome would be demerged to small pieces.

![notebook17](https://static.igem.wiki/teams/5480/notebook/notebook-17.png)

 *PccdK- up - Kan- down enzyme digestion verification.*

Lane 2 and 3 are between 8000 and 12000 base pairs. In the Snapgene simulation, the desired
lengths are 9155 bp. The electrophoresis result corroborates the restriction enzyme digestion.

![notebook17](https://static.igem.wiki/teams/5480/notebook/notebook-18.png)

*pYB1s - ndmDBCAE enzyme digestion verification.*

4. RED homologous recombination through electroporation

Goal: knocking out the gene of *guaB* in the desired plasmid

Method: Homologous recombination using RED system

RED system construction

Flanking: introduce a up-kan-down(5'-3') to substitude up-*guaB*-down fragment.
penetrating the cell membrane: by using 1400 v 3 ms electroporation, a small amount of
up-kan-down
would have an opportunity to transpassing the phospholipid layer of the Bateria successively.
construction of main components
functional plasmid PKD46 will offer Exonuclease, Annealing Protein, and Inhibition of Host
Exonucleases for the flaking fragment to bind on the plasmid and knock out the *guaB* gene
with an
antibiotic.

![notebook19](https://static.igem.wiki/teams/5480/notebook/notebook-19.png)

*Electroporator*

Fig 1: Electroporator

Finally, 100 ul of electropored bacteria was spread evenly on the agar plate and 50 ul of
xanthine
was added to test the conditional growth and the success of target fragment incorporation.

- Week 7 (7.1 Mon-7.7 Sun) -

1. Phenotypic verification preparation(gel of anything with xanthine and caffeine)

Bacterial Petri dishes made of different materials, from left to right are oolong tea, jasmine
oolong tea, green tea, coffee, creatine, and C4 - Preworkout.

![notebook20](https://static.igem.wiki/teams/5480/notebook/notebook-20.png)

![notebook21](https://static.igem.wiki/teams/5480/notebook/notebook-21-1.png)

*Growth curve on the Microplate reader.*

2. Transfer three types of strains into three types of nutrient medium at aseptic
bench(LB,LB+Xan,LB+caf)

3. 37 degrees celsius shake cultivation

4. Growth curve tested by Microplate reader.

- Week 8 (7.8 Mon-7.14 Sun) -

1. PCR amplification of the plasmid of the Temperature-Controlled Cell-Free Expression System,
including vector, restriction enzyme *DpnI*, and lambda repressor (Control/regulation)

2. Electrophoretic PCR products

3. PCR products recycling

![notebook22](https://static.igem.wiki/teams/5480/notebook/notebook-22.png)

*PCR samples*

4. Product concentration test

- Week 9 (7.15 Mon-7.21 Sun) -

1. The construction of plasmid of pY38a-*DpnI*(Temperature-Controlled Cell-Free Expression
System)
through Gibson method as directed.

2. PCR the plasmid constructs

3. Electrophoretic PCR products

![notebook23](https://static.igem.wiki/teams/5480/notebook/20240929224439.jpg)

4. PCR products recycling

5. Product concentration test

![notebook23](https://static.igem.wiki/teams/5480/notebook/notebook-23.png)

![notebook24](https://static.igem.wiki/teams/5480/notebook/notebook-24.png)

6. Bacterial conversion of py38a-*DpnI*(Temperature-Controlled Cell-Free Expression System)
product, the bacteria were cultured to the next day for growth curve and phenotype verification,
took one night for the colonies to appear.

- Week 10 (7.15 Mon-7.21 Sun) -

1.No colony grows at the first connection

![notebook27-1](https://static.igem.wiki/teams/5480/notebook/notebook-25.png)

2. It is connected again and then chemically transformed

![notebook29-1](https://static.igem.wiki/teams/5480/notebook/notebook-26.png)

3. Growth curve and phenotypic prediction

![notebook31-1](https://static.igem.wiki/teams/5480/notebook/notebook-27.png)

4.Colony PCR did not produce the correct bands

![notebook31-2](https://static.igem.wiki/teams/5480/notebook/notebook-28.png)

5.It is connected again and then chemically transformed

![notebook29-1](https://static.igem.wiki/teams/5480/notebook/notebook-29.png)
![notebook31-3](https://static.igem.wiki/teams/5480/notebook/notebook-30.png)

7.Perform colony PCR

![notebook29](https://static.igem.wiki/teams/5480/notebook/notebook-31.png)

*Graph of electrophoresis for colony PCR.*

![notebook30](https://static.igem.wiki/teams/5480/notebook/notebook-32.png)

8.Growth curve and phenotypic prediction

![notebook31](https://static.igem.wiki/teams/5480/notebook/notebook-33.png)

*Schematic diagram of temperature sensitivity experiment of *E. coli*.*

- Week 11 (7.29 Mon-8.4 Sun) -

1. Plasmid extraction

![notebook32](https://static.igem.wiki/teams/5480/notebook/notebook-34.png)

*Check plasmid concentration.*

2. Enzyme digestion verification

![notebook33](https://static.igem.wiki/teams/5480/notebook/notebook-39.png)

3. Phenotype verification

![notebook34](https://static.igem.wiki/teams/5480/notebook/notebook-40.png)

# Menu

* [##### ⚪ Week 1 (5.20 Mon-5.26 Sun)](#a1)
* [##### ⚪ Week 2 (5.27 Mon-6.2 Sun)](#a2)
* [##### ⚪ Week 3 (6.3 Mon-6.9 Sun)](#a3)
* [##### ⚪ Week 4 (6.10 Mon-6.16 Sun)](#a4)
* [##### ⚪ Week 5 (6.17 Mon-6.23 Sun)](#a5)
* [##### ⚪ Week 6 (6.24 Mon-6.30 Sun)](#a6)
* [##### ⚪ Week 7 (7.1 Mon-7.7 Sun)](#a7)
* [##### ⚪ Week 8 (7.8 Mon-7.14 Sun)](#a8)
* [##### ⚪ Week 9 (7.15 Mon-7.21 Sun)](#a9)
* [##### ⚪ Week 10 (7.15 Mon-7.21 Sun)](#a10)
* [##### ⚪ Week 11 (7.29 Mon-8.4 Sun)](#a11)

![](https://static.igem.wiki/teams/5480/head-banner/10.png)

- Overview -

Our project follows the Design-Build-Test-Learn cycle, centered on secure information encoding
using genetically modified *E. coli*. The design is divided into three main modules: first, we
created *E. coli* with a *guaB* gene knockout, allowing selective growth under xanthine or caffeine.
Second, we introduced the DeCaf pathway, enabling the use of caffeine to increase transmission
stealth. Third, we implemented a temperature-sensitive self-destruct mechanism to prevent
information leakage by degrading DNA under non-ideal conditions. Each module was rigorously
tested and improved through iterative feedback.

![engineering1](https://static.igem.wiki/teams/5480/engineering/engineering-1.png)

Cycle 1:

- *guaB* Gene Knockout -

· Design

We designed the knockout of the *guaB* gene to prevent *E. coli* from synthesizing GTP and dGTP,
thereby restricting the growth of the strain. This design aims to ensure that the engineered
strain can only grow under specific conditions (e.g., with the addition of exogenous xanthine),
thereby enhancing the security of information transmission. The *guaB* gene, located in the E.
coli genome, encodes guanine nucleotide reductase, a crucial enzyme in the synthesis of GTP and
dGTP. We initially constructed a targeting fragment, pccdk-up-kana-down, and then used RED
homologous recombination to knock out the *guaB* gene in *E. coli* BW25113, replacing it with a
resistance gene (KanR) for selection.

![engineering2](https://static.igem.wiki/teams/5480/engineering/engineering-2.png)

![engineering3](https://static.igem.wiki/teams/5480/engineering/engineering-3.png)

· Build

1. Plasmid extraction
2. Standard PCR
3. Gel recovery
4. Golden Gate assembly
5. Transformation into DH5α
6. Colony PCR
7. Enzyme digestion verification
8. DNA sequencing
9. Transformation into BW25113 cells using Pulse Cell Transfection System
10. Colony PCR

![engineering4](https://static.igem.wiki/teams/5480/engineering/engineering-4.png)

![engineering5](https://static.igem.wiki/teams/5480/engineering/engineering-5.png)

· Test

We first cultured the BW25113 strain and BW-*ΔguaB* strain in LB medium, using a microplate reader
to measure cell density every hour. The growth curve in LB medium showed that the BW25113 strain
grew well, while the BW-*ΔguaB* knockout strain showed significantly impaired growth, confirming
the loss of *guaB* function. We also cultured the engineered strain in M9 medium with and without
xanthine. Colony formation experiments showed that the *guaB* knockout strain’s growth was
significantly inhibited in the absence of xanthine, but normal growth was restored when xanthine
was added.

![engineering6](https://static.igem.wiki/teams/5480/engineering/engineering-6.png)

· Learn

The experimental results validated our design hypothesis: knocking out the *guaB* gene effectively
limits the growth of the strain. This outcome demonstrates that by controlling the availability
of exogenous xanthine, selective growth control of the engineered strain can be achieved,
thereby enhancing the security of information transmission. Furthermore, the successful
application of the RED homologous recombination method highlights the reliability of our
gene-editing approach, setting the stage for further modifications in subsequent cycles.

- Cycle 2: Introduction of the DeCaf Pathway -

· Design

The goal of this design is to engineer a bacterial strain that can only grow in the presence of
caffeine, providing a controllable mechanism for both information encryption and biological
containment. To achieve this, we introduced the caffeine degradation pathway (DeCaf Pathway)
into the *guaB* knockout strain. Since caffeine is more readily available than xanthine, this
modification allows the strain to grow selectively in caffeine-containing environments. The
pathway, derived from Pseudomonas putida CBB5, degrades caffeine into xanthine, compensating for
the GTP and dGTP synthesis deficiency caused by the *guaB* knockout. Using synthetic biology
methods, we introduced the relevant genes of the DeCaf Pathway (ndm DBCAE) into the BW-*ΔguaB*
strain, constructing a new strain, BW-*ΔguaB*-DeCaf.

![engineering7](https://static.igem.wiki/teams/5480/engineering/engineering-7.png)

![engineering8](https://static.igem.wiki/teams/5480/engineering/engineering-8.png)

· Build

1. Plasmid extraction
2. Standard PCR
3. Gel recovery
4. Golden Gate assembly
5. Transformation into DH5α cells
6. Colony PCR
7. Enzyme digestion verification
8. DNA sequencing
9. Transformation into BW25113 cells

![engineering9](https://static.igem.wiki/teams/5480/engineering/engineering-9.png)

· Test

We verified the successful construction through resistance screening and gene expression
analysis. The constructed BW-*ΔguaB*-DeCaf strain was inoculated in LB medium containing either
caffeine or xanthine, with BW25113 and BW-*ΔguaB* strains as controls. The results showed that
BW-*ΔguaB*-DeCaf grew well in caffeine medium, while the BW-*ΔguaB* strain could not survive.
Additionally, we tested the ability of various caffeinated beverages to support the growth of
the strain, demonstrating that high-caffeine beverages effectively supported the strain’s
growth.

![engineering9-2](https://static.igem.wiki/teams/5480/engineering/engineering-10.png)

· Learn

Through this cycle, we confirmed that the introduction of the DeCaf pathway enables the *guaB*
knockout strain to grow specifically in caffeine-containing environments, successfully
overcoming the growth deficiency caused by the *guaB* knockout. In our project, this system
achieved the goal of controlling bacterial growth through caffeine, validating our core design
concept of using caffeine as a selective growth condition to ensure the security and
controllability of information transmission. By verifying the system's effectiveness with
naturally occurring caffeine sources (such as treated beverages), we fulfilled the design
objectives, demonstrating that this system can selectively grow under specific conditions. This
not only met the project's expectations but also provided a reliable solution for biological
encryption and controlled information transmission.

![engineering9-3](https://static.igem.wiki/teams/5480/engineering/engineering-11.png)

Cycle 3:

- Implementation of the Self-Destruction Mechanism -

· Design

To prevent information leakage, we designed a self-destruction mechanism that enables the
engineered strain to express DNase under normal cultivation conditions at 37°C, thereby
degrading its own DNA and leading to cell death. We chose a temperature-sensitive promoter
combined with the *DpnI* gene to achieve this mechanism. The *DpnI* gene was cloned into an
expression vector under the control of the temperature-sensitive promoter and introduced into
the BW-*ΔguaB*-DeCaf strain, constructing a strain capable of self-destruction at elevated
temperatures.

![engineering10](https://static.igem.wiki/teams/5480/engineering/engineering-12.png)

![engineering11](https://static.igem.wiki/teams/5480/engineering/engineering-13.png)

· Build

1. Plasmid extraction
2. Standard PCR
3. Gel recovery
4. Gibson assembly (C116)
5. Transformation into DH5α cells
6. Colony PCR
7. Enzyme digestion verification
8. DNA sequencing
9. Transformation into BW25113 cells

![engineering12](https://static.igem.wiki/teams/5480/engineering/engineering-14.png)

· Test

We validated the mechanism through temperature gradient experiments. In the test, the
constructed strain was cultured at 30°C and 37°C. The results showed that the strain grew
normally at 30°C but could hardly survive at 37°C, indicating the successful activation of the
self-destruction mechanism.

![engineering13](https://static.igem.wiki/teams/5480/engineering/engineering-15.png)

· Learn

The experimental results demonstrate that the designed self-destruction mechanism can be
effectively triggered under the specified temperature conditions, ensuring that the engineered
strain cannot be misused or leaked after information transmission. This mechanism provides a
final safeguard for information security, making the entire information transmission system more
robust. Additionally, the success of this cycle highlights the effectiveness of integrating
temperature-sensitive elements in synthetic biology designs, offering insights into future
strategies for controlled gene expression in various applications.

# Menu

* [##### ⚪ Overview](#one)
* [##### ⚪ Cycle 1: *guaB* Gene Knockout](#two)
* [##### ⚪ Cycle 2: Introduction of the DeCaf Pathway](#three)
* [##### ⚪ Cycle 3: Implementation of the Self-Destruction Mechanism](#four)


![](https://static.igem.wiki/teams/5480/head-banner/5.png)

- Overview -

This study is divided into three main experimental components: bacterial transmission, gene
privacy, and self-destruction mechanisms. First, we designed experiments to explore bacterial
transmission through physical contact, specifically handshakes, where we observed that bacteria
could indeed be transmitted, albeit with a decreasing quantity over repeated interactions.
Second, to enhance information privacy, we knocked out the *guaB* gene in the BW25113
strain and
introduced a caffeine dependency, ensuring that the engineered bacteria could only survive in
caffeine-rich environments. This modification provided a controllable growth mechanism,
strengthening the security of the genetic information stored in the bacteria. Finally, the
self-destruction mechanism, regulated by the *DpnI* gene and triggered at higher
temperatures, was
designed to inhibit bacterial growth, acting as a final layer of protection for information
security by ensuring the bacteria are destroyed after transmission. Each of these components
plays a critical role in ensuring the overall security and functionality of the system, with
bacterial transmission enabling the spread of information, caffeine dependency providing
selective growth conditions, and the self-destruction mechanism preventing unauthorized access.

Module 1:

- Information encoding and transmission -

In this experiment, we investigated the potential of bacterial transmission through
handshakes. This first part of our study includes two key sections: Encoding and
transmission possibility. In the Encoding section, we developed a coding method that embeds
information into bacterial DNA using synthetic biology techniques. The goal of this method
is to encode complex information securely and ensure that it can be transmitted biologically
without being easily deciphered. For the transmission possibility, we smeared bacterial
strains onto a hand and then observed their transfer across multiple handshakes to evaluate
the effectiveness of bacterial transmission. Figure 1 displays the bacterial distribution in
culture dishes at various stages of the experiment. For more details on the Encoding process
and the software used for our experiments
[(click run to Software)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D)

![result1-1](https://static.igem.wiki/teams/5480/results/result-1.png)

*Figure 1:

(00) Initial bacterial smear on the hand.

(01) After first handshake.

(02) After second handshake.

(03) After third handshake.

(04) After fourth handshake.*

The results clearly demonstrate that bacteria can be transferred through multiple consecutive
handshakes. While the bacterial load decreases with each interaction, the presence of bacteria
is still evident after several handshakes, highlighting the efficiency of this transmission
method. This illustrates how information, in the form of bacterial cells, can be repeatedly
passed between individuals.

Module 2:

- INFORMATION PRIVACY -

The experimental design of this module consists of two main parts: *guaB* gene knockout and
the
introduction of the Decaf metabolic pathway. First, we used the Lambda-Red recombination method
to construct a *guaB* gene knockout strain, verifying its impact on bacterial growth
through the
disruption of the purine synthesis pathway, achieving the goal of preventing growth in standard
media. Next, we introduced the Decaf metabolic pathway into the *guaB* knockout strain,
using
caffeine as a selective metabolite to ensure that the bacteria can only grow in
caffeine-containing environments. These two components together form a highly controllable
bacterial information transmission system, enhancing information security and privacy
protection.

· 1. Experimental results of *guaB* gene knockout in BW25113 strain using Lambda-Red
recombination method.

The purpose of this experiment was to construct the pccdk-up-Kan-down plasmid via the Lambda-Red
recombination method, knocking out the *guaB* gene in the BW25113 strain to investigate the
impact
of this gene on bacterial growth and phenotype.

![result2](https://static.igem.wiki/teams/5480/results/result-2.png)

*Figure 2:

(A) Schematic diagram of the pccdk-up-Kan-down plasmid construction.

(B) PCR amplification results showing *guaB* upstream fragment, KanR fragment, and
*guaB*
downstream fragment.

(C) Enzyme digestion verification showing 2654 bp and 2103 bp fragments, confirming the
correct plasmid construction.

(D) Colony PCR showing a 2106 bp band, verifying successful gene knockout.

(E) DNA sequencing results confirming the correct insertion and orientation of the
fragments.*

Using the Red recombination method, we successfully constructed the pccdk-up-Kan-down plasmid
containing the *guaB* upstream, KanR, and *guaB* downstream fragments. PCR
amplification, colony
PCR, enzyme digestion, and DNA sequencing all confirmed the successful knockout of the
*guaB* gene
and validated the accuracy of the plasmid construction.

The objective of knocking out the *guaB* gene was to study its crucial role in bacterial
metabolism and growth. The correct construction and validation of the plasmid provide a solid
foundation for subsequent functional analyses, allowing us to further investigate the specific
role of the *guaB* gene in physiological processes.

![result3](https://static.igem.wiki/teams/5480/results/result-3.png)

*Figure 3:

(A) Cellex pulse cell transfection system, used to introduce the plasmid into the
BW25113 strain.

(B) Colony PCR gel electrophoresis results showing a 1662 bp band, confirming successful
*guaB* gene knockout.

(C) Growth curves in LB medium, with blue representing the BW25113 strain and green
representing the *guaB* knockout strain.

(D) Phenotype verification, where the *guaB* knockout strain does not grow on M9
medium
but shows growth on M9 medium supplemented with 0.5 mM xanthine.*

After successfully introducing the plasmid into the BW25113 strain using the pulse cell
transfection system, colony PCR results confirmed the *guaB* gene knockout. Growth curve
analysis
shows that the loss of the *guaB* gene significantly affects the growth of the strain,
which is
further supported by phenotype verification. The restoration of growth upon the addition of
xanthine suggests the crucial role of the *guaB* gene in purine biosynthesis.

· 2. Decaf pathway results

The purpose of this experiment is to construct a plasmid carrying the Decaf metabolic pathway
genes and to verify its accuracy. Through PCR amplification, enzyme digestion verification, and
DNA sequencing, we ensured that the target gene fragments were successfully inserted into the
plasmid and could be effectively expressed in the host bacteria.

![result4](https://static.igem.wiki/teams/5480/results/result-4.png)

*Figure 4:

(A) Plasmid map of the Decaf metabolic pathway genes.

(B) PCR amplification results show the successful amplification of the pYB1s, ndmBCAE,
and ndmD fragments.

(C) Enzyme digestion shows a 9155 bp fragment, confirming the correct construction of
the plasmid.

(D) Five colonies were selected for colony PCR.

(E) Colony PCR shows a 1767 bp target fragment, demonstrating that the plasmid was
successfully transferred into the host bacteria.

(F) DNA sequencing results matched the expected fragments, confirming the correct
plasmid construction.*

Through PCR amplification, enzyme digestion verification, and DNA sequencing, we successfully
constructed a plasmid carrying the Decaf metabolic pathway genes. All verification steps
indicated that the target genes were successfully integrated into the plasmid and effectively
transferred to the host bacteria. This experiment provides a solid foundation for further
studies on the role of the Decaf metabolic pathway in bacterial metabolism.

The purpose of this experiment was to introduce the Decaf metabolic pathway plasmid into the
BW-ΔguaB strain, generating the BW-ΔguaB-Decaf strain. This aimed to investigate whether the
strain would become dependent on caffeine for growth after the Decaf metabolic pathway was
added. By observing the growth curves of the strain under different conditions (normal LB, LB
supplemented with xanthine, and LB supplemented with caffeine), we sought to verify the role of
the Decaf pathway in bacterial metabolism.

![result5](https://static.igem.wiki/teams/5480/results/result-5.png)

*Figure 5:

(A) Under normal LB conditions, the blue curve shows the rapid growth of the original
BW25113 strain; the green curve (BW-ΔguaB) and the orange curve (BW-ΔguaB-Decaf) show
significantly limited growth.

(B) Under LB conditions with 0.5 mM xanthine, the blue curve shows continuous good
growth of the original strain, while the green and orange curves show some recovery of
growth in the *guaB* knockout strains, though not to the level of the original strain.

(C) Under LB conditions with 0.5 mM caffeine, the blue curve still shows a high growth
rate, while the green curve shows almost no growth. The orange curve shows a significant
recovery in growth, indicating that the Decaf-introduced strain can rely on caffeine for
growth.*

By introducing the Decaf metabolic pathway into the BW-ΔguaB strain, the experiment demonstrated
that the strain’s growth was limited in normal LB medium and LB supplemented with xanthine,
falling short of the growth observed in the original strain. However, in LB medium supplemented
with caffeine, the strain with the Decaf pathway (BW-ΔguaB-Decaf) showed significant recovery in
growth, suggesting that the strain could rely on caffeine for growth through the introduced
Decaf pathway.

This demonstrates that the introduction of the Decaf metabolic pathway successfully altered the
metabolic process of the strain, making it dependent on caffeine. This provides experimental
evidence for the potential use of commonly available caffeine as a metabolic supplement in place
of xanthine and showcases the potential of genetic engineering to adjust metabolic pathways.

The purpose of this experiment was to investigate the growth of the strain in media made from
common beverages containing different concentrations of caffeine through phenotype verification.
The goal was to test whether the strain can rely on caffeine for growth in a high-caffeine
environment.

![result6](https://static.igem.wiki/teams/5480/results/result-6.png)

*Figure 6: Shows the growth of the strain in media made from different caffeine-containing
beverages.*

![result7](https://static.igem.wiki/teams/5480/results/result-7.png)

*Figure 7: Shows the comparison between the control group (M9 + caffeine and M9 media) and
the test group (media made from different beverages) in terms of strain growth.*

The results clearly show that the strain exhibited superior growth in high-caffeine beverages
(such as pre-workout), while no growth was observed in other beverages with lower caffeine
content (such as green tea, jasmine tea, oolong tea, and coffee). This indicates that a high
concentration of caffeine is necessary for the strain’s growth.Although coffee contains
caffeine, no growth was observed in the media made from brewed coffee. This may be due to
insufficient caffeine concentration or the complex chemical composition of the coffee, which may
have inhibited the strain’s normal metabolic functions. This suggests that, in addition to
caffeine concentration, the complexity of the beverage’s composition may also negatively affect
the strain’s growth.Comparing the growth results between the control group (M9 + caffeine) and
the different beverage-based media further supports the critical role of caffeine concentration.
Pre-workout, with its high caffeine content, was the only test group that supported the strain’s
growth. This also suggests that high-caffeine products can be considered as a substitute for
laboratory-prepared caffeine solutions when optimizing the strain’s growth conditions.

Overall, the experimental results demonstrate that the strain with the introduced Decaf pathway
exhibits caffeine dependency in a high-caffeine environment, while beverages with lower caffeine
concentrations or more complex compositions are not suitable for its growth. This provides new
insights for further research into the metabolic mechanism of the Decaf pathway and how to use
commonly available high-caffeine products to optimize strain growth.

Module 3:

- SELF-DESTRUCTION -

The purpose of this experiment was to construct a plasmid containing the *DpnI* gene and
use
temperature regulation to control its expression, thereby achieving a self-destruction mechanism
in bacteria to ensure information security. We designed a plasmid regulated by the pR and pL
promoters, which activates the *DpnI* gene under specific temperature conditions, leading
to
bacterial death. This self-destruction mechanism ensures that the genetic information carried by
the bacteria is destroyed under certain conditions, preventing information leakage or misuse.
The experimental results demonstrated the effectiveness of this system, laying a foundation for
future applications in information protection and biosecurity.

![result8](https://static.igem.wiki/teams/5480/results/result-8.png)

*Figure 8:

(A) Plasmid map.

(B) PCR amplification shows the target fragment.

(C) Enzyme digestion verifies the correct construction of the plasmid.

(D) Ten colonies were selected for colony PCR.

(E)Colony PCR shows successful amplification of the target fragment.

(F)DNA sequencing results confirm the accuracy of the inserted fragment.*

This experiment successfully constructed a plasmid carrying the *DpnI* gene, which is
regulated by
the pR and pL promoters, and includes an ampicillin resistance gene as a selection marker. The
results from PCR amplification, enzyme digestion, and DNA sequencing confirmed the correctness
of the plasmid construction and the accurate insertion of the fragment.

The primary function of the *DpnI* gene is to trigger a self-destruction mechanism under
specific
temperature conditions. When the temperature rises to 37°C, the expression of the *DpnI*
gene is
activated, leading to the inability of the host bacteria to grow. This characteristic can be
used to control bacterial survival and reproduction under specific conditions. Conversely, at
30°C, the expression of the *DpnI* gene is inhibited, allowing the bacteria to grow
normally. This
temperature-dependent gene expression regulation mechanism offers the potential for developing a
controllable self-destruction system.

![result9](https://static.igem.wiki/teams/5480/results/result-9.png)

*Figure 9:

(A) At 30°C, the expression of the *DpnI* gene is inhibited, allowing the bacteria
to grow
normally.

(B) At 37°C, the expression of the *DpnI* gene is activated, and bacterial growth
is
severely restricted.*

The experimental results validated the effect of *DpnI* gene expression on bacterial growth
under
different temperature conditions. At 30°C, *DpnI* expression is inhibited, allowing the
bacteria
to grow normally. However, at 37°C, *DpnI* is activated, leading to a significant
inhibition of
bacterial growth. This demonstrates the feasibility of a temperature-dependent self-destruction
mechanism.

This self-destruction mechanism greatly enhances information security, as it ensures that
information encoded in the genome cannot be easily retrieved, while also providing a
“self-destruct” feature. Once the temperature changes, the expression of the *DpnI* gene
triggers
bacterial self-destruction, ensuring that the information is not disseminated or misused in
uncontrolled environments. This gene-based self-destruction mechanism offers an innovative
solution for secure information transmission and lays the foundation for future applications in
biosecurity and information protection.

Conclusion

Throughout this study, we successfully achieved the key objectives of our project: constructing
a controllable bacterial information transmission system. By knocking out the *guaB* gene
and
introducing the caffeine-dependent Decaf metabolic pathway, we demonstrated selective bacterial
growth based on environmental caffeine levels. Furthermore, the introduction of the
temperature-controlled *DpnI*-based self-destruction mechanism ensured the secure and
controlled
destruction of encoded information under specific conditions. These results validate the overall
design of our project, showcasing a novel and reliable approach for encrypted biological
information transmission and protection, providing a solid basis for future advancements in
biosecurity and information encryption technologies.

# Menu

* [##### ⚪ Overview](#a1)
* [##### ⚪ Module 1:Information encoding and transmission](#a2)
* [##### ⚪ Module 2:Information PRIVACY](#a3)
* [##### ⚪ Module 3:SELF-DESTRUCTION](#a4)
* [##### ⚪ Conclusion](#a5)