iGEM 2023 — Team jlu-china

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Notebook

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Engineering~~~

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Experiments

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~~Results~~

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# **Overview**

Our goal was to utilize E. coli engineered bacteria to produce the DMBT1 protein to inhibit the growth of Pseudomonas aeruginosa, so we constructed a protein expression system, and at the same time regulated the growth state of the engineered bacteria through a population sensing system, a toxin-antitoxin system, and an auto-inducible system to ensure that it performs its specific function under the appropriate conditions. Through this integrated approach, we aim to reduce the risk of Pseudomonas aeruginosa infections in contact lens users and to safeguard the safety and health of contact lens users.

Our validation experiments are divided into four segments, one is the protein expression system, the second is the population sensing system, the third is the hicA-hicB system, and the fourth is the self-induced system

The experiments are divided into plasmid extraction, plasmid transformation, plasmid sequencing,Corneal epithelial cell and Pseudomonas aeruginosa culture , and validation experiments for each of the four sections. The experiments are shown below.

1. Plasmid extraction

2. transformation

3. Plasmid sequencing

4. Corneal epithelial cell and Pseudomonas aeruginosa culture

5. validation: protein expression system

6. Validation: self-induction system

7. Validation: Population Induction System

8. Validation:Toxin-antitoxin system

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# **1、Plasmid extraction concentration experiments**

1. Our various experimental and control groups had a total of 9 plasmids as follows, and their respective numbers and concentrations after bulk extraction are shown below.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cc1.png)

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# **2、Transformation**

We successfully transformed 2/3/6 and 4+5/7+8/6+7/6+8 plasmids after teamwork and experimental division of labor, and the experimental results are as follows

![col-four-group](https://static.igem.wiki/teams/4609/wiki/tr1.png)
![col-four-group](https://static.igem.wiki/teams/4609/wiki/tr2.png)

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# **3、Plasmid sequencing**

### Design plasmid with hsp gene.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu1.jpg)

Figure 1 Amplified hsp27 plasmid

### Sequencing results:

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu2.jpg)

Figure 2 PET-30a-DMBT1-sfGFP Sequencing results

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu3.jpg)

Figure 3 PET-30a-LuxR-RFP Sequencing results

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu4.jpg)

Figure 4 pET30a-HicA Sequencing results

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu5.jpg)

Figure 5 pET30a-HicB Sequencing results

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cexu6.jpg)

Figure 6 PET-21a-HSPB1(HSP27) Sequencing results

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# **4、HCE-T corneal epithelial cell and Pseudomonas aeruginosa culture**

### 4.1 HCE-T culture

We obtained HCE-T primary cryopreserved cells from our company and cultured them in 5% CO2 incubator using DMEM complete medium supplemented with relevant growth factors and nutrients, and every 2~3 days, cells were passaged and some cells were frozen at -80s℃ for storage. Cells were digested and counted before use and used for cell spreading in 96-well plates. The cell morphology was normal and free of contamination during the culture process.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/hce-t-day1.png)

![col-four-group](https://static.igem.wiki/teams/4609/wiki/day3-4-x10-chuan.png)

### 4.2 Pseudomonas aeruginosa culture

We cultured Pseudomonas aeruginosa in the BSL-2 laboratory by resuscitating and inoculating the purchased bacterial solution in LB broth medium and incubating it using a thermostatic shaker at 200 rpm with shaking at 37°C. Subsequently, the overnight solution was doubly diluted and then the plates were coated, and several dilutions of the plates were separately placed in a 37°C thermostatic incubator to culture the bacteria, and the plates in which 30-300 colonies were present were chosen to count the colonies.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/tonglv.jpg)

![col-four-group](https://static.igem.wiki/teams/4609/wiki/tl2.jpg)

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# **5、Validation: Protein Expression System**

### 5.1 DMBT1 protein expression validation

#### 5.1.1 Fluorescence photo shooting

When we designed the DMBT1 protein expression system, in order to reduce the difficulty of detecting the expression of the target protein, we fused the DMBT1 protein with the sfGFP fluorescent protein, and utilized the cytation3 fluorescent enzyme marker to take fluorescent photographs to detect whether the DMBT1 protein is expressed or not, and we obtained the following results of the pictures

![col-four-group](https://static.igem.wiki/teams/4609/wiki/yg.png)

![col-four-group](https://static.igem.wiki/teams/4609/wiki/image.png)

#### 5.1.2Western Blot Validation

We fused DMBT1 protein and sfGFP protein, took the E. coli bacterial fluid of pET30a-DMBT1-sfGFP, extracted the protein in the engineered bacteria by using the whole protein extraction reagent, and verified whether the DMBT1 protein was expressed or not by incubating with the antibody against sfGFP protein, and got the results in the figure below, and we got that the size of sfGFP was about 24kDa by checking the related papers. The size of sfGFP is about 24kDa, and we successfully verified the expression of DMBT1 protein by comparing the corresponding size of marker.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/dmbt1wb.png)

#### 5.1.3 Validation of Pseudomonas aeruginosa co-culture with HCE-T corneal epithelial cells

We wished to determine the cellular activity of HCE-T human corneal epithelial cells infiltrated by Pseudomonas aeruginosa by cck8 and inhibited the treatment of Pseudomonas aeruginosa using our production of DMBT1 protein.

Before the formal experiments began we conducted a part of the pre-experiment to explore the optimal incubation time and the number of cells inoculated in 96-well plates The results are shown below, where the optimal number of inoculated cells was obtained from the paper as 10,000 cells per null. The incubation time was about 2.5 hours

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cell.png)

In addition, we performed bacterial enumeration of overnight P. aeruginosa bacterial fluids using plate colony counting, and obtained a P. aeruginosa density of approximately 9.6\*10^8CFU/mL, with a counting petri dish as follows

![col-four-group](https://static.igem.wiki/teams/4609/wiki/tlj.jpg)

We then centrifuged the Pseudomonas aeruginosa bacterial fluid after overnight incubation, centrifuged it in a centrifuge, discarded the supernatant and resuspended it in complete cell culture solution, and added DMBT1 protein extracted from the engineered bacteria for incubation, added it to a 96-well plate with the cells spread according to the ratio of bacteria/cells of 100/1, and incubated it overnight in an incubator, and then washed the 96-well plate with antibiotic-containing PBS to remove the Pseudomonas aeruginosa bacteria. Aeromonas aeruginosa was removed. Finally, complete cell culture medium containing cck8 was added and incubated in the cell culture incubator for 2.5 hours for measurement. The experimental group treated with DMBT1 and the control group without any treatment were set up, and the following results were obtained based on the analysis of the data results, which proved that DMBT1 could prevent P. aeruginosa from infesting the human corneal epithelial cells.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/cva.png)

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# 6、Validation: Self-inducing system

### 6.1 Quantitative determination of fluorescence signal

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res6-1.png)

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res6-2.png)

In order to compare the effect of hsp27 induction with IPTG
induction, we set up three control groups and one experimental
group with the same concentration, and put four tubes of bacterial
fluids into the shaker at the same time to incubate, and detected
the fluorescence intensity of the bacterial fluids every 1 hour, and
then fitted them to make a curve after the data were obtained. The
results are shown in the figure below, the fluorescence intensity
indirectly reflected that the protein expression of the group adding
0.5 mmol IPTG was less than PET-21a-HSPB1(HSP27) group, while hsp27
was approximate to the protein expression of the group adding 1
mmol IPTG.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res6-3.png)

### 6.2 Western Blot Validatio

We extracted the proteins from the engineered bacteria using
whole protein extraction reagent with pET-30a-DMBT1-sfGFP and
pET28a-Hsp27 bacterial fluids and pET-30a-sfGFP and pET-28a-sfGFP bacterial fluids, respectively, and incubated with antibodies
against sfGFP and Hsp27 proteins, respectively, to verify whether
the Hsp27-SILEX self-inducible system was expressed or not.
Hsp27-SILEX auto-inducible system is expressed or not, and the
results are shown in the figure below. After checking the related
papers, we found that the size of sfGFP is about 24kDa and the
size of Hsp27 is about 28kDa, and the Hsp27-SILEX auto-inducible system was successfully verified by comparing with the
corresponding size of the markers.

Hsp27 antibody results verification, the following results prove that
Hsp27 protein is successfully expressed in the self-inducible
system, and pET30-a-Hsp27 plasmid successfully expresses the
protein gene of Hsp27, which proves that it can be expressed in
the E. coli system.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res-6-4.png)

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res6-5.png)

In addition, we verified that Hsp27 protein could induce the expression of target protein genes on other plasmids using the sfGFP antibody results, and the analyzed images proved that
Hsp27 protein promotes the expression of target genes successfully, based on the size of the sfGFP protein of about 24 kDa.

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# 7 Validation: group sensing syste

### 7.1 Fluorescent Photo Shooting

When we designed the group sensing system, in order to
determine the strength of the group sensing corresponding to the
downstream E-cleavage protein gene expression, we E-cleavage
protein fused with the mCherry red fluorescent protein, and used
the cytation3 fluorescence enzyme marker to take fluorescent
photographs to detect whether the E-cleavage protein is
expressed or not, so as to verify whether the group sensing
system works or not, and we got the results of the pictures as
follows.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res7-1.png)
![col-four-group](https://static.igem.wiki/teams/4609/wiki/res7-2.png)

### 7.2 Growth curves

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res7-3.png)

We set up three experimental groups and one control group
this time, respectively, we tested the growth curves of the
population sensing plasmid again, placed in a shaker when all
4 tubes of bacterial solution had an OD600 value of 0.05, and
measured the OD value every 2.5 hours, and the results are
shown below.

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res7-4.png)

Growth curves of four groups of bacterial solutions, OD values over time

With the increase of inducer concentration, the peak and decrease
of E. coli concentration increased significantly compared to the
control group, which proved the success of our improved colony
induction system and had more obvious results.

# 8.Validation: toxin-antitoxin system

we set up two groups of bacterial fluids i.e. control
group (BL21 E. coli) as well as experimental group (hicA-hicB
plasmid cotransformation fluids).The two groups were diluted
to the appropriate concentration and coated plates were
made.The plates of the control group were free of antibiotics
whereas in experimental group due to plasmid having
kanamycin resistance, the plates contained a certain
concentration of kanamycin.The plates were divided into equal
number of four groups and were placed into the incubator

![col-four-group](https://static.igem.wiki/teams/4609/wiki/res8-1.png)
![col-four-group](https://static.igem.wiki/teams/4609/wiki/res8-2.png)

The results showed that both experimental and
control groups grew very well at 37°C, and the
control group grew better at 23°C, while few
colonies were found in the experimental group at
23°C, which proved that the dormant system
basically worked, and after observing the results,
the plates of the experimental group incubated at
23°C were put into the incubator at 37°C, and the
number of colonies increased significantly after 16
hours, and the growth was good, which proved that
the resuscitation system also worked. This proved
that the resuscitation system was also effective.

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# Conclusion

In this project, we focused on building a
comprehensive DMBT1 production system to deal
with the potential Pseudomonas aeruginosa
infections associated with contact lens use.

Through the design and validation of four key
systems, we successfully realized the project's
vision of providing contact lens users with a more
comprehensive eye care and cleaning function to
reduce the risk of eye infections and inflammation,
thereby enhancing the user's eyewear experience.

First, we successfully constructed a protein
expression system, and by monitoring the
expression of green fluorescent protein, as well as
validation experiments, we realized the efficient
expression of DMBT1 protein and verified that it
was effective in inhibiting bacteria. The
establishment of this system provides a solid
foundation for the large-scale production of DMBT1,
which lays an important technical support for the
subsequent spectacle cleaning process.

The introduction of the population sensing
system enabled us to control the bacterial
population density and trigger bacterial lysis when
needed. Through the introduction of lysis genes and
red fluorescence genes of phage and the
measurement of growth curves under AHL induction,
we successfully realized the function of the colony
sensing system and verified its proper operation.
This provides a key mechanism for maintaining a
constant density of bacterial populations and
ensures homeostasis of the entire system.

The role of the toxin-antitoxin system in the overall
system is to keep the bacteria dormant and trigger
their revival when needed. The successful
realization of this system relies on the synergistic
action of HicA toxin and HicB antitoxin, as well as
the modulation of the temperature-sensitive
promoter. We verified the normal expression of the
HicA system by observing the growth of plate
colonies at different temperatures and the change in
the number of colonies before and after placing a
23 degree Celsius plate into a 37 degree Celsius
case plate. This system provided us with precise
control over the bacterial growth conditions and
ensured the controllability of the whole system.

Finally, the introduction of the self-inducible
system provided us with a new pathway for
spontaneous expression of target proteins.The
SILEX system is unique in that it does not require
any media adaptation and makes protein self-induced expression possible through metabolic
modifications driven by interactions with metabolic
E. coli GAPDH enzymes. We basically affirmed its
role as a tractable expression by its systematic
comparison with IPTG. The successful application
of this system provides an effective way for the
spontaneous expression of DMBT1, while avoiding
the toxic side effects that may be caused by the
previous use of inducers such as IPTG. In addition,
the introduction of the self-inducible system
increased the stability of the whole system and
ensured the continuous and efficient expression of
DMBT1.

Taken together, the successful integration of these
four systems provides a solid foundation for our
comprehensive DMBT1 production system. The
protein expression system ensures efficient
expression of DMBT1, the population sensing
system maintains a constant density of bacterial
populations, the toxin-antitoxin system provides a
precise means of growth state regulation, and the
self-induced system makes spontaneous
expression of DMBT1 possible

Our system is not just a simple combination of
four independent parts, but rather, through careful
design and commissioning, these parts are
organically combined to form an integrated system
with synergistic effects. The synergistic effect of
these systems fulfills our initial experimental vision
of building a comprehensive and efficient DMBT1
production system, providing an innovative and
viable solution to our goal, which is expected to
reduce the risk of Pseudomonas aeruginosa
infection in contact lens users, and improve ocular
health and experience. This will provide new
strategies for preventing eye infections caused by
Pseudomonas aeruginosa, bringing new
innovations and advances to the field of eyewear
care. Through this project, we validated our initial
experimental concept and provided important
technical support for future eye care product
development.

This research is not only important for the fields of
biopharmaceuticals and protein expression, but also
contributes a strong practical example for the
development of synthetic biology. The success of
this project highlights the importance of
interdisciplinary collaboration, combining
microbiology, bioengineering, and ophthalmology
for eye health, and its success brings new ideas to
the field of eye care, laying a solid foundation for
future research and innovation.

However, in order to apply it successfully in real
production, further experiments and analyses are
needed to ensure the feasibility and efficiency of the
system, and more in-depth discussions may be
required to clarify the interactions between the
systems and the room for improvement. In the
future, we can further optimize the parameters of
the system to improve its efficiency and stability.
Meanwhile, we can also consider applying this
integrated system to actual contact lens care
products to provide users with more comprehensive
eye care and cleaning functions, and we look
forward to bringing new progress and
breakthroughs to this field.

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