iGEM 2024 — Team jlu-nbbms

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

![](https://static.igem.wiki/teams/5304/loading0.gif)

![](https://static.igem.wiki/teams/5304/totop.webp)

![](https://static.igem.wiki/teams/5304/pagemain/notebook0000.webp)

* Stage 1 (1.8~1.14)
* Stage 2 (1.14~1.27)
* Stage 3 (1.28~2.11)
* Stage 4 (3.1~3.27)
* Stage 5 (7.31~9.10)
* Stage 6 (8.15~current)

# Stage 1 (1.8~1.14)

Construction of the ΔmsbB::T7 RNA polymerase Strain χ11803 in Salmonella χ11802

1. We used PCR to amplify the T7 RNA polymerase gene and purify the PCR product for ligation
and transformation into Escherichia coli. We verified the success of vector construction by PCR and sequencing.

![](https://static.igem.wiki/teams/5304/notebook/note3.webp)

2. We knocked out the msbB gene in attenuated Salmonella strain χ11802 and inserted T7 RNA
polymerase at the same site. We named the modified strain as χ11803.

3. We identified χ11803 colonies PCR products through agarose gel electrophoresis. By
designing primer, preparing individual PCR systems including pre-denaturation, denaturation, annealing,
extension and agarose gel electrophoresis.

![](https://static.igem.wiki/teams/5304/notebook/note4.webp)

# Stage 2 (1.14~1.27)

Electroporation of Designed Plasmids into χ11803

1. We transformed the plasmid pSilencer-CLDN6 into χ11803 through electroporation. The steps
included cell preparation, preparation of DNA solution, preparation and disinfection of electrodes,
pre-treatment of culture medium and electroporation experiment.

![](https://static.igem.wiki/teams/5304/notebook/note7.webp)

2. We transfected pCLDN6-EGFP into MCF-7 through electroporation to establish
CLDN6-overexpressing cell lines. The steps included cell preparation, preparation of DNA solution, preparation
and disinfection of electrodes, pre-treatment of culture medium and electroporation experiment.

![](https://static.igem.wiki/teams/5304/notebook/note8.webp)

# Stage 3 (1.28~2.11)

Functional Validation of Plasmids in χ11803

1. Validation of the Delayed Lysis System: We used LB media, ara media with and without carb
to evaluate the growth of attenuated Salmonella strain χ11802. We did this both in liquid medium and in solid
medium.

![](https://static.igem.wiki/teams/5304/notebook/note1.webp)

![](https://static.igem.wiki/teams/5304/notebook/note2.webp)

2. Validation of Anaerobic-Induced hlyA Expression by Western Blot Anaerobic Treatment: We
detected the expression levels of hlyA protein in χ11803 under hypoxic and non-hypoxic conditions through
Western Blot. The steps included cell lysis, protein quantitation, SDS-PAGE electrophoresis, membrane transfer,
blocking, primary antibody incubation, membrane wash, secondary antibody incubation, wash the membrane again and
visualization and detection.

![](https://static.igem.wiki/teams/5304/notebook/note6.webp)

3. Validation of RGD Peptide Expression on Salmonella by Flow Cytometry: We analyzed the
expression of RGD peptide on the cell wall of χ11803 using flow cytometry. The steps included preparation of
solutions and reagents, cell fixation, staining, obtaining and maintaining a single-cell suspension, antibody
incubation and analyzing stained bacteria with a flow cytometer and using FlowJo software to analyze the data.

![](https://static.igem.wiki/teams/5304/notebook/note05.webp)

# Stage 4 (3.1~3.27)

Validation of Drug Resistance-caused Gene Knock-down by Salmonella-mediated RNAi

We co-cultured χ11803/pSilencer-CLDN6 with MCF-7/MDR and MCF-7/CLDN6 (MCF-7 Cell Transfection
with pCLDN6-GFP), and then used Western Blot to detect the expression of CLDN6 in both cell lines. The Western
Blot steps included cell lysis, protein quantitation, SDS-PAGE electrophoresis, membrane transfer, blocking,
primary antibody incubation, membrane wash, secondary antibody incubation, wash the membrane again and
visualization and detection.

![](https://static.igem.wiki/teams/5304/notebook/note9.webp)

# Stage 5 (7.31~9.10)

Efficacy validation of modified Salmonella

IC50

We selected the multiple drug-resistant human breast cancer cell line MCF-7/MDR to verify
that Bio-TARGET can reduce the resistance of drug-resistant tumor cells. Stable cell clones were screened by
transfection of empty plasmids (MCF-7/MDR-sh-k) as control group. We used CCK8 to detect the sensitivity of
MCF-7/MDR cells to ADM, 5-FU, and DDP before and after the addition of χ11803/pSilencer-CLDN6.

![](https://static.igem.wiki/teams/5304/notebook/note10.webp)

Expression levels of drug resistance-related proteins

We used Western Blot to detect the expression of P-gp, BCRP, and MRP-1, three drug
resistance-related proteins, in MCF-7/MDR before and after the addition of χ11803/pSilencer-CLDN6.

![](https://static.igem.wiki/teams/5304/notebook/note11.webp)

Chemotherapy-induced apoptosis in tumor cells

1. We measured the apoptosis rate of MCF-7/MDR cells before and after the addition of
χ11803/pSilencer-CLDN6 using Annexin V/PI double staining. First, set up control tubes, including blank tubes,
single-staining tubes, experimental tubes, and negative tubes, then perform cell processing and staining.

![](https://static.igem.wiki/teams/5304/notebook/note12.webp)

2. We detected the expression of apoptotic proteins in MCF-7/MDR before and after the
addition of χ11803/pSilencer-CLDN6 using Western Blot. The steps included cell lysis, protein quantitation,
SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, membrane wash, secondary
antibody incubation, wash the membrane again and visualization and detection.

![](https://static.igem.wiki/teams/5304/notebook/note13.webp)

We used the same efficacy validation methods to test the sensitivity of ovarian cancer cell
lines SKOV3/CDDP and A2780/CDDP to DDP before and after the addition of χ11803/pSilencer-PGC1α.

![](https://static.igem.wiki/teams/5304/notebook/note14.webp)

# Stage 6 (8.15~current)

Construction of Drug-loaded Nanoparticles

1. We added propyl acetate/ethyl acetate, PEG, and catalyst to a heat-resistant glass tube,
flushed with nitrogen, heated to dissolve and vacuumed for 2 hours, and then sealed the tube to prepare
mPEG-PLGA.

![](https://static.igem.wiki/teams/5304/notebook/note15.webp)

2. We dissolved mPEG-PLGA in dry organic solvent, added BOC-L-phenylalanine and
N,N-dicyclohexylcarbodiimide, slowly added 4-dimethylaminopyridine, protected by nitrogen, stirred at room
temperature for 1-3 days, filtered, washed with alkaline water, concentrated, precipitated, filtered, and vacuum
dried to prepare mPEG-PLGA-Boc(Z).

![](https://static.igem.wiki/teams/5304/notebook/note16.webp)

3. We dissolved mPEG-PLGA-Boc(Z) in dry organic solvent, protected by nitrogen, slowly added
dry trifluoroacetic acid at 0℃, continued the reaction, removed the solvent and unreacted trifluoroacetic acid
by rotary evaporation, dissolved the residue in organic solvent, precipitated, filtered, and vacuum dried to
prepare PEG-PLGA-NH2.

![](https://static.igem.wiki/teams/5304/notebook/note17.webp)

4. We dissolved PEG-PLGA-NH2 in dry organic solvent, added N-carboxylic anhydride, protected
by nitrogen, reacted at room temperature, concentrated, precipitated, filtered, and vacuum dried to prepare
mPEG-PLGA-PZLL.

5. We dissolved mPEG-PLGA-PZLL in a quantified amount of trifluoroacetic acid at 0°C,
reacted, precipitated, filtered, and vacuum dried to prepare mPEG-PLGA-PLL.

![](https://static.igem.wiki/teams/5304/notebook/note18.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5304/loading0.gif)

![](https://static.igem.wiki/teams/5304/totop.webp)

![](https://static.igem.wiki/teams/5304/pagemain/engineering0000.webp)

* Target module
* + Cycle1
  + Cycle2
  + Cycle3
* Safety module
* + Cycle1
  + Cycle3
  + Cycle3
* Gene regulation module
* + Cycle1
  + Cycle2
  + Cycle3
* Drug delivery module
* + Cycle1
  + Cycle2

Target module

Cycle 1: The Preliminary Construction of RGD Surface Display on Salmonella

Design

During a discussion with Professor Quan from the College of Basic Medical Sciences, Jilin
University, he suggested that we could engineer bacteria to enhance their tumor-targeting capability. After
reviewing the literature, we discovered that the Lpp-OmpA structure can be used to display RGD peptides on
the outer membrane of Salmonella, thereby enhancing the bacteria's tumor-targeting specificity.

Build

We searched for the sequences of lpp, ompA, and RGD peptides and combined them, naming the
construct Lpp-OmpA-RGD.

Test

Using Alphafold3 for structural simulation, we found that the protein structure scored an
ipTM of less than 0.5, indicating that the structure is unlikely to exist stably.

![](https://static.igem.wiki/teams/5304/engineering/tm001.webp)

Figure 1. Protein Structure Simulation of Different Types of
RGD Peptides Linked to Lpp-OmpA Structure

Learn

Upon further review of the literature, we discovered that the Lpp-OmpA surface display
system is actually composed of the signal peptide and the first nine residues of Braun's lipoprotein,
referred to as Lpp', and five of the eight membrane-spanning segments of the OmpA porin (residues 46-159).

Cycle2: The Structural Optimization of RGD Surface Display on Salmonella

Design

After discovering the instability of the full-length OmpA structure in Cycle 1, we adjusted
our design by utilizing the Lpp' and residues 46-159 of OmpA for RGD peptide display. To further validate
the stability and display efficiency of this new design, we decided to assess it through molecular docking
and other in silico methods.

Build

We reconstructed the correct Lpp'-OmpA(46-159)-RGD sequence.

Test

We performed modeling again using Alphafold3, and found that the structure scores (ipTM) of
the newly constructed fusion protein were all above 0.5, indicating improved structural stability. However,
through structure simulation alone, we could not yet assess the affinity differences.

![](https://static.igem.wiki/teams/5304/engineering/tm002.webp)

Figure 2. Protein Structure Simulation of Different Types of
RGD Peptides Linked to Lpp'-OmpA(46-159) Structure

Therefore, we decided to further investigate this issue using molecular docking. We
downloaded the integrin structure file from the PDB database and performed molecular docking using PDBePISA.
The results showed that Lpp'-OmpA(46-159)-RGD exhibited the best docking performance.

![](https://static.igem.wiki/teams/5304/engineering/tm003.webp)

Figure 3. Molecular Docking Visualization of
Lpp'-OmpA(46-159)-RGD with Integrin

To further validate that Lpp'-OmpA-RGD exhibited the highest affinity, we proceeded with
molecular dynamics simulations. The results showed that the radius of gyration (Rg) distribution peak of
Lpp'-OmpA-RGD in complex with integrin was the narrowest, indicating the highest structural rigidity
post-binding, thus confirming its strongest affinity.

![](https://static.igem.wiki/teams/5304/engineering/tm004.webp)

Figure 4. Distribution of the radius of gyration (Rg)

Learn

Before conducting wet lab experiments, it is crucial to perform dry lab predictions and
simulations. Through protein structure prediction and molecular docking analysis, we can evaluate the
structural stability of proteins and their binding potential with target molecules in advance. This approach
not only helps optimize and adjust our designs but also increases the feasibility and success rate of the
experiments. By doing so, we can minimize potential errors during the experimental phase, avoid unnecessary
resource waste, and provide a solid theoretical foundation for subsequent wet lab work. This process has
allowed us to deeply appreciate the significance of dry lab modeling tools in bioengineering, as they
greatly enhance the efficiency and accuracy of experimental design.

Cycle 3: Validation of RGD Expression

Design

After reviewing previous iGEM parts, we found that the part BBa\_K2632008 designed by
iGEM18\_HZAU-China includes the LPP'-OmpA(46-159)-RGD-His sequence. Given that this design closely aligns
with our requirements, we decided to directly adopt this part. However, the original part used the plac
promoter to regulate RGD expression, which we did not require. Therefore, we modified the part by removing
the lac operator region while retaining the positive regulation by the CAP protein, effectively converting
the plac promoter into a constitutive one. Additionally, the part had a His tag added to the N-terminus of
RGD, which facilitated the detection of RGD expression. We decided to keep this feature in our design for
ease of subsequent validation.

Build

We successfully constructed the modified sequence into a plasmid and transformed it into
the Salmonella strain. Next, we prepared to validate the expression of RGD to confirm whether the modified
design could effectively display RGD on the bacterial surface.

![](https://static.igem.wiki/teams/5304/engineering/tm005.webp)

Figure 5. Schematic Representation of Lpp-OmpA-RGD

Test

Using the His tag, we detected the expression of RGD on the surface of Salmonella through
flow cytometry. The results showed successful display of RGD on the bacterial surface, and the flow
cytometry data demonstrated expression levels consistent with our expectations, further validating the
effectiveness of our design.

![](https://static.igem.wiki/teams/5304/engineering/stm006.webp)

Figure 6. Validation of RGD expression using flow cytometry

Learn

By referencing and modifying existing iGEM parts, we learned that utilizing established
designs in genetic engineering can significantly accelerate the experimental process and help avoid
redundant work. However, we also realized that adapting these designs to meet our specific needs is
essential. For instance, removing unnecessary regulatory elements to achieve constitutive expression proved
crucial in our case. Additionally, incorporating a His tag for protein detection greatly simplified
experimental procedures, demonstrating that introducing detectable tags at the design stage can enhance both
experimental efficiency and result reproducibility. This experience gave us a deeper understanding of how to
make reasonable adjustments to mature designs, enabling faster and more accurate experimental validation.

Safety module

Cycle 1:Development of a Regulated Delayed Lysis System for Salmonella

Design

During a brainstorming session, our PI, Prof. Ming Yang, suggested the need to enhance the
safety of Salmonella, and he provided us with the delayed lysis strain χ11802. We used this delayed lysis
strain, χ11802, where the asd and murA genes were knocked out, preventing the bacteria from synthesizing an
intact cell wall. These two genes were introduced into a corresponding plasmid and placed under the control
of the araC pBAD promoter, ensuring that the bacteria could only survive in the presence of arabinose.

Build

We inserted the sequences for asd, murA, and araC pBAD into the plasmid. After
transformation, we constructed the Salmonella strain carrying the plasmid.

![](https://static.igem.wiki/teams/5304/engineering/sm001.webp)

Figure 1. Schematic Representation of araC pBAD-asd-murA

Test

We cultured the transformed Salmonella in test tubes and media with and without arabinose.
The results showed that bacterial growth occurred only in the arabinose-containing medium, while the
bacteria could not survive in the absence of arabinose.

![](https://static.igem.wiki/teams/5304/engineering/sm002.webp)

Figure 2. Bacterial Growth in Test Tubes with LB, Arabinose,
and Kanamycin

![](https://static.igem.wiki/teams/5304/engineering/sm003.webp)

Figure 3. Bacterial Growth on LB Agar Plates with and without
Arabinose

Learn

This method allowed us to precisely control the survival conditions of Salmonella and
ensure that the bacteria undergo lysis and die after their therapeutic function is fulfilled. This design
significantly enhances the safety of the therapy, effectively preventing bacterial persistence and potential
risks post-treatment.

Cycle 2: Knockout of msbB to Reduce Bacterial Toxicity

Design

To reduce the toxicity of Salmonella and enhance its safety for
use in humans, we decided to knock out the msbB gene. The msbB gene encodes a lipid A myristoyl transferase
involved in the modification of lipid A in lipopolysaccharides (LPS). Knocking out msbB results in the
production of structurally defective LPS, significantly reducing the endotoxin's pyrogenicity and immune
stimulation.

Build

We successfully knocked out the msbB gene in Salmonella χ11802
using homologous recombination.

Test

PCR and gel electrophoresis confirmed the successful knockout of the msbB gene, and we successfully constructed the ΔmsbB χ11802
strain. This lays the groundwork for reducing strain toxicity and improving its safety.

![](https://static.igem.wiki/teams/5304/engineering/smgai004.webp)

Figure 4. BPCR Validation of msbB Gene Knockout in Salmonella

Learn

By successfully knocking out the msbB gene, we learned the
effectiveness of gene editing techniques in reducing bacterial toxicity and enhancing safety. This process
highlighted the potential of precise genetic modifications to regulate the pathogenicity of Salmonella, providing a safer option for its application in tumor therapy.

Cycle 3: Hypoxia-Induced Expression for Tumor-Specific Targeting

Design

To further enhance the specificity of Salmonella in targeting tumor tissues, we decided to
exploit the hypoxic characteristics of the tumor microenvironment. We designed a gene expression system that
is specifically activated under low oxygen conditions. We chose to use the nirB promoter, which is activated
in hypoxic environments. Our plan was to use the nirB promoter to drive the expression of the hlyA gene, ensuring that hlyA would only be expressed in the
low-oxygen tumor microenvironment, minimizing its effects on normal tissues.

Build

We placed the hlyA gene under the control of the nirB promoter.
This plasmid was then transformed into our previously constructed Salmonella strain.

![](https://static.igem.wiki/teams/5304/engineering/sm005.webp)

Figure 5. Schematic Representation of pnirB-hlyA

Test

The modified Salmonella was cultured under both hypoxic and normoxic conditions. We used
Western blot analysis to assess the expression of the hlyA gene (encoding
listeriolysin O) under these conditions. The results showed significant expression of listeriolysin O under
hypoxic conditions, while its expression was either minimal or undetectable under normal oxygen levels. This
confirms that the nirB promoter effectively drives hlyA expression in hypoxic environments.

![](https://static.igem.wiki/teams/5304/engineering/sm006.webp)

Figure 6. Listeriolysin O Expression under Hypoxic Conditions

Learn

Through this experiment, we successfully validated the inducibility of the nirB promoter
under hypoxic conditions, achieving tumor-specific expression of the therapeutic gene. This experiment
highlighted the potential of utilizing the hypoxic nature of tumors to enhance the specificity of treatment,
reducing side effects on normal tissues. We learned the importance of environment-inducible promoters in
gene therapy, providing valuable insight for future designs.

Gene regulation module

Cycle 1: Design of Bacteria-Delivered shRNA Expression Plasmid

Design

Building on last year's work, we continued to use bacteria as a vector to deliver shRNA
expression plasmids.

Build

We used an shRNA design tool to preliminarily construct an shRNA expression plasmid
targeting the CLDN6 gene.

Learn

We learned how to properly design shRNA sequences. The key to shRNA design lies in
selecting appropriate targets, avoiding off-target effects, and ensuring the stability and efficiency of the
shRNA sequence. We realized that the careful selection of sequences not only enhances gene knockdown
efficacy but also significantly reduces non-specific effects and uncertainties during the experiment. This
experience provided us with valuable foundational knowledge for future shRNA sequence design.

Cycle 2: Development of Trans-kingdom RNAi

Design

After discussing with the BUNZH-CHINA 2023 team, we learned about their trans-kingdom RNAi
(tkRNAi) system. This system enables bacteria to express shRNA, allowing the bacteria to enter tumor cells
and release shRNA to induce gene silencing. Based on our communication and their experimental results, we
concluded that the tkRNAi system might achieve better gene silencing compared to simply using bacteria as a
vector to deliver shRNA expression plasmids. Therefore, we decided to adopt their tkRNAi approach to enhance
gene silencing efficiency.

Build

We modified our shRNA expression system by incorporating BUNZH-CHINA 2023's strategy and
constructed a tkRNAi plasmid.

![](https://static.igem.wiki/teams/5304/engineering/rm001.webp)

Figure 1. Schematic Representation of tkRNAi components

Learn

Throughout this process, we learned the design principles and applications of tkRNAi. We
realized the unique advantage of tkRNAi, which uses bacteria as an RNAi delivery vector, enabling
cross-species shRNA transfer to regulate target genes. This approach offered a new perspective for our
research, highlighting the flexibility and potential of bacteria-mediated gene delivery systems in gene
regulation. The introduction of tkRNAi significantly enhanced the gene silencing effect and opened up
broader potential for further applications.

Cycle 3: Improvements for tkRNAi

Design

While designing the tkRNAi plasmid, we realized that the T7 RNA
Polymerase gene was too large, causing space limitations on the plasmid. To ensure that the
tkRNAi system would work effectively in our study, we decided to insert the T7 RNA
Polymerase gene into the genome of the previously constructed ΔmsbB
χ11802 Salmonella strain. This would free up plasmid space for other critical elements.

Build

Using gene recombination techniques, we inserted the T7 RNA
Polymerase gene into the Salmonella genome. See the experiment section
for detailed procedures.

![](https://static.igem.wiki/teams/5304/engineering/rm002.webp)

Figure 2. Schematic Representation of Genetic Modification of
Salmonella

Test

We validated the successful integration of the T7 RNA Polymerase gene into the Salmonella
genome through PCR and gel electrophoresis.

![](https://static.igem.wiki/teams/5304/engineering/srm003.webp)

Figure 3. PCR Validation of T7 RNA
Polymerase Gene Knock-in in Salmonella

Learn

By integrating the T7 RNA Polymerase gene into the Salmonella genome, we successfully constructed a new strain capable of expressing any
desired gene placed under the control of the T7 promoter, enabling efficient gene expression. This
modification not only expanded the functionality of our gene regulation module but also provided greater
flexibility for future experiments. It allows us to express multiple genes on the same platform,
significantly enhancing the system's adaptability and scalability.

Drug delivery module

Cycle 1: Design of Bacteria-Mediated Chemotherapy Drug Delivery via Nanoparticles

Design

During our initial brainstorming, Professor Ming Yang from the College of Basic Medical
Sciences at Jilin University suggested that if the bacteria only carried the RNAi module, its modularity and
functionality would be limited, and it would lack true innovation as an “engineered bacteria.” Since our
original goal was to address the issue of chemotherapy resistance, Yang proposed that delivering
chemotherapy drugs directly to the tumor via the engineered bacteria could be a valuable enhancement. This
led us to the idea of using nanoparticles for drug delivery.

We explored various methods for attaching nanoparticles to bacteria, including chemical
bonds, ionic bonds, or biotin-based linkages. After consulting with Professor Caina Xu from the College of
Basic Medical Sciences at Jilin University, we concluded that chemical bonding would provide the highest
feasibility and stability. Given the abundance of carboxyl groups on the surface of Salmonella, and referencing previous studies (we will add a citation later), we chose
to use chemical bonds to link the nanoparticles to the bacteria.

Build

Based on this, we decided to modify the surface of the nanoparticles with amino groups,
allowing them to form amide bonds with the carboxyl groups on the bacteria. Additionally, considering safety
and cost factors, we opted to use PLGA as the drug carrier material, with PEG-NH2 serving as the linker to
connect the nanoparticles to Salmonella.

Test

As we refined the nanoparticle preparation and linkage protocols, we discovered that using
PEG with dual modifications might cause the nanoparticles to link to one another, resulting in reduced
material efficiency.

Learn

We learned that if we could avoid using an external linker and instead treat the bacteria
itself as a functional linker, we could potentially improve the material efficiency and resolve the issue of
low material utilization.

Cycle 2: Redesign and Optimization of Nanoparticles for Chemotherapy Drug Delivery

Design

To address the issues identified in Cycle 1, we consulted with Professor Yuetao Zhang from
the College of Chemistry at Jilin University to redesign the nanoparticles and optimize their functionality.
The primary issue in Cycle 1 was that the NH2 group was located on the linker, rather than directly on the
nanoparticles. If we could modify or attach NH2 directly to the nanoparticles, this problem could be
resolved.

Build

Based on this new approach, we decided to use multi-block supramolecular polymers to
prepare the nanoparticles. To ensure safety while having the amino groups exposed on the nanoparticle
surface, we chose to utilize the amino side chains on lysine. Additionally, due to the presence of
phagocytes in the bloodstream, we needed to prevent the nanoparticles from being engulfed before reaching
the tumor tissue. Therefore, we decided to use mPEG to prevent phagocytosis. Considering these factors, we
opted to construct nanoparticles using mPEG-PLGA-PLL triblock copolymers.

Test

To ensure professionalism and safety, our team conducted experiments under the supervision
of experts from the State Key Laboratory of Supramolecular Structure and Materials at Jilin University and
professionals from the chemistry department. Ultimately, we successfully obtained the mPEG-PLGA-b-PLL block
copolymer.

![](https://static.igem.wiki/teams/5304/engineering/dm001.webp)

Figure 1. mPEG-PLGA-b-PLL in DMSO-d6

Learn

We successfully synthesized the nanoparticles. To prepare for future experiments, we loaded
cisplatin into the nanoparticles to validate their effectiveness in subsequent trials.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5304/loading0.gif)

![](https://static.igem.wiki/teams/5304/totop.webp)

![](https://static.igem.wiki/teams/5304/pagemain/experiments0000.webp)

* Cell Line and Cell
  Culture
* + Cell line
  + Cell culture
* Plasmid and Bacteria
* Construction of
  χ11803
* + msbB Knockout
  + T7 RNAP Knock-in
  + PCR Validation
* Electroporation
* + Bacteria Preparation
  + Electroporation
  + Cleaning and storage
* Plasmid Function
  Test
* + Delayed Lysis
  + hlyA Expression
  + RGD Expression
* MCF-7 Transfection
* Knock-down Validation
* Drug-resistance
  Gene
* + CLDN6 Knockdown
  + PGC1α Knockdown
* Nanoparticles
* + mPEG-PLGA-PLL
  + Drug-loaded Nps

Cell Line and Cell Culture

Cell line

The human breast cancer cell line MCF-7, the multidrug-resistant human breast cancer cell line
MCF-7/MDR, the cisplatin-resistant human ovarian cancer cell line SKOV3/CDDP and the cisplatin-resistant human
ovarian cancer cell line A2780/CDDP were provided by The Key Laboratory of Pathobiology of the Ministry of
Education, Jilin University.

Cell culture

Cell line revival

1. Remove the RPMl-1640 medium containing 10% fetal bovine serum and 1% penicilin-streptomycin
from the 4°C refrigerator. Allow it to reach room temperature. Before the experiment, sterilize the laminar flow
hood, 10 cm culture dishes pipette tips, and EP tubes by irradiating with a UV lamp. Clean the surface of the
laminar flow hood with a cotton ball soaked in 75% alcohol.

2. Retrieve the frozen cells from liquid nitrogen and quickly thaw them in a 37°C water bath.
Ignite the alcohol lamp in the laminar flow hood, then add the culture medium to the frozen cells in the laminar
flow hood. After mixing, transfer the mixture to a centrifuge and spin at 1050 rpm for 5 minutes.

3. Discard the supernatant and resuspend the pellet in the culture medium. Transfer the cells
to a 10 cm culture dish containing 6-7 mL of culture medium. Label the culture dish with the cell line and
revival date, and place it in a cell incubator at 5% CO2 and 37°C for cultivation.

Cell media exchange

1. Prepare as mentioned above.

2. When the cells have not yet fully covered the culture dish, transfer the culture dish from
the cell incubator to the laminar flow hood. Use a pipette to remove the old culture medium and wash the culture
dish with 1-2 mL of 0.01M PBS buffer. After removing the buffer, add 6-7 ml of fresh culture medium to the dish,
and transfer it back to the cell incubator for cultivation.

Cell passing

1. Prepare as mentioned above.

2. When the cell confluence reaches 80%-90%, wash the culture dish with 1-2 mL of 0.01M PBS
buffer. After removing the buffer, add 1 mL of 0.25% trypsin digestion solution to the dish for cell digestion.
Under the microscope, observe until 60%-70% of the cells detach from the dish. Add 1 mL of culture medium to
stop digestion, and use a pipette to gently detach the cells from the dish. Collect the cell suspension and
centrifuge at 1050 rpm for 5 minutes.

3. Discard the supernatant and resuspend the cells in 1 mL of fresh culture medium. Transfer an
appropriate volume of the cell suspension to a new culture dish containing culture medium. Place the dish in the
cell incubator for further experiments, or take an appropriate amount of cells for cell cryopreservation.

Cell Cryopreservation

1. Prepare as mentioned above.

2. Take the cells in logarithmic growth phase as mentioned above. Digest and collect them by
centrifugation, discard the supernatant, add a certain amount of cell cryopreservation solution, store in
cryovials, seal with plastic wrap and medical adhesive tape, label the type of cryopreservation, date, and user,
and store at -80°C freezer or in liquid nitrogen.

Plasmid Construction and Bacteria

The plasmid was designed with the following key components:

1. pnirB::HlyA::HA tag::terminator: Hypoxia-induced expression of the hlyA (encoding
listeriolysin O) fused with an HA tag under the control of the nirB promoter, allowing selective expression in
tumor microenvironments.

2. T7 promoter::shRNA::terminator: The T7 promoter drives expression of shRNA targeting the
CLDN6 gene (sh-CLDN6). The shRNA sequence (caGTGCAAGGTGTACGACTCATTCAAGAGATGAGTCGTACACCTTGCACtg) was based on a
previously validated sequence used in the lab. ShRNA sequences with no homology to human genes were used as
controls.

3. plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD::His tag: The plac(ΔlacO) promoter, a constitutive
version with the lac operator binding site deleted, ensures continuous expression of the Lpp’::OmpA(46-159)-RGD
fusion protein. This protein contains an RGD peptide for tumor targeting and a His tag for detection and
purification.

4. araC PBAD-murA-asd: Essential for survival of the delayed lysis strain χ11802, this system
is controlled by the araC pBAD promoter, driving expression of the murA and asd genes.

![](https://static.igem.wiki/teams/5304/experiments/expfig1.webp)

Figure 1. pSliencer-CLDN6

Precautions:

1. The plasmid carries a kanamycin resistance marker (KanR) for selection.

2. The plasmid was synthesized by Yunzhou Biosciences (Guangzhou) Co., Ltd, based on the
design described above. The sequence was verified by the company through sequencing, and the final construct was
delivered in lyophilized form.

3. The bacterial strain used for the experiments was the attenuated regulated delayed lysis
Salmonella enterica subspecies Serovar Typhimurium strain χ11802. The plasmids were electroporated into the
Salmonella before use.

Construction of the ΔmsbB::T7 RNA Polymerase Strain χ11803 in Salmonella χ11802

Construction of the ΔmsbB χ11802 attenuated Salmonella strain

1. Inoculate bacteria (ΔmsbB-pRE112 χ7213; χ11802) into 5 mL LB culture medium and culture on a
shaker at 200 rpm, 37℃ for 16 h.

2. Mix 400 μl of recipient strain with 800 μl of donor strain in a 1.5 mL Eppendorf tube by
pipetting. (Note: Recipient and donor strains are mixed in a 1:2 ratio)

3. Transfer the entire mixed culture onto a DAP-LB plate. Gently swirl (no need to spread with
a spreader), and incubate in the incubator at 37℃ for 20 h. (Note: The plate should be incubated upright. Due to
excess liquid, it cannot be inverted for incubation)

4. From the bacterial mat formed on the DAP-LB plate in step 3, take a small amount of the
bacterial mat with a pipette tip and perform a three-zone streak on the Cm+-LB plate, then incubate in the
incubator at 37℃ for 20 hours.

5. From the Cm+-LB plate in step 4, use a pipette tip to pick a single colony and perform
purification culture on the Cm+-LB plate. Incubate at 37℃ for 20 hours.

6. Place 1 μl of Salmonella serum on a clean slide. Use a toothpick to pick a single colony
from the Cm+-LB plate in step 5, smear it in the serum, gently mix and then observe.

7. If granular phenomena appear, which indicates Salmonella, take 200 μl of LB culture medium
into a 1.5 mL Eppendorf tube, use a pipette tip to pick a verified purified colony from the Cm+-LB plate in step
6, and culture in a shaker at 200 rpm, 37℃ for 1 h. (Note: If a uniformly turbid phenomenon appears, it
indicates Escherichia coli.)

8. Dilute the bacterial solution 1:103 and 1:104, and take 100 μl of the bacterial solution
onto a 10% sucrose-LB plate, perform uniform plating, and incubate in the incubator at 37℃ for 20 h.

9. Use a toothpick to pick a single colony from the 10% sucrose-LB plate in step 8, and
simultaneously perform corresponding spot inoculation on the Cm+-LB plate and LB plate, incubating in the
incubator at 37℃ for 20 hours. (Note: First streak the Cm+-LB plate, then streak the LB plate.)

10. If no bacteria growth appears on the Cm+-LB plate, but bacteria grow at the corresponding
position on the LB plate, it indicates the presence of target recombinant bacteria. Perform PCR on the target
recombinant bacteria (primer: msbB homologous arm), and then identify by electrophoresis. (Note: Pick the target
colony from the LB plate.)

11. If the identification in step 10 is successful, further purify the target colony on the LB
plate. Incubate in the incubator at 37℃ for 20 hours, and perform PCR and electrophoresis identification (repeat
step 10).

12. If step 11 is also successfully identified, incubate the target colony overnight and freeze
it for storage at -80℃.

Construction of the ΔmsbB::T7 RNA polymerase χ11802 attenuated Salmonella strain

1. Take bacteria (:: Ptrc T7 RNA polymerase - pRE112 χ7213; ΔmsbB-χ11802 ) in 5 mL LB culture
medium, cultured on a shaker at 200 rpm, 37℃ for 16 h.

2. Mix 400 μL of recipient strain with 800 μL of donor strain in a 1.5 mL Eppendorf tube by
pipetting. (Note: The recipient and donor strains are mixed in a 1:2 ratio)

3. Transfer the entire mixed culture onto a DAP-LB plate. Gently swirl (no need to spread with
a spreader), and incubate in the incubator at 37℃ for 20 h. (Note: The plate should be incubated upright. Due to
excess liquid, it cannot be inverted for incubation)

4. From the bacterial mat formed on the DAP-LB plate in step 3, take a small amount of the
bacterial mat with a pipette tip and perform a three-zone streak on the Cm+-LB plate, then incubate in the
incubator at 37℃ for 20 hours.

5. From the Cm+-LB plate in step 4, use a pipette tip to pick a single colony and perform
purification culture on the Cm+-LB plate, then incubate in the incubator at 37 ℃ for 20 hours.

6. Place 1 μL of Salmonella serum on a clean slide. Use a toothpick to pick a single colony
from the Cm+-LB plate in step 5, smear it in the serum, gently mix and then observe.

7. If granular phenomena appear, which indicates Salmonella, take 200 μL of LB culture medium
into a 1.5 mL Eppendorf tube, use a pipette tip to pick a verified purified colony from the Cm+-LB plate in step
6, and culture in a shaker at 200 rpm, 37℃ for 1 h. (Note: If a uniformly turbid phenomenon appears, it
indicates Escherichia coli.)

8. Dilute the bacterial solution 1:103 and 1:104, and take 100 μL of the bacterial solution
onto a 10% sucrose-LB plate, perform uniform plating, and incubate in the incubator at 37℃ for 20 h.

9. Use a toothpick to pick a single colony from the 10% sucrose-LB plate in step 8, and
simultaneously perform corresponding spot inoculation on the Cm+-LB plate and LB plate, incubating in the
incubator at 37℃ for 20 hours. (Note: First streak the Cm+-LB plate, then streak the LB plate.)

10. If no bacteria growth appears on the Cm+-LB plate, but bacteria grow at the corresponding
position on the LB plate, then it is the target recombinant bacteria. Perform PCR on the target recombinant
bacteria (primers: msbB-arms), and then identify by electrophoresis. (Note: The target colony is picked from the
LB plate.)

11. Select the target colony based on the PCR results from step 10, and perform PCR again
(primers: Ptrc T7 RNA polymerase), then identify by electrophoresis.

12. If the identification in step 11 is successful, further purify and culture the target
colony on an LB plate, incubate at 37℃ for 20 hours, and repeat PCR and electrophoresis identification (steps 10
and 11).

13. If step 12 is also successfully identified, culture the target colony overnight and stored
at -80℃ in the freezer.

14. Following the gene knockout of msbB and insertion of the T7 RNA polymerase, the modified
strain was designated as χ11803, distinguishing it from its precursor strain, χ11802.

PCR Validation of msbB Knockout and T7 RNA Polymerase Insertion

1. Primer treatment:

1.1 According to the relevant information of the primer wall, add buffer to the primer to a
final concentration of 100 μM (μmol/L), and store it in a -20℃ refrigerator as a storage solution for storage.
If used as simple cloning primers, quantitative primers, and primers for double-stranded interference, the stock
primer can be diluted to 10 μM with ddH2O2 and stored as a working solution.

1.2 Befobre adding the solution, centrifuge the prime tube briefly at 3000-4000 rpm for about 1
mintue to collect the contents at the botton of the tube.

1.3 After centrifugation, open the tube cap slightly, insert the pipette tip, and add the
solution. Deionized water or DNA-free water that has been autoclaved is acceptable, as is autoclaved ddH2O, but
it must be autoclaved. The volume of water added should be 10 times the volume of the primer tube. For example,
if the primer indicates 37uL of water to 100uM, we need to add 370uL of water to 10uM.

1.4 After adding the water, shake the primer for 10-20 minutes to ensure it is fully dissolved.

2. Prepare individual PCR systems.

3. Set up the PCR program on the machine.

3.1 Pre-denaturation: Denature double-stranded DNA to single-stranded DNA. This step is
typically performed at 95°C for a specific duration, which can be found in the reagent instructions.

3.2 Denaturation: Separate double-stranded DNA into single-stranded DNA, preparing for primer
annealing, and its usually placed at 95℃ for 30s or 15s (specific time can be referred to the reagent
instructions).

3.3 Annealing: Anneal the primer to the template, typically setting annealing temperature based
on the primer's Tm value. For example, if the primer Tm value is 60℃, the annealing temperature can initially be
set to 60℃. If the result is not satisfactory, other temperatures can be explored.

3.4 Extension: Generally performed at 72℃, DNA polymerase extends from the binding site of the
primer on the template, and synthesize a new DNA strand from the 5' end to the 3' end.

3.5 Complete extension: Extend the reaction at 72°C for 5-10 minutes to ensure that all DNA
strands are fully synthesized, completing the extension of all double-stranded DNA.

Place the sample into the PCR instrument and set up the program. The specific settings should
be based on the specific circumstances.

![](https://static.igem.wiki/teams/5304/experiments/exptable1.webp)

4. Agarose Gel Electrophoresis

4.1 Preparation of Electrophoresis buffer: 1xTAE (take 20 mL of 50xTAE and add ddH2O to make a
final volume of 1L).

4.2 Preparation of agarose gel: Choose an appropriate gel concentration based on the desired
size of the DNA fragment (or the amplified DNA fragment). Generally, prepare a 1% agarose gel. For example,
dissolve 0.2g of agarose in 20 mL of 1xTAE by heating, and dissolve 0.4g of agarose in 40 mL of 1xTAE by
heating. Heat the mixture in a microwave until boiling, then cool it down to a temperature that is not hot to
touch.

4.3 Add nucleic acid dye:

 &nbspa. For small wells, add 0.2g of agarose, 20 mL of 1xTAE, and 2μL of nucleic acid dye.

 &nbspb. For medium wells, add 0.4g of agarose, 40 mL of 1xTAE, and 4μL of nucleic acid
dye.

 &nbspc. For large wells, add 0.8g of agarose, 80 mL of 1xTAE, and 8μL of nucleic acid dye.

4.4 Install combs and slide gently pour the gel solution into the wells from the bottom right,
cooling it down. Try to avoid trapping air bubbles and wait for solidification (about 20 minutes). Remove the
gel after solidification.

4.5 Loading samples: Place the gel with the plate into an appropriate electrophoresis chamber,
then pour in 1xTAE buffer, ensuring that the gel is submerged by 1mm. Choose an appropriate sample volume:

 &nbspPCR products: 2μL for a 20μL reaction system, and 4μL for a 50μL reaction system.

 &nbspDNA extraction products: Mix 5μL of the product with 1μL of 6x loading buffer (to
ensure a 1x concentration of loading buffer). Carefully pipette 2μL of the mixture into a sample well.

 &nbspMarker: Choose a marker of approximately 100bp (within 1000bp) or 1000bp (within
10000bp) based on the size of the amplified products and add 1μL.

4.6 Run the gel: After covering the chamber and connecting the power supply (pay attention to
the polarity), set the voltage to 60-100V and let it run for 20-40 minutes.

4.7 After completion, observe the gel bands and their positions using a gel imaging system to
determine if the desired bands have been amplified.

Electroporation of Designed Plasmids into χ11803

Preparation of Electrocompetent Bacterial Cells

Day 1:

1. Using a pipette tip or inoculation loop, pick a single colony and inoculate it into a
culture tube containing 5mL of LB liquid medium. Prepare two tubes to ensure a backup. Incubate overnight (14-16
hours) at 37°C with shaking at 220 RPM to allow for bacterial growth.

2. Sterilize large centrifuge bottles (250-500mL) and several 50mL centrifuge tubes for use the
next day to collect the bacteria by centrifugation.

3.Prepare several bottles of sterilized water (note: use ultrapure water, total volume of about
1.5 liters),
and store them in the freezer for use the next day to resuspend the bacterial cells at a lower
temperature.

4. Prepare 10% glycerol using ultrapure water.

Day 2:

1. Take 2-5mL of overnight culture and inoculate into 500mL of LB (or 2XTV) liquid medium,
controlling the initial OD600 to be between 0.03 and 0.05 for uniform growth. Incubate at 37°C with shaking at
220 RPM for 2-4 hours, measure the OD every half hour, and stop the culture when the OD value reaches 0.5.

2. Pre-chill the bacterial solution on ice for 30 minutes. At the same time, turn on the
centrifuge and pre-cool it to 4°C to maintain low temperatures during centrifugation.

3. Transfer the bacterial solution into pre-chilled 250mL or 500mL centrifuge bottles and
centrifuge at 4°C, 4000 RPM for 15 minutes.

4. Discard the supernatant. Firstly resuspend the pellet with a small amount (e.g., 20-50mL) of
sterilized ice water. Then dilute with water to about 2/3 of the volume of the centrifuge bottle, fully
resuspending the cells (can be done by shaking the bottle by hand). Centrifuge at 4°C, 4000 RPM for 15
minutes.

5. Discard the supernatant, add a small amount of sterilized water to resuspend the bacterial
cells, then add water to suspend all cells in about 500mL of ice water. Centrifuge at 4°C, 4000 RPM for 15
minutes.

6. Discard the supernatant, add a small amount of sterilized 10% glycerol (pre-cooled) to the
centrifuge tube, resuspend the bacterial cells. Then add 10% glycerol to a final volume of about 20mL.
Centrifuge at 4°C, 4000 RPM for 10 minutes.

7. Carefully discard the supernatant (the pellet may be very loose), add 2mL of sterilized 10%
glycerol (pre-cooled) to resuspend the cells.

8. Dispense the resuspended bacterial solution into 1.5mL EP centrifuge tubes at 200μL/tube,
quickly freeze in liquid nitrogen, and then store at -70°C freezer.

9. To test the transformation efficiency, take 100μL of freshly prepared competent cells, add
0.01ng of known concentration plasmid DNA, electro-transform, and then plate 10μL (1%) and 100μL (10%) of the
cells resuspended in 1mL of SOC medium to estimate the transformation efficiency.

wqhdsg

Brief Process for Preparing Competent Cells:

Collect cells, rinse with ice water twice, rinse with 10% glycerol once, resuspend cells in 10%
glycerol, and dispense.

Electroporation

1. Remove competent cells from the -80°C freezer and thaw on ice.

2. Place the sterile electroporation cuvette on ice to pre-cool.

3. Transfer the thawed competent cells to a pre-cooled 1.5mL centrifuge tube at a volume of
40~100µL per tube, mix gently, and place on ice.

4. Take 1-2µL of purified plasmid or cloning ligation product into a 1.5mL centrifuge tube, and
place on ice for 10 minutes. If the volume of added DNA is too large, the salt in it can cause electric sparks
during electroporation!

5. Turn on the electroporator [BIO-RAD GENE PULSER SYSTEM], set to MANUAL, and adjust the
parameters to 25µF, 200 Ohms, with a voltage of 2.0KV (these settings can be slightly adjusted based on
experience and the specific protocol).

6. Transfer the mixture to the pre-cooled electroporation cuvette, gently tap the cuvette to
ensure the mixture evenly enters the bottom of the electrode cuvette. Wipe the outer wall of the cuvette with a
piece of absorbent paper to remove any condensation.

7. Push the cuvette into the electroporation apparatus, press the PULSE button, and after
hearing the beep, quickly add 2X500µl of SOC liquid medium to the cuvette, resuspend the cells, and transfer to
a 1.5mL centrifuge tube.

8. Resuscitation at 37°C, 220-250RPM for 1 hour.

9. Centrifuge, plate, incubate at 37°C overnight. Check the transformation results the next
day.

Cleaning and storage of electroporation cuvettes

1. Rinse the used cuvettes with tap water, add 75% isopropyl alcohol to rinse the cuvettes.

2. Discard the alcohol, rinse 2~3 times with distilled water, and then use a 1mL pipette to
repeatedly blow and suck the cuvette with ultrapure water more than 10 times.

3. Add 1-2mL of anhydrous ethanol to the cuvette, soak for 5 minutes.

4. Discard the anhydrous ethanol, and invert the cuvette on absorbent paper to allow the
ethanol to fully evaporate.

5. Cover the cleaned and dried cuvette and store it for future use.

6. The RP4 plasmid is large, with a size of 60KPB, and the quality of the plasmid needs to be
of a certain standard. It is best to desalt the extracted plasmid.

Functional Validation of Plasmids in χ11803

Validation of the Delayed Lysis System

1. Liquid Culture Growth:

1.1. Inoculate χ11803/pSilencer-CLDN6 (Salmonella strain χ11803 with plasmid pSilencer-CLDN6)
into three sterile test tubes, one containing 5 mL of LB medium, one with 5 mL of ara medium (LB with
arabinose), and one with 5 mL of carb medium (LB with carbenicillin).

1.2. Incubate the cultures at 37°C for 24 hours with shaking (220 RPM).

1.3. After 24 hours, visually inspect the turbidity of each tube to assess bacterial growth.

1.4. Record the growth observations for each medium.

2. Solid Culture Validation:

2.1. Prepare LB agar plates, ara plates (containing arabinose), and carb plates (containing
carbenicillin).

2.2. Inoculate χ11803/pSilencer-CLDN6 onto the surface of each type of agar plate using the
streak plate method.

2.3. Incubate the plates at 37°C for 24 hours.

2.4. After 24 hours, observe bacterial colony formation on each plate. Record which plates show
growth and which do not.

Validation of Anaerobic-Induced hlyA Expression by Western Blot

Anaerobic Treatment

1. Bacterial Culture Preparation:

1.1. Add 5 mL of LB liquid medium and 50 μL of bacterial solution to a sterile 12 mL culture
cube.

1.2. Incubate the culture for 5 hours at 37°C with shaking at 220 RPM.

2. Prepare Low-Oxygen Conditions:

2.1. Prepare a 100 g/L Na₂SO₃ solution using sterilized water, filter it through 0.2 μm
membranes for sterilization.

2.2. Prepare 5 mL of LB liquid medium containing 1 g/L Na₂SO₃.

2.3. Pre-reduce the medium by placing it in an anaerobic tank with anaerobic bags at room
temperature for at least 10 hours to absorb dissolved oxygen thoroughly.

3. Centrifugation:

3.1. After the initial culture period, centrifuge the bacterial culture at 3000×g at room
temperature for 5 minutes.

3.2. Discard the supernatant.

4. Transfer Bacteria:

4.1. Resuspend the bacteria in the low-oxygen medium prepared in step 2.2.

4.2. Transfer the resuspended bacteria into the medium treated for hypoxia as quickly as
possible.

5. Anaerobic Incubation:

5.1. Place the culture cube containing the resuspended bacteria in an anaerobic tank with
anaerobic bags.

5.2. Incubate overnight at 37°C with shaking.

6. Sample Collection for Western Blot:

6.1. After incubation, collect 100 μL of the bacterial solution for Western blotting to
validate hlyA gene expression.

Western Blot Analysis of hlyA (Listeriolysin O) Expression

1. Prepare TBST buffer

1.1 Take a sterile container and add 1L of 10X TBS (10X TBS buffer (pH 7.4): 1.37 M NaCl, 27 mM
KCl, 100 mM Tris buffer)

1.2 Dilute the 10X PBS buffer to a 1X concentration using distilled water, and stir well.

1.3 Add 0.1% Tween-20 (the “0.1%” refers to the volume fraction, meaning add 1 mL of Tween-20
to 1L of 1X PBS buffer), continue stirring to mix thoroughly.

1.4 Filter or centrifuge the prepared TBST buffer to remove any particles or impurities that
may be present.

1.5 Aliquot the buffer into small sterile bottles to avoid repeated freeze-thaw cycles, and
store in a cool place at room temperature (or 4 degrees Celsius).

2. Sample preparation

Direct lysis method:

2.1. Pre-cool all reagents and consumables in an ice box.

2.2. Take out the culture dish with 80~90% cell confluence. Wash the cells three timeswith PBS,
and use a pipette to remove the PBS, then add 0.5ml of protein lysis buffer containing PMSF. Transfer to an EP
tube.

2.3. Incubate the EP tube on ice for 30 minutes, shaking every 5 minutes EP tube, then
centrifuge at maximum speed for 15 minutes at 4℃.

2.4. Take the supernatant and transfer it to a new pre-chilled EP tube, label it, and prepare
for BCA protein concentration measurement.

3. BCA protein concentration measurement

Perform protein concentration determination according to the instructions of the BCA protein
quantification kit from Biotime.

3.1. Take the extracted protein and protein standards out of the refrigerator and place them in
a pre-prepared ice box.

3.2. Dilute the thawed protein at a ratio of 1:10 (cells are 1:10), then centrifuge the diluted
protein to mix.

3.3. Prepare the solution by mixing reagent A and reagent B from the BCA kit according to the
ratio specified in the instructions.

3.4. Take out the 96-well plate and add 20μL to each well in the order of protein standards and
the protein to be measured.

3.5. Add 200μL of the A and B mixed solution to each well, protect from light, and incubate at
37℃ for 30 minutes. Measure the OD value at 560nm using a microplate reader.

3.6. Create a standard curve and calculate the concentration of the protein to be measured
based on the standard curve and the OD value of the sample.

Note: The optimal protein concentration is 3-5μg/μL

4. Protein denaturation

4.1. Take the extracted protein out of the refrigerator and place it in a freezing box to thaw.

4.2. Calculate the required amount of protein and lysis buffer based on the protein
concentration, and add loading buffer for quantification.

4.3. Place the protein in a constant temperature device at 100℃ and boil for 5 minutes.

4.4. Allow the boiled protein to cool to room temperature, then place it on ice for later use.

5. Gel preparation

Prepare 12% separation gel or 5% concentration gel according to the table below, and pour into
the electrophoresis solution. The sample loading amount should be suitable at 50~80μg;

![](https://static.igem.wiki/teams/5304/experiments/exptable2.webp)

5.1. After assembling the gel pouring device, water can be poured onto the glass plates to
check for leaks, then invert and air dry for about 10 minutes.

If there is a gel leak: check if the device is tightly secured and if the gel strips are aged;

If the glass plates are not cleaned properly, it may lead to uneven gel or trailing bands.

5.2. Choose an appropriate concentration of separation gel based on the size of the proteins to
be run.

5.3. With the long plate inside and the short plate outside, after adding the separation gel,
add 1 ml of water or anhydrous ethanol to seal it. After 30 minutes, pour out the sealing liquid, and once the
gel is dry, pour out the excess. After adding the concentration gel, wait for 30 minutes until the gel is
completely solidified for subsequent electrophoresis.

6. Electrophoresis

6.1. Take the cooked protein sample out of the refrigerator and place it in a box to thaw.

6.2. Heat the thawed protein again at 100℃ for 5-10 minutes.

6.3. Add protein maker and protein samples into the SDS-PAGE gel.

6.4. 80V constant voltage electrophoresis, wait for the bromophenol blue to reach the interface
of the two layers, then switch to 120V constant voltage electrophoresis.

7. Transfer membrane (PVDF membrane)

7.1. First, take out the gel and cut it as needed. Assemble the transfer device according to
the filter paper-gel-membrane-filter paper sandwich structure.

7.2. Add pre-cooled transfer buffer, ice pack, and place it in the ice box.

7.3. For wet transfer, set the power supply to a constant voltage of 100V for 90 minutes. For
semi-dry transfer, set the current to 2.5 times the membrane area (mA).

Tips: Pay attention to the assembly order of the transfer device and check whether the positive
and negative power supply is correct; otherwise, it may result in no bands.

8. Milk blocking

8.1. Take out the PVDF membrane.

8.2. Place it in the incubation box pre-filled with milk, and slowly shake at room temperature
for 1 hour.

8.3. Rinse the hybridization membrane with TBST buffer to remove residual blocking solution.

Tips: Common blocking agents: 5% non-fat milk, 3% BSA.

9. Antibody hybridization

9.1. Primary antibody hybridization: incubate at room temperature overnight at 4°C.

9.2. Discard the primary antibody or recover the primary antibody solution, add TBST to wash
the membrane, 10 min each time, three times.

9.3. Secondary antibody hybridization: place the secondary antibody in the incubation box and
incubate at room temperature for 1 hour.

10. Development

10.1. Discard the secondary antibody solution, wash the membrane with TBST three times, 10 min
each time.

10.2. Remove excess liquid, place the membrane in the chemiluminescence imaging system, and
evenly add ECL detection solution.

10.3. Analyze using Genesys analysis software.

Tips: Before using BST, ensure pH is between 7.4-7.6, and ECL luminescent solution must evenly
cover the membrane surface.

Precautions:

1. After electrophoresis, the electrophoresis buffer in the tank can be recovered (be careful
to distinguish it from new buffer).

2. After cleaning the gel plates, dry them and place them on the gel plate rack. Return the
electrophoresis tank to its original position and turn off the electrophoresis apparatus.

3. The transfer tank should be cleaned promptly after use, and it can be returned to the drawer
only after drying.

4. Dispose of staining solutions, gels, membranes, and waste promptly after the experiment.

5. After the experiment, return all items to their original positions and wipe off any water
stains caused by the experiment.

Validation of RGD Peptide Expression on Salmonella by Flow Cytometry

1. Grow the Salmonella strain expressing RGD peptide in LB medium to an OD600 ≈ 0.8.

2. Take 1 mL of the bacterial culture (approx. 1.0 × 10⁹ CFU) and centrifuge at 4000 × g for 10
minutes to harvest the cells.

3. Wash the bacterial cells twice with PBS. Fix the cells with 4% paraformaldehyde for 15-20
minutes at room temperature.

4. After fixation, incubate the bacterial cells overnight at 4°C with a mouse anti-His tag
monoclonal antibody (A00186, GenScript) diluted in PBA (PBS with 3% BSA).

5. Wash the cells thoroughly with PBS to remove the primary antibody. Resuspend the cells in
PBA containing 3% BSA and incubate with goat anti-mouse IgG Alexa Fluor 488-conjugated antibody (A-11001,
Invitrogen) for 1 hour at room temperature. Protect from light.

6. Wash the cells again with PBS and resuspend them in 500 μL of PBS for flow cytometry
detection.

7. Detect the fluorescence signal using the FITC channel of the flow cytometer (NovoCyte 1030,
Agilent, USA). Set the forward scatter (FSC) detection threshold to 1000 and record 100,000 events for analysis.

MCF-7 Cell Transfection with pCLDN6-GFP

The plasmid pCLDN6-GFP was provided by The Key Laboratory of Pathobiology of the Ministry of
Education, Jilin University.

![](https://static.igem.wiki/teams/5304/experiments/expfig2.webp)

Figure 2. pCLDN6-GFP

1. Cell Preparation

Plate the MCF-7 cells in a 24-well plate with 70%‐80% confluence.

2. Preparation of DNA Solution

Dilute the plasmid DNA with EP buffer to a concentration of 1 μg/μL. Then, take a sterile 1.5mL
centrifuge tube, adding 30μL of plasmid DNA stock solution and 470μL of EP buffer to obtain a final volume of
500 μL of DNA dilution solution. Each well of cells will be transfected with 300 μL of DNA dilution solution.

3. Preparation and Disinfection of Electrodes

3.1 Take a clean 24-well plate and prepare 70% ethanol disinfectant in one well, leaving one
well empty, and filling two wells with EP buffer.

3.2 Pre-UV irradiate the electrodes for 5-10 minutes in the workspace.

3.3 Immerse the electrodes in 70% ethanol disinfectant to disinfect and sterilize them.

3.4 Transfer the electrodes to an empty well to air dry.

3.5 Transfer the electrodes to EP buffer, and gently shake them to clean them from ethanol.

3.6 Transfer the electrodes to another well of EP buffer and repeat step 5 to clean off any
remaining ethanol. Leave the electrodes in the well for later use.

4. Pre-treatment of Culture Medium: Prior to the experiment, preheat EP buffer and culture
medium to 37℃.

5. Electroporation Experiment

5.1 Wash the cells with EP buffer 1-2 times.

5.2 Remove the EP buffer from the targeted well and add 300 μL of DNA dilution solution.

5.3 Place the electrode at the bottom of the targeted well and stabilize it with your fingers.

5.4 Set the electroporation parameters, then measure and record the resistance. The conditions
are 2500-3000 V, 200 Ω, 25 μF. and start the electroporation program.

5.5 Lift the electrode to separate it from the cells in the well, and rotate it 90°, then place
it back in the well to increase the transfection area.

5.6 Measure and record the resistance again, and perform the electroporation program. Record
the current, energy, and other parameters displayed by the instrument after the second electroporation program.
These data can be used for troubleshooting. Collect and save the DNA dilution solution in the original 1.5mL
centrifuge tube.

5.7 Perform electroporation for the next targeted well, following steps 1-7.

5.8 After the experiment, clean the electroporation cuvette: rinse with ddH2O first, then rinse
with 75% ethanol, and finally soak in 75% ethanol. Typically, the electroporation cuvette is soaked in 75%
ethanol. Before use, the electroporation cuvette needs to be exposed to UV irradiation for 15-30 minutes.

Validation of Drug Resistance-caused Gene Knock-down by Salmonella-mediated RNAi

Bacterial Infection

1. Inoculate CLDN6-overexpressing MCF-7 cells in a culture dish and culture them until they
reach an appropriate state and density (approximately 70-80% confluency).

2. Take 10 μL of χ11803/pSilencer-CLDN6 bacteria from glycerol preservation and add it to 5 mL
of LB liquid medium. Incubate at 37°C with shaking at 220 rpm for 12 hours to allow bacterial expansion.

3. Transfer the bacterial solution to 50 mL LB liquid medium and grow until the OD600 reaches
0.5 (approximately 1.6×10⁸ CFU/mL).

4. At least 30 minutes before co-incubation, replace the cancer cell medium with DMEM basic
culture medium containing 10% FBS but without antibiotics.

5. Take the appropriate amount of bacterial solution (based on the required MOI value),
centrifuge the solution, and wash the bacteria twice with 1×PBS. Resuspend the bacteria in DMEM without
antibiotics or serum.

6. Add the resuspended bacterial solution to the cancer cells at the desired MOI. Co-incubate
the bacteria and cancer cells at 37°C for 2 hours.

7. After 2 hours of co-incubation, wash the cancer cells twice with 1× PBS. Wash once with DMEM
culture medium containing antibiotics.

8. Continue to culture the cancer cells in a 37°C, 5% CO2 incubator with saturated humidity for
24 hours.

CLDN6 Protein Expression Analysis

After the 24-hour incubation, collect the cancer cells for Western Blot analysis.

Effects on Drug-Resistant Tumor Cells After Gene Knockdown

Effects on MCF-7/MDR After CLDN6 Knockdown

Co-Culture of χ11803/pSilencer-CLDN6 and MCF-7/MDR Cells

1. Seed MCF-7/MDR cells in 24-well plates at a density of 5×10⁵ cells per well and incubate for
12 hours.

2. Take 10 μL of χ11803/pSilencer-CLDN6 bacteria from glycerol preservation and add to 5 mL LB
liquid medium. Incubate at 30°C with shaking at 200 rpm for 12 hours.

3. Transfer the bacterial solution to 50 mL of LB liquid medium and grow until OD600 reaches
0.5.

4. Add the χ11803/pSilencer-CLDN6 bacteria to the MCF-7/MDR cell culture at a MOI (Multiplicity
of Infection) of 1:150.

5. Co-culture the cells and bacteria for 5 hours. After 5 hours, wash the cells three times
with sterile PBS.

6. Replace the culture medium with complete DMEM containing 5% fetal bovine serum (FBS) and 50
μg/mL gentamicin, and continue culturing the cells for an additional 24 hours.

CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to Chemotherapy Drugs

1. Cell Preparation:

1.1 Prepare a cell suspension and perform cell counting.

1.2 Adjust the cell density to 3000 cells/100 μL.

1.3 Seed 100 μL of cell suspension per well into a 96-well plate.

1.4 Incubate the cells overnight in a 37°C incubator with 5% CO2.

2. Drug Treatment:

2.1 On the following day, add 100 μL of various concentrations of the chemotherapy drugs to
each well.

2.2 Continue to incubate the cells for 48 hours at 37°C.

3. CCK-8 Reagent Addition:

3.1 After 48 hours of incubation, carefully remove the medium from each well.

3.2 Prepare the CCK-8 working solution by diluting CCK-8 reagent with serum-free medium at a
ratio of 9:1.

3.3 Add 100 μL of the diluted CCK-8 working solution to each well.

4. Incubation and Measurement:

4.1 Incubate the plate for 1-2 hours at 37°C.

4.2 Measure the absorbance at 450 nm using a microplate reader.

5. Data Analysis:

5.1 Calculate the cell growth inhibition rate for each group.

5.2 Use GraphPad Prism software to calculate the IC50 value, which reflects the sensitivity of
the cells to the drugs and indicates chemotherapy resistance.

Western Blot Analysis of Drug Resistance-Related Protein Expression

The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug
resistance-associated protein 1 (MRP-1) was validated through Western blot after co-culture. (The detailed
procedure for Western blot has been described previously under the section 'Validation of Anaerobic-Induced hlyA
Expression by Western Blot.' Please refer to that section for specific steps.)

Annexin V/PI Staining to Assess the Effect on Cell Apoptosis

1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a
concentration of 1 x 10^6 cells/ml.

2. Transfer 100 µl of the solution (1 x 10^5 cells) to a 5 ml culture tube.

3. Add 5 µl of FITC Annexin V and 5 µl PI.

4. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.

5. Add 400 µl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

Western Blot Analysis of Apoptosis-Related Protein Expression

The expression levels of cleaved-PARP and cleaved-caspase-9 were analyzed in MCF-7/MDR cells
with different treatments using Western blot, following the steps previously described. (The detailed procedure
for Western blot has been described previously under the section 'Validation of Anaerobic-Induced hlyA
Expression by Western Blot.' Please refer to that section for specific steps.)

Effects on SKOV3/CDDP and A2780/CDDP After PGC1α Knockdown

To further validate the efficacy of our system, we selected two additional cisplatin-resistant
ovarian cancer cell lines, SKOV3/CDDP and A2780/CDDP. We targeted the PGC1α gene (shRNA sequence:
GTTATACCTGTGATGCTTT) to evaluate its role in chemotherapy sensitivity, drug resistance, and its effect on
apoptosis in SKOV3/CDDP and A2780/CDDP cells. Chemotherapy sensitivity was assessed using the CCK-8 assay to
measure cell viability after treatment with cisplatin. Apoptosis was evaluated using Annexin V/PI staining to
quantify the apoptotic rate. Western blot analysis was performed to detect the expression of apoptosis-related
proteins, including BAX, caspase-9, cleaved-caspase-3, and the anti-apoptotic protein BCL-2. The detailed
experimental procedures have already been described in earlier sections.

Construction of Drug-loaded Nanoparticles

Preparation of mPEG-PLGA-PLL

1. Preparation of mPEG-PLGA:

Vacuum heat-dry a heat-resistant glass tube, add a certain molar mass ratio of lactide /
glycolide raw materials
(ratios of 8:2, 7:3, 6:4, 5:5), then add PEG accounting for 1% to 20% of the total mass of the raw materials,
with a
molecular weight range of 350 to 5000, followed by the addition of a catalyst, nitrogen gas purging, heating to
dissolve, vacuum extraction, cooling and solidifying, vacuum sealing the tube for 2 hours, and reacting at
120-150°C for
8-50 hours.

2. Preparation of mPEG-PLGA-Boc(Z):

A certain amount of mPEG-PLGA is dissolved in a dry organic solvent, stirred, and
BOC-L-phenylalanine (1~15 eqv.) and N, N-dicyclohexylcarbodiimide (1~15 eqv.) are added dropwise at a slow rate
while maintaining a temperature of 0~40℃. Afterward, 4-dimethylaminopyridine is added, nitrogen protection is
applied, and the mixture is stirred at room temperature for 1~3 days. The solution is then filtered, washed with
alkaline water (or dialyzed), concentrated, and the product is precipitated by adding ice methanol or ice ether,
followed by filtration and vacuum drying.

3. Preparation of PEG-PLGA-NH2:

Dissolve mPEG-PLGA-Boc(Z) in a dry organic solvent under nitrogen protection, stir at 0℃ while
adding dry trifluoroacetic acid dropwise over 10 to 60 minutes, continue the reaction for 1 to 3 hours, and
remove the solvent and unreacted trifluoroacetic acid by rotary evaporation. Dissolve the residue in an organic
solvent, precipitate with ice methanol or ice ether, filter, and dry under vacuum.

4. Preparation of mPEG-PLGA-PZLL:

Dissolve PEG-PLGA-NH2 in a dry organic solvent, add N-carboxyanhydride (NCA) (1 to 60 eqv.),
protect under nitrogen, react at room temperature for 1 to 5 days, concentrate, precipitate with ice methanol or
ice ether, filter, and dry under vacuum.

5. Preparation of mPEG-PLGA-PLL:

Dissolve mPEG-PLGA-PZLL in a measured amount of trifluoroacetic acid, add a small volume
fraction of 33% hydrobromic acid (HBr) acetic solution, react at 0℃ for 0.5-8 hours, precipitate with ice
methanol or ice ether, filter, and vacuum dry. Grafting of Arginine-Glycine-Aspartic acid (RGD): Dissolve
mPEG-PLGA-PZLL in dimethyl sulfoxide, then add RGD and N,N'-carbonyldiimidazole (CDI), stir and react for 2-72
hours. After the reaction, dialyze the solution in a dialysis bag for 3-96 hours, then freeze-dry for storage.

Preparation of drug-loaded mPEG-PLGA-PLL nanoparticles

Prepared by the emulsification-evaporation method:

1. Dissolve the material mPEG-PLGA-PLL in a mixed solvent of dichloromethane or a mixture of
dichloromethane and acetone, and add a solution of cisplatin.

2. After ultrasonic emulsification, add to a solution containing F68, and sonicate again.

3. Stir at room temperature for 0.5-5 hours to remove the organic phase, yielding mPEG-PLGA-PLL
nanoparticles loaded with cisplatin.

4. ADM and 5-FU are encapsulated in the same manner.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5304/loading0.gif)

![](https://static.igem.wiki/teams/5304/totop.webp)

![](https://static.igem.wiki/teams/5304/pagemain/results0000.webp)

* Construction of the ΔmsbB::T7 RNA
  Polymerase Strain χ11803 in Salmonella χ11802
* Assessment
  of LPS Activity in χ11803
* Functional Validation of Plasmids in χ11803
* + Delayed Lysis System
  + Anaerobic-Induced hlyA Expression
  + RGD Peptide Expression
* Validation
  of Drug Resistance-caused Gene Knock-down by Salmonella-mediated RNAi
* Effects on MCF-7/MDR Cells After Gene Knockdown
* + CCK-8 Assay to Assess MCF-7/MDR Cells
    Sensitivity to Chemotherapy Drugs
  + Western Blot Analysis of Drug
    Resistance-Related Protein Expression
  + Apoptosis Rate Detection by Annexin V/PI
    Staining
  + Western Blot Analysis of
    Apoptosis-Related Protein Expression
* Effects on SKOV3/CDDP and A2780/CDDP After PGC1α Knockdown
* + Chemosensitivity Evaluation by CCK-8
    Assay
  + Apoptosis Rate Detection by Annexin V/PI
    Staining
  + Western Blot Analysis of
    Apoptosis-Related Protein Expression
* Construction of Drug-loaded Nanoparticles
* + Synthesis of Hydroxyl-Terminated
    mPEG-PLGA
  + Synthesis of Boc-L-Phe-Terminated
    mPEG-PLGA
  + Removal of the Boc Protecting Group
  + Synthesis of
    mPEG-PLGA-b-Poly(Nε-(Z)-L-lysine)
  + Removal of the Nε-(Carbobenzyloxy)
    Protecting Group
  + Characterization of mPEG-PLGA-b-PLL
    Block Copolymer
  + Preparation and Characterization of
    Chemotherapeutic Drug-Loaded mPEG-PLGA-PLL Nanoparticles

# Construction of the ΔmsbB::T7 RNA Polymerase Strain χ11803 in Salmonella χ11802

A delayed-lysis attenuated strain of Salmonella χ11802 was used as the
foundation. The strain has deletions in the asd and murA genes,
which are critical for bacterial cell wall synthesis. These genes were reintroduced on a plasmid regulated by the
arabinose operon. Building on χ11802, we generated the new χ11803 strain by knocking out the msbB gene and inserting the T7 RNA polymerase gene at the same locus
using homologous recombination. This modification further reduces the strain's virulence while allowing it to
express T7 RNA polymerase. PCR analysis confirmed the successful construction. As shown
in Figure 1, the msbB band (~982 bp) is absent in the modified strain, while a distinct band corresponding to T7
RNA polymerase (~2749 bp) is present, confirming the gene replacement.

These results validate the successful creation of the ΔmsbB::T7 RNA
Polymerase strain χ11803.

![](https://static.igem.wiki/teams/5304/result/res001.webp)

Figure 1. Colony PCR was used to confirm the knockout of msbB and its
replacement with T7 RNA polymerase
① WT; ② △msbB; ③ ΔmsbB::T7 RNAP; ④ marker
The PCR product length of
msbB is approximately 982 bp, while that of T7 RNA polymerase is approximately 2749 bp.

# Assessment of LPS Activity in χ11803

The LPS activity has been implicated in the safety concerns associated with Salmonella. To
assess LPS activity, we measured the ability of endotoxin to stimulate the production of pro-inﬂammatory cytokine
TNF-α. LPS was isolated from Salmonella mutants and used to induce TNF-α production in
the macrophage-like cell line RAW264.7. Stimulation of RAW264.7 cells with LPS from the △msbB χ11802 strain resulted in lower levels of TNF-α production compared to LPS from χ11802,
across concentration ranging from 10-3 to 103 pmol/L. As expected, the ability to induce TNF-α was no
significantly altered in the χ11803 (ΔmsbB::T7 RP) mutant compared to the parent strain
△msbB χ11802.

![](https://static.igem.wiki/teams/5304/result/res002.webp)

Figure 2. Analysis of the TNF-α level induced by purified LPS derived
from the Salmonella mutant strains in RAW264.7

# Functional Validation of Plasmids in χ11803

We constructed the plasmid pSilencer-CLDN6 and transformed it into the Salmonella strain χ11803
to validate the functionality of the plasmid.

![](https://static.igem.wiki/teams/5304/result/res003.webp)

Figure 3. Schematic diagram of plasmid pSilencer-CLDN6

Delayed Lysis System

To verify that the growth of the engineered bacteria can be regulated by arabinose, the
transformed bacteria were cultured in LB medium, LB medium containing arabinose, and LB medium containing
carbenicillin at 37°C for 24 hours. As expected, bacterial growth was observed only in the LB medium supplemented
with arabinose, confirming the system’s dependence on arabinose for bacterial survival and replication.

![](https://static.igem.wiki/teams/5304/result/res004.webp)

Figure 4. Growth of bacteria in LB, LB with arabinose, and LB with
carbenicillin media

Additionally, the transformed bacteria were plated on LB and arabinose-containing LB agar
plates. Consistent with the broth culture results, bacterial colonies were only observed on the plates containing
arabinose, further supporting the effective functionality of the delayed lysis system.

The results indicated that our engineered Salmonella strain dissolves
and dies as the concentration of arabinose gradually decreases, demonstrating strong safety and targeting
capabilities.

![](https://static.igem.wiki/teams/5304/result/res005.webp)

Figure 5. Growth of bacteria on LB plates and LB plates with
arabinose

Anaerobic-Induced hlyA Expression

To validate the hypoxia-inducible expression system, we cultured Salmonella χ11803 harboring the plasmid pSilencer-CLDN6 under anaerobic conditions. The
bacterial culture was transferred to a low-oxygen environment using Na₂SO₃ and pre-reduced LB medium, followed by
incubation at 37°C for 24 hours. After incubation, bacterial lysates were collected, and Western blot analysis was
performed to detect the expression of listeriolysin O, the product of the hlyA gene.

The results showed a marked increase in listeriolysin O expression in bacteria exposed to
hypoxic conditions, while the normoxic control group exhibited minimal or no expression. This confirms the
successful induction of hlyA expression in response to low-oxygen environments, validating the system’s
functionality under hypoxic conditions similar to those in tumor tissues.

![](https://static.igem.wiki/teams/5304/result/res006.webp)

Figure 6. Listeriolysin O Expression under Hypoxic Conditions

RGD Peptide Expression

To confirm the surface expression of the RGD peptide on Salmonella
χ11803, flow cytometry analysis was performed. Bacterial cultures were grown to an OD600 ≈ 0.8, and approximately
1.0 × 10⁹ CFU of bacterial cells were harvested by centrifugation. The bacterial cells were washed with PBS and
fixed using 4% paraformaldehyde. For immunofluorescence staining, the cells were incubated overnight at 4 °C with
a mouse anti-His tag monoclonal antibody (diluted in PBA containing 3% BSA), followed by incubation with a goat
anti-mouse IgG antibody conjugated with Alexa Fluor 488 for 1 hour at room temperature.

The fluorescence signal was detected using a flow cytometer in the FITC channel, with a
detection threshold of forward scatter (FSC) set to 1000 and 100,000 events recorded. The control group (χ11803
without the RGD construct) exhibited low fluorescence intensity, while the experimental group (χ11803 carrying the
RGD construct) showed a significant increase in fluorescence. This indicates successful surface expression of the
RGD peptide on the bacterial membrane.

These results validate the functional design of the system, confirming that the RGD peptide was
effectively displayed on the bacterial surface through the use of immunofluorescence staining and flow cytometry
detection.

![](https://static.igem.wiki/teams/5304/result/res007.webp)

Figure 7. Validation of RGD expression using flow cytometry

# Validation of Drug Resistance-caused Gene Knock-down by Salmonella-mediated RNAi

To establish a CLDN6-overexpressing MCF-7 cell line, we transfected MCF-7 cells with the pCLDN6-GFP plasmid using electroporation. The GFP reporter gene on the plasmid allowed us to
assess transfection efficiency via fluorescence microscopy.

![](https://static.igem.wiki/teams/5304/result/res008.webp)

Figure 8. plasmid pCLDN6-GFP

Successful transfection was confirmed by the observation of strong GFP fluorescence and gene
expression in the transfected cells, indicating effective introduction and expression of the pCLDN6-GFP plasmid in
the MCF-7 cells.

![](https://static.igem.wiki/teams/5304/result/res009.webp)

Figure 9. Fluorescence image of MCF-7 cells with plasmid pCLDN6-GFP

Before conducting the Salmonella-mediated RNA interference (RNAi) knockdown experiment, we
assessed the expression of CLDN6 protein in three cell lines: MCF-7, CLDN6-overexpressing MCF-7 (MCF-7/CLDN6), and
MCF-7/MDR through Western blot analysis. The results demonstrated that CLDN6 expression was significantly higher
in the MCF-7/CLDN6 cells compared to the parental MCF-7 cells. Additionally, CLDN6 expression was also elevated in
the MCF-7/MDR drug-resistant cells.

![](https://static.igem.wiki/teams/5304/result/res010.webp)

Figure 10. Western blot analysis of CLDN6 expression in MCF-7,
MCF-7/CLDN6, and MCF-7/MDR cell lines

To assess the efficacy of Salmonella-mediated RNA interference (RNAi) in knocking down drug
resistance-related genes, MCF-7/CLDN6 cells and MCF-7/MDR cells were co-cultured with the engineered
χ11803/pSilencer-CLDN6 bacteria. The goal was to determine whether the bacteria could successfully deliver shRNA
targeting CLDN6 into tumor cells, thereby reducing the expression of CLDN6.

Western blot analysis was performed to evaluate the expression of CLDN6 in the treated
MCF-7/CLDN6 cells and MCF-7/MDR cells. The results, as shown in the figure, indicate that the group co-cultured
with χ11803/pSilencer-CLDN6 showed a significant reduction in CLDN6 expression compared to the control group and
other experimental conditions. Specifically, the χ11803/pSilencer-CLDN6 treatment group exhibited a marked
decrease in CLDN6 levels, demonstrating the successful shRNA delivery into tumor cells by Salmonella and effective
gene knockdown.

![](https://static.igem.wiki/teams/5304/result/res011.webp)

Figure 11. Western Blot Analysis of CLDN6 Knockdown in MCF-7/CLDN6

![](https://static.igem.wiki/teams/5304/result/res012.webp)

Figure 12. Western Blot Analysis of CLDN6 Knockdown in MCF-7/MDR

# Effects on MCF-7/MDR Cells After Gene Knockdown

CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to Chemotherapy Drugs

To evaluate whether knocking down the CLDN6 gene in drug-resistant tumor cells would enhance
their sensitivity to chemotherapy drugs, a CCK-8 assay was performed. MCF-7/MDR cells were co-cultured with
χ11803/pSilencer-CLDN6, followed by treatment with three chemotherapy drugs: ADM, 5-FU, and DDP. The results
showed that cells treated with χ11803/pSilencer-CLDN6 exhibited significantly reduced IC50 values for all three
drugs compared to the control group. This indicates that knocking down CLDN6 successfully enhanced the drug
sensitivity of tumor cells.

![](https://static.igem.wiki/teams/5304/result/res013.webp)

Figure 13. CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to
Chemotherapy Drugs

Western Blot Analysis of Drug Resistance-Related Protein Expression

After co-culturing χ11803/pSilencer-CLDN6 with drug-resistant tumor cells, Western blot
analysis was performed to assess the expression of drug resistance-related proteins: P-glycoprotein (P-gp), breast
cancer resistance protein (BCRP), and multidrug resistance-associated protein 1 (MRP-1). As shown in the figure,
the expression of both P-gp and BCRP was significantly reduced in the group treated with χ11803/pSilencer-CLDN6
compared to the negative control (shRNA-NEG). No significant change in MRP-1 expression was observed. These
results suggest that the knockdown of CLDN6 reduces the expression of key drug resistance proteins, thereby
potentially enhancing the sensitivity of tumor cells to chemotherapy.

![](https://static.igem.wiki/teams/5304/result/res014.webp)

Figure 14. Western Blot Analysis of Drug Resistance-Related Protein
Expression

Apoptosis Rate Detection by Annexin V/PI Staining

To examine the effects of CLDN6 knockdown on apoptosis, Annexin V/PI staining followed by flow
cytometry analysis was conducted. Tumor cells treated with χ11803/pSilencer-CLDN6 and cisplatin (DDP) showed a
higher apoptosis rate compared to other groups. These results suggest that the knockdown of CLDN6 enhances the
ability of chemotherapy drugs to induce apoptosis in drug-resistant tumor cells.

![](https://static.igem.wiki/teams/5304/result/res015.webp)

Figure 15. Annexin V/PI Staining to Assess the Effect on Cell
Apoptosis

Western Blot Analysis of Apoptosis-Related Protein Expression

The expression of cleaved-PARP and cleaved-caspase-9, markers of apoptosis, was significantly
increased in the χ11803/pSilencer-CLDN6 treated group, especially when combined with ADM, further validating the
effect of CLDN6 knockdown on promoting apoptosis in MCF-7/MDR cells.

![](https://static.igem.wiki/teams/5304/result/res016.webp)

Figure 16. Western Blot Analysis of Apoptosis-Related Protein
Expression

Effects on SKOV3/CDDP and A2780/CDDP After PGC1α Knockdown

We further validated the system’s effects using SKOV3/CDDP and A2780/CDDP ovarian cancer cells,
focusing on their drug resistance gene PGC1α.

Chemosensitivity Evaluation by CCK-8 Assay

The sensitivity of the tumor cells to chemotherapy drugs was assessed using CCK-8 assays. The
χ11803/pSilencer-PGC1α treated group displayed significantly lower IC50 values for cisplatin (CDDP) in both
SKOV3/CDDP and A2780/CDDP cells, indicating enhanced chemotherapy sensitivity.

![](https://static.igem.wiki/teams/5304/result/res017.webp)

Figure 17. CCK-8 Assay to Assess SKOV3/CDDP and A2780/CDDP Cells
Sensitivity to Cisplatin

Apoptosis Rate Detection by Annexin V/PI Staining

Apoptosis was measured using Annexin V/PI staining and flow cytometry. Tumor cells treated with
χ11803/pSilencer-PGC1α and cisplatin showed a higher apoptosis rate compared to other groups, demonstrating the
knockdown's effect on promoting apoptosis.

![](https://static.igem.wiki/teams/5304/result/res018.webp)

Figure 18. Annexin V/PI Staining to Assess the Effect on Cell
Apoptosis

Western Blot Analysis of Apoptosis-Related Protein Expression

Western blot analysis showed that the significant increase in caspase-9, cleaved-caspase-3,
and BAX expression, along with the decrease in BCL-2 expression, was primarily observed when
χ11803/pSilencer-PGC1α was combined with cisplatin. This further highlights the synergistic effect of PGC1α
knockdown and cisplatin in inducing apoptosis in cisplatin-resistant ovarian cancer cells.

![](https://static.igem.wiki/teams/5304/result/res019.webp)

Figure 19. Western Blot Analysis of Apoptosis-Related Protein
Expression

# Construction of Drug-loaded Nanoparticles

Synthesis of Hydroxyl-Terminated mPEG-PLGA

Through the ring-opening polymerization (ROP) reaction of D,L-lactide and glycolide catalyzed
by mPEG and stannous octoate (Sn(Oct)2), we successfully synthesized hydroxyl-terminated mPEG-PLGA. This process
was carried out in an oil bath at 130°C for 8 hours. The structural integrity of the product was confirmed by ^1H
NMR characterization (Figure 1).

![](https://static.igem.wiki/teams/5304/result/res020.webp)

Figure 20. mPEG-PLGA in CDCl3

Synthesis of Boc-L-Phe-Terminated mPEG-PLGA

Using the hydroxyl-terminated mPEG-PLGA mentioned above, we successfully converted the hydroxyl
end group into Boc-L-Phe by reacting it with Boc-L-Phe in anhydrous dichloromethane at 0°C for 48 hours, using DCC
and DMAP as reagents. After the reaction, the product was purified by filtration and washing, and recovered by
precipitation from the dichloromethane solution. This process resulted in Boc-L-Phe-terminated mPEG-PLGA (Figure
2).

![](https://static.igem.wiki/teams/5304/result/res021.webp)

Figure 21. Boc-L-Phe end-capped mPEG-PLGA in CDCl3

Removal of the Boc Protecting Group

To further functionalize the polymer, we treated Boc-L-Phe-terminated mPEG-PLGA with
trifluoroacetic acid under nitrogen protection, removing the Boc protecting group and obtaining amine-terminated
mPEG-PLGA. The product was concentrated and precipitated in methanol (Figure 3).

![](https://static.igem.wiki/teams/5304/result/res022.webp)

Figure 22. amino-terminated mPEG-PLGA in CDCl3

Synthesis of mPEG-PLGA-b-Poly(Nε-(Z)-L-lysine)

Using amine-terminated mPEG-PLGA as an initiator, Nε-(Z)-L-lysine N-carboxyanhydride (NCA)
underwent ring-opening polymerization (ROP) in dry chloroform to synthesize the block copolymer
mPEG-PLGA-b-poly(Nε-(Z)-L-lysine). The reaction was carried out at room temperature for 72 hours, and the product
was separated by precipitation in cold ether, yielding the block copolymer (Figure 4).

![](https://static.igem.wiki/teams/5304/result/res023.webp)

Figure 23. mPEG-PLGA-b-poly(Nε-(Z)-L-lysine) in DMSO-d6

Removal of the Nε-(Carbobenzyloxy) Protecting Group

The aforementioned block copolymer was treated with hydrogen bromide/acetate to remove the
Nε-(carbobenzyloxy) protecting group, yielding the mPEG-PLGA-b-PLL block copolymer. The reaction mixture was
precipitated in cold ether, resulting in the final product (Figure 5).

![](https://static.igem.wiki/teams/5304/result/res024.webp)

Figure 24. mPEG-PLGA-b-PLL in DMSO-d6

Characterization of mPEG-PLGA-b-PLL Block Copolymer

The mPEG-PLGA-b-PLL block copolymer was thoroughly characterized by FTIR and GPC, confirming
its chemical structure and molecular weight characteristics (Figures 24, 25).

![](https://static.igem.wiki/teams/5304/result/res025.webp)

Figure 25. The FTIR spectra of mPEG-PLGA-b-PLL

![](https://static.igem.wiki/teams/5304/result/res026.webp)

Figure 26. The GPC traces of mPEG-PLGA-b-PLL(Mn=41400 g/mol,
PDI=2.26)

Preparation and Characterization of Chemotherapeutic Drug-Loaded mPEG-PLGA-PLL
Nanoparticles

mPEG-PLGA-PLL nanoparticles were prepared using the ultrasonic emulsification method and
successfully loaded with cisplatin. The resulting nanoparticles exhibited a uniform spherical shape with good
dispersibility, an average particle size of 140.2 nanometers, a zeta potential of +12.1 millivolts, and a loading
efficiency of 88.45%.

{% endblock %}