iGEM 2024 — Team hubu-4-chn

## 1. Wetlab

**March 2024**

1. We learned the basic theory of synthetic biology and the experimental scheme of strain modification.
2. We received basic experimental skills training such as bacterial culture, plasmid construction, SDS-PAGE and Western blot.
3. Through literature review and discussion, we studied how to improve the targeting and adaptability of OMVs to adapt to different application scenarios.
4. We learned from the previous team's work in ensuring safety in the project and learned from it to better manage the safety management issues in the project.

**April 2024**

1. We conducted pre-experiments to explore the suitable conditions for preparing protoplasts using liposome extruders.
2. Based on previous work in the laboratory, we constructed self-assembled plasmids of Cas9 RNP and transferred them to EcN for expression (Fig.1).

![](https://static.igem.wiki/teams/5382/notebookfig1.png)

**Fig.1** Schematic diagram of Cas9 RNP synthesis process by E. coli biological self-assembly.

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

[Overview](#section1)

[Cycle 1](#section2)

[Cycle 2](#section3)

[Cycle 3](#section4)

[Cycle 4](#section5)

[Cycle 5](#section6)

[References](#section7)

![](https://static.igem.wiki/teams/5382/liu-rui.png)

# . Engineering .

## Overview

![](https://static.igem.wiki/teams/5382/file/engineer1-1.png)

Synthetic biology is an interdisciplinary science that seeks to design and build new biological parts and systems to perform specific functions. To ensure that a product meets final expectations, it needs to go through an engineering cycle of design, build, test, and learn. Our team applied this philosophy to the project by designing a targeted gene-edited delivery system based on the outer membrane vesicles of the probiotic  *Escherichia coli*  Nissle 1917 (EcN). By engineering EcN, using "self-assembly" biosynthesis to produce CRISPR/Cas9 ribonucleoprotein complex (RNP), displaying pairing elements or targeting elements on its surface, and then processing the constructed strains into protoplasts.The ideal size of bacterial outer membrane vesicles (OMVs) carrying Cas9 RNP can be obtained by liposome extruder to achieve the targeted delivery of Cas9 RNP, providing new possibilities for gene therapy.

## Cycle 1

### Self-assembly synthesis of spCas9 RNP in wild-type EcN

#### Design

The CRISPR-Cas9 system can be delivered into an organism for gene editing in the form of a plasmid vector, mRNA or Cas9 RNP. Cas9 RNP, which has advantages due to its fast editing, low off-target rate and weak immune response, has been used to develop gene therapy tools for genetic diseases. However, the traditional in vitro assembled form of synthetic Cas9 RNP is inefficient, costly, and its activity cannot be maintained stably. Here, we propose a one-step biosynthesis of Cas9 RNP via wild-type EcN using the laboraty-pioneered  *E. coli*  expression synthesis method[1] (Fig. 1).

![](https://static.igem.wiki/teams/5382/file/engineer2-2.jpg)

**Fig.1** Schematic diagram of Cas9 RNP synthesis process of  *E. coli*  by "biological self-assembly"

**Build**
Based on previous work in the laboratory, we constructed in vivo self-assembled plasmids of Cas9 RNP, in which both the expression of Cas9 protein and the transcription unit of gRNA utilize Tac promotors that can be recognized by  *E. coli*  RNA polymerase. The plasmids were transferred into wild-type EcN for expression.

**Tests**
We conducted protein purification and in vitro activity verification of EcN-expressed Cas9 RNP (Fig. 2) to test its efficiency in cutting target plasmids.

![](https://static.igem.wiki/teams/5382/file/engineer3.png)

**Fig.2** Purification effect and in vitro activity verification of Cas9 RNP

**Learning**
The experimental results show that the yield of Cas9 RNP synthesized by Tac promoter is about 1 mg per liter of fermentation medium, which is far lower than the experimental expectation. The analysis may be due to the fact that Tac promoter is not a strong promoter, and the transcriptional effect of gRNA is poor, resulting in a low level of fully assembled RNP with enzyme activity.

## Cycle 2

#### Design

We learned that wild EcN does not have a T7 RNA polymerase that can recognize the T7 strong promoter. Therefore, in order to improve the expression of gRNA and the assembly efficiency of Cas9 RNP, we designed an experiment to tap into the T7 RNA polymerase gene in the EcN genome.

**Build**
By phage homologous recombination, we inserted T7 RNA polymerase gene into EcN genome and reconstructed the expression plasmid of Cas9 RNP, in which the Cas9 protein was expressed by Tac promoter, and the transcription products of gRNA were obtained by T7 activation, and the Cas9 RNP was synthesized by self-assembly in EcN bacteria.

**Tests**
We utilized SDS-PAGE to compare the expression levels of Cas9 RNP in the newly constructed EcN strain using the T7 promoter with those of the previous cycle using the tac promoter. The results indicated that the protein expression level of the EcN strain with the T7 promoter was significantly higher than that with the tac promoter (Fig.3). Additionally, we successfully validated the gene editing activity of the Cas9 RNP expressed by the newly constructed EcN strain through in vitro cleavage assays (Fig.4).

![](https://static.igem.wiki/teams/5382/file/engineer13.jpg)

**Fig.3** Results of SDS-PAGE experiment on expression of purified CAS9 RNP

![](https://static.igem.wiki/teams/5382/file/engineer12-2.png)

**Fig.4**  In vitro cleavage of the target plasmid
Lane M: DNA Marker
Lane 1: 5483bp Target Plasmid
Lane 2: Xba I Single-Enzyme Restriction
Lane 3: spCas9 RNP Exogenous Enzyme Cleavage

In addition, OMVs were prepared by liposome extruder, purified and characterized by transmission electron microscopy and dynamic light scattering. The results showed that OMVs containing Cas9 RNP was successfully obtained by EcN self-assembly (Fig. 5), and the particle size was about 100 nM. The expression level of Cas9 RNP reached 8 mg per liter of fermentation medium, which had excellent enzyme digestion activity.

![](https://static.igem.wiki/teams/5382/file/engineer4.png)

**Fig.5** TEM image analysis and DLS analysis results of OMVs

**Learning**
The results show that EcN constructed by genetic engineering can be used as chassis cells for the subsequent preparation of OMVs delivery vector experiments.

## Cycle 3

#### Design - Path 1

The dissociation constant Kd of the Colicin E7 DNase (CE7) domain with its immune protein 7 (Im7) reaches 10^-14 to 10^-17 M. CL7, an engineered variant of CE7, loses DNase activity but maintains a high affinity with Im7. We designed this pairing element to enable easy access to a variety of targeting plug-ins (Fig.6).

![](https://static.igem.wiki/teams/5382/file/design-3new.png)

**Fig.6** Schematic diagram of the targeting plug-in pairing system for Im7 protein to CL7 fusion pair displayed on the EcN surface.

Firstly, we designed two plasmids. One displayed Im7 protein on the surface of EcN membrane, and Im7 was anchored to the cell surface by the extracellular membrane anchor protein InaK. Secondly, we constructed a CL7-fused fluorescent protein sfGFP to verify the successful display of the foreign target protein on the EcN surface. Among them, we designed 1xlinker and 3xlinker.

**Build**
We transferred the pCold-Inak-Im7 plasmid into EcN and pET23a-CL7-sfGFP into *Escherichia coli* BL21. Then, it was verified whether EcN successfully expressed Im7 protein and BL21 successfully expressed CL7-sfGFP. The engineered EcN was prepared into protoplasts, and OMVs with different pore sizes were obtained by liposome extruder.

**Tests**
We first incubated EcN engineered bacteria with Im7 protein displayed on their surfaces with purified CL7-sfGFP fluorescent protein and washed away the non-specifically bound GFP. Under a fluorescence microscope, the results (Fig.7) showed that Im7 successfully displayed and was able to bind specifically to CL7-fused proteins, and the results of experiments containing 3xlinker were significantly better than those containing 1xlinker.

![](https://static.igem.wiki/teams/5382/file/engineer6.png)

**Fig.7** (a) Bright field of view of negative control;(b) the image of the negative control under 488 nm excitation light;(c)Bright field of view in EcN/pCold-InaK-Im7 (1xlinker) experimental group;(d)Images of EcN/pCold-InaK-Im7 (1xlinker) experimental group under 488 nm excitation light,(e)bright field of EcN/pCold-InaK-Im7 (3xlinker) experimental group;(f)Images of EcN/pCold-InaK-Im7 (3xlinker) experimental group under 488 nm excitation light

Next, we incubated the OMVs obtained from the liposome extruder with purified CL7-sfGFP fluorescent protein, and washed away the non-specifically bound GFP. Observation under a fluorescence microscope showed that the results (Fig.8) were also consistent with expectations, indicating that the prepared OMVs delivery vector was able to bind the CL7 fusion protein.

![](https://static.igem.wiki/teams/5382/file/engineer7.png)

**Fig.8** (a) negative control under 488 nm excitation light, and (b) OMVs/pCold-ClyA-Im7 experimental group under 488 nm excitation light

#### Design - Path 2

In addition to the above Im7-CL7 based superaffinity pairing system, we also learned through literature review that some bacterial proteins such as *Staphylococcus aureus* protein A, Streptococcal protein G, etc., can bind[3] specifically to the Fc end of antibodies (such as IgG). Based on this, we designed the second path as follows: express these bacterial proteins on the membrane surface of EcN, and then incubate the OMVs with these proteins displayed on the membrane surface directly with the antibodies with Fc domain purchased from the company to realize the display of antibodies on the surface of OMVs. Therefore, we chose z domain as EcN surface display protein here.

![](https://static.igem.wiki/teams/5382/file/design-4new.png)

**Fig.9** Schematic diagram of the EcN surface displaying the Z domain or 4Zd domian for binding to commercial antibodies

**Build**
Plasmid construction has been completed for this part of the EcN and expression testing is under way.

**Learning**
The experimental results show that the EcN outer membrane display system constructed by genetic engineering can meet the expectations, and can display the designed paired binding elements on the surface, and verify the physical extrusion method to obtain OMVs, which displays the targeted element binding plug-in on the surface.

## Cycle 4

#### Design

EcN two-plasmid system for co-expressing Cas9 RNP and surface display elements.

**Build**
We transferred the expression plasmid of ampicillin resistant Cas9 RNP and the display plasmid of kanamycin resistant Im7 into the engineered EcN strain for expression (Fig.10), and investigated their concentration ratios in order to obtain the best protein expression levels.

![](https://static.igem.wiki/teams/5382/file/engineer9.png)

**Fig.10** Schematic diagram of the double plasmid expression system

**Tests**
By SDS-PAGE and western-blot, we demonstrated that Cas9 RNP and Im7 proteins could be successfully expressed by this two-plasmid expression system (Fig.11), and the optimal transfection volume of the plasmid was about Cas9 RNP: Im7=1:10.

![](https://static.igem.wiki/teams/5382/file/engineer10.png)

**Fig.11** Cas9 RNP and Im7 proteins were successfully expressed by the dual-plasmid expression system

## Cycle 5

#### Design

Cell genome editing experiment and targeted delivery system test.

**Build**
First, we delivered OMVs coated with Cas9 RNP into HeLa cells for testing, targeting PRDX4 gene, and verifying Indel gene editing activity; Next, the transmembrane peptide and monoclonal antibody expressed by fusion of CL7 protein were incubated with it to verify its cell targeting and gene editing activity.

**Tests**
Through the classic T7E1 experiment, we have verified that the prepared OMVs-Cas9 RNP has comparable cellular genome editing activity to the commercial liposomal transfection reagent(Fig.12).

![](https://static.igem.wiki/teams/5382/file/engineer11.png)

**Fig.12** Verification of genome editing activity in T7E1 cells

At present, we are verifying the results of cell gene editing after the insertion of the targeted element.

## References:

1. Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y. Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun Biol. 2019; 2:161.Published 2019 May 3. doi:10.1038/s42003-019-0402-x
2. Zhao, M., Cheng, X., Shao, P. et al. Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages For enhanced cancer immunotherapy. Nat Commun 15, 950 (2024).
3. Wiklander, O.P.B., Mamand, D.R., Mohammad, D.K. et al. Antibody-displaying extracellular vesicles for targeted cancer therapy. Nat. Biomed. Eng (2024).

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Hubei University, Wuhan, China
No.368,Youyi Avenue,Wuhan, P.R.China,430062

© 2024 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2024/hubu-4-chn](https://gitlab.igem.org/2024/hubu-4-chn).

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments&Results{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

[xxxxxxx](#section1)

[xxxxxxx](#section2)

[xxxxxxx](#section3)

[xxxxxxx](#section4)

[xxxxxxx](#section5)

[xxxxxxx](#section6)

[xxxxxxx](#section7)

![](https://static.igem.wiki/teams/5382/chen-yuyanyan.png)

# . Experiments&Results .

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Hubei University, Wuhan, China
No.368,Youyi Avenue,Wuhan, P.R.China,430062

© 2024 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2024/hubu-4-chn](https://gitlab.igem.org/2024/hubu-4-chn).

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

[Purification and In Vitro Activity Verification of Cas9 RNP in EcN](#section1)

[Im7 Protein Display on the Surface of EcN-Engineered Bacteria](#section2)

[Testing OMV Diameters After Extrusion](#section3)

[Validation of the Dual-Plasmid Co-Expression Module](#section4)

[Verification of Anchoring Effect After Extrusion](#section5)

[Verification of OMV Targeting Efficiency](#section6)

[Testing Cas9 RNP Gene Editing Efficiency](#section7)

![](https://static.igem.wiki/teams/5382/tian-siyi.png)

# . Results .

## 1. Purification and In Vitro Activity Verification of Cas9 RNP in EcN

![](https://static.igem.wiki/teams/5382/file/result1.png)

**Fig.1** Purification and In Vitro Activity
Verification of Cas9 RNP

a. Lane M: three-color prestained protein Marker;
Lane 1: Bacteriostatic precipitation;
Lane 2: supenatarnt with cleaved bacteria;
Lane 3: Flow-through eluent after binding to nickel beads;
Lanes 4-7: Gradient eluents of different concentrations of imidazole;
b. Lane M: DNA Marker;
Lane 1: target plasmid pCDNA3.1-Flag-PRDX4;
Lane 2: pCold-Cl7-Cas9 -P4 experimental group;
Lane 3: SPEL single digestion;

As shown in Figure 1, the purified RNPs have friendly cleavage
activity against the target plasmid.

## 2. Im7 Protein Display on the Surface of EcN-Engineered Bacteria

This experiment aimed to display the Im7 protein on the surface of
EcN cells using the affinity protein InaK (Fig.2). First, the *Escherichia coli* gene-editing
plasmid system pKD46 was used to integrate the T7 RNA polymerase gene, controlled by the lac-uv5
promoter, into the attB site of the EcN genome. As a result, an EcN strain compatible with the pET
expression system was successfully engineered. Next, the cryoprotein InaK was fused with the IM7
protein to construct a fusion protein expression plasmid, which was then introduced into the
engineered EcN strain. IPTG was added to induce protein expression, and successful production of the
InaK-Im7 fusion protein was confirmed by SDS-PAGE electrophoresis (Fig.2).

![](https://static.igem.wiki/teams/5382/file/result2.png)

**Fig.2** Induction of InaK-Im7 Fusion Protein
Expression in EcN-Engineered Bacteria

Lanes 1, 3, 5: Pre-induction; Lanes 2, 4, 6: Post-induction with
IPTG.

## 3. Testing OMV Diameters After Extrusion

To determine whether vesicles of the desired size could be obtained
through physical extrusion, we characterized the particle sizes using transmission electron
microscopy (TEM) and dynamic light scattering (DLS).

![](https://static.igem.wiki/teams/5382/file/result3.png)

**Fig.3** TEM and DLS Analysis of OMVs

The OMVs containing Cas9 RNP, obtained through EcN self-assembly
(Fig.3), were successfully characterized with a particle size of approximately 100 nm.

## 4. Validation of the Dual-Plasmid Co-Expression Module

The expression levels of Cas9 RNP and Im7 proteins were analyzed by
SDS-PAGE and western blotting.

![](https://static.igem.wiki/teams/5382/file/result4.png)

**Fig.4** Co-Expression of Cas9 RNP and Im7 Proteins
Using the Dual-Plasmid System

The results showed that the Cas9 RNP and Im7 proteins were
successfully expressed by this double plasmid expression system (Fig.4) and that the amount of
plasmid transfection was optimal at approximately Cas9 RNP:IM7=1:10.

## 5. Verification of Anchoring Effect After Extrusion

To assess the impact of physical extrusion on the anchoring of
proteins, we validated the system using Dil and sfGFP staining, followed by laser confocal
microscopy.

![](https://static.igem.wiki/teams/5382/file/result-5.png)

**Fig.5** EcN pCold-Inak-lm7 800 nm Extruded OMVs
Laser Confocal Characterization a. Dil staining localization. b. CL7-sfGFP c.
Colocalization

Due to limitations in the resolution of the equipment, we
characterized vesicles extruded at 800 nm. Approximately 40% of the vesicles showed successful
attachment of elements within the same field of view. We predict that vesicles extruded at 100 nm
would exhibit similar ligation efficiency.

## 6. Verification of OMV Targeting Efficiency

The primary component of OMVs is a phospholipid bilayer membrane,
which allows for membrane fusion with mammalian cells, facilitating intracellular delivery. To
verify the efficiency of OMVs in delivering target proteins, OMVs containing CL7-mCherry red
fluorescent protein were mixed with Im7-attached OMVs in EP tubes for 15 minutes. The mixture was
then transferred to HeLa cells for incubation. After approximately 4 hours, fluorescence microscopy
at an excitation wavelength of 534 nm was used to visualize the results.

![](https://static.igem.wiki/teams/5382/file/result-6.png)

**Fig.6** Delivery of CL7-Fused Red Fluorescent
Protein into HeLa Cells Using Im7-Attached OMVs

## 7. Testing Cas9 RNP Gene Editing Efficiency

OMVs containing Cas9 RNPs were delivered into HeLa cells to assess
their gene-editing activity. The editing efficiency of the OMV-Cas9 RNPs was compared with that of a
commercial liposome transfection reagent using a T7E1 assay (Fig.6).

![](https://static.igem.wiki/teams/5382/file/result-7.png)

**Fig.7** T7E1 Assay Validation of Cas9 RNP Genome
Editing Activity

The results showed that the Cas9 RNP-loaded OMVs exhibited similar
gene-editing efficiency to the commercial liposome transfection reagent, with both achieving
approximately 40% cleavage of the target gene.

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Hubei University, Wuhan, China
No.368,Youyi Avenue,Wuhan, P.R.China,430062

© 2024 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2024/hubu-4-chn](https://gitlab.igem.org/2024/hubu-4-chn).

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yiliu0825@hubu.edu.cn

{% endblock %}