iGEM 2024 — Team bnu-china

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

[Mono-MSP & Nanodiscs](#0)

[Multi-Polymerized MSP](#1)

[Membrane Protein Polymerization](#2)

[Functional Verification](#3)

## Mono-MSP & Nanodiscs

## Multi-Polymerized MSP

## Membrane Protein Polymerization

## Functional Verification

{% endblock %}

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

[Overview](#0)

[Production of MSP](#1)

[Membrane Protein Polymerization](#2)

[Fabrication and characterization of monomer MSP nanodiscs](#3)

[Functional Verification](#4)

[References](#5)

## Overview

"Failure is central to engineering." This statement has always inspired our team.

To realize the vision of NanoDisguiser, the wet lab group members were divided into different teams to construct different parts of NanoDisguiser sequentially, and finally complete the assembly of the nanodisc. We hope to engineer the assembly process of the nanodisc to make it more repeatable. For example, we conducted a large number of repeat experiments in the purification of MSP to explore the best experimental conditions (see [Notebook page](https://2024.igem.wiki/bnu-china/notebook)). After the assembly was completed, we conducted a rich characterization and measurement of NanoDisguiser and carried out functional verification. In short, our engineering design revolves around the following four parts:

1. Express MSP, using models to help us refine the protein structure design step by step.
2. Use sGFP technology to construct stable membrane protein dimers.
3. Assemble NanoDisguiser and conduct multifaceted characterization and measurement.
4. Verify the antiviral function of NanoDisguiser using virus-like particles (VLPs).

Our entire engineering process is logically carried out under the DBTL (Design, Build, Test, Learn) principles recommended by iGEM, continuously modifying and iterating to improve our project.

## Production of MSP

### Research

The inspiration for nanodiscs comes from high-density lipoprotein (HDL) in the human body that transports cholesterol. By engineering the membrane scaffold protein (MSP) through the modification of ApoAI, it has become a common tool in structural biology. Whether in the laboratory or on the production line, MSP faces engineering issues in terms of length design, purification process, and cyclization methods. We aim to conduct comprehensive engineering iterations on MSP, starting with the common MSP1E3D1. Through literature research, we learned that adding SpyTag and SpyCatcher at both ends of MSP can achieve self-cyclization of MSP, enhancing its stability and reducing the difficulty of purification. We refer to these monomer MSPs as Mono-MSP. During the purification of Mono-MSP, we encountered some difficulties, which forced us to consider how to more effectively produce large nanodiscs, leading us to create Multi-Polymerized MSP, which expresses three MSPs that can link to each other, greatly reducing the difficulty of induction expression and protein extraction.

### Cycle1 Design and presentation validation of Mono-MSP

#### 1. Design

MSP has a very high degree of editability. To achieve project goals, we need to produce different types of nanodiscs, characterize their properties and functions. Starting with cyclization methods and length design, we have designed experiments for four types of Mono-MSP: MSP1E3D1 – for making the most basic and most fluid nanodiscs; spMSP1D1 and spNW15 – for making more stable and controllable self-cyclized nanodiscs; spNW50 – for making self-cyclized nanodiscs with a larger membrane area.

#### 2. Build

We obtained plasmids and gene sequences from Addgene and synthesized plasmids in GENEWIZ (Figure 1). BL21(DE3) containing plasmids were induced with 0.2 mM IPTG and cultured at 16°C for 16 hours.

![](https://static.igem.wiki/teams/5301/engineer/1.png)

Figure 1: Plasmid maps of spNW15(a), spMSP1D1(b), spNW50(c), MSP1E3D1(d).

Next, we need to further extract and purify Mono-MSP. Although MSP has amphiphilic properties similar to membrane proteins, due to its special conformation and support from related literature, we simplified the purification process of amphiphilic proteins, assuming that MSP could be purified in a manner similar to water-soluble proteins, while controlling some conditions to stabilize MSP. We used affinity chromatography to purify the bacterial liquid, and then further purified it by size exclusion chromatography (SEC).

#### 3. Test

We verified the results of induction and purification of Mono-MSP through SDS-PAGE. We found that the initial induction effects of spNW50 and MSP1E3D1 were not good. For spNW15, spMSP1D1, and spNW50, we observed impurities with a molecular weight twice that of these three, and the amount of this impurity for spNW50 was particularly high, leading us to speculate that MSP might have undergone dimerization, and spNW50 is more prone to dimerization.

#### 4. Learn

In the SDS-PAGE results after the purification of MSP1E3D1, we did not observe dimerization bands, which convinced us that the occurrence of dimerization was due to the interaction between the tag and catcher. For the phenomenon that spNW50 is more prone to dimerization, we believe it is because the longer the MSP, the farther the distance between the tag and catcher, and the lower the probability of self-cyclization.

We found that the purification process of MSP1E3D1 was very difficult, and we could only obtain a protein with a very low concentration. We analyzed that this was because MSP could not be simply approximated as a soluble protein, and the purification of MSP protein might need to partially refer to the purification methods of membrane proteins. In contrast, the purification of MSP with tags and catchers was more successful, with a higher yield, which is consistent with the phenomena reported in the literature[1].

Further literature review revealed that during the purification process of MSP with tags/catcher, dimerization and oligomerization phenomena do occur (Figure 2), and the literature characterized this[1]. However, we do not want MSP to oligomerize too much, leading to protein loss and the inability to form the expected size of nanodiscs.

![](https://static.igem.wiki/teams/5301/engineer/2.png)

Figure 2: Representative SDS-PAGE of proteins purified from cells induced with 1 mM IPTG at 37°C. M, monomer; D, dimer[1].

### Cycle 2 Exploration of the dimerization of spNW15, spMSP1D1 and spNW50

#### 1. Design

The literature[1] mentioned that to reduce the formation of oligomers, they chose to induce protein expression at 16°C, and our induction temperature is consistent with theirs, but still cannot avoid the combination between MSP molecules. Therefore, we further pondered how to make MSP have a preference for the latter between molecular combination and molecular self-cyclization?

In response to this question, we first designed simple control experiments and western blot to further verify the existence of intermolecular interactions; secondly, we optimized the overall induction and purification conditions.

#### 2. Build and Test

We purified the bacterial liquid without IPTG induction under the same conditions and did not observe any protein bands through SDS-PAGE, which proved that only when the protein was successfully expressed, this large molecular weight protein would appear, and the molecular weight of this protein was an integer multiple of the monomer protein, which was enough to prove the occurrence of dimerization. We verified the bacterial liquid induced by IPTG through western blot, but unfortunately, we did not observe the bands of dimerization. We placed the protein at different temperatures and verified through SDS-PAGE after 24 hours, but did not find obvious differences between the bands of different temperature groups.

#### 3. Learn

We successfully demonstrated the existence of protein dimers through a control experiment, but the western blot was not successful. However, it should be noted that errors may occur during the western blot process, such as insufficient transfer time. We verify that storage temperature is not responsible for the dimerization of MSP. It has been hypothesized that proteins themselves are relatively prone to dimerization, and that the dimerization of proteins intensifies with longer storage time.

The purification of spNW50 was very difficult, which inspired us that to build large nanodiscs, we cannot simply increase the length of MSP. We then turned to design a new Polymer Linker Systems, creating a new efficient method for building large nanodiscs.

### Cycle 3 Testing of Polymer Linker Systems

#### 1. Design

Next, we aim to connect MSP monomers through protein linker systems, which enables flexible control over MSP length, and facilitates the construction of large nanodiscs.

Given the efficient protein ligation properties of the SpyTag-SpyCatcher system and successful examples of adding linkers to the N- and C-termini of monomeric MSP to enhance self-cyclization[2], we aim to test the feasibility of using these linkers to create multi-polymerized MSP. Additionally, to achieve the connection of multiple MSP segments, we have also identified linker systems with similar properties- SnoopTag/SnoopCatcher and Sdytag/SdyCatcher, and initially investigated their performance through literature review.

To rapidly characterize linker interactions, we turn to the BiFC system- by constructing fusion proteins of the test proteins and fluorescent protein fragments, fluorescence could be observed when the test proteins interact. Meanwhile, to avoid potential interference of MSP cyclization characterization from the characterization of dimeric membrane proteins, we have selected the split-mCherry system for fluorescent labeling, distinguishing it from the GFP fluorescence associated with membrane proteins.

#### 2. Build

To initially assess the feasibility of achieving cyclization using the tag/catcher systems, we constructed MSP fused with SpyCatcher/SpyTag or SpyCatcher/SpyTag variants, SnoopCatcher/SnoopTag, and SdyCatcher/SdyTag at both ends as the basic model. mCherry fragments were inserted into these constructs, and successful cyclization of monomeric MSP was used as the criterion for validation (validation of mCherry takes SpyTag/SpyCatcher system as an example).

![](https://static.igem.wiki/teams/5301/engineer/eng-3new.png)

Figure 3: Schematic Diagram of the Test Sequence SpyCatcher-mCherry[1-10]-MSP-mCherry[11]-SpyTag (Contiguous)

#### 3. Test

Due to time constraints, we utilized AlphaFold 2 to model and validate these constructs, obtaining protein models for the different fusion proteins. Observations revealed that SpyCatcher-SpyTag and SpyCatcher-SpyTag variants, SnoopCatcher-SnoopTag, and SdyCatcher-SdyTag, could all interact normally, with the conserved regions of MSP exhibiting regular and stable α-helix structures. However, when each components of SpyCatcher-mCherry[1-10]-MSP-mCherry[11]-SpyTag were directly concatenated, there was a high confidence in the model that the SpyCatcher-SpyTag system interfered with the split-mCherry system, resulting in cross-linking as shown in the diagram, which made it impossible to ensure normal interactions.

![](https://static.igem.wiki/teams/5301/engineer/4.png)

Figure 4: Alpha Fold 2 Predicted Model for the Test Sequence SpyCatcher-mCherry[1-10]-MSP-mCherry[11]-SpyTag (Contiguous), Demonstrating Predicted Conformational Binding Between SpyTag and Split-mCherry[1-10]

#### 4. Learn

The modeling results indicated that direct connection between components might interfere with the interaction of linkers, thus failing to ensure the correct conformation of the protein. Therefore, we introduced GSlinker—a flexible linker peptide composed of Gly and Ser residues which connects different components to reduce interference.

![](https://static.igem.wiki/teams/5301/engineer/5.png)

Figure 5: Amino Acid Sequence of GSlinker

The Alpha Fold 2 prediction results showed that after the use of GSlinker, mCherry[1-10] and mCherry[11] could correctly assemble into a β-barrel conformation. This demonstrated that the flexible connection between structural domains plays a crucial role in the function of our MSP, advancing our design towards a mature MSP tag/catcher universe.

![](https://static.igem.wiki/teams/5301/engineer/6.png)

Figure 6: Alpha Fold 2 Predicted Model for the Test Sequence SpyCatcher - mCherry[1-10] – MSP - mCherry[11] - SpyTag (Linked) (Showing the Predicted Binding Between SpyCatcher-SpyTag System and between Split-mCherry System, in Accordance with Theoretical Expectations)

### Cycle 4 Design of Multi-polymerized MSP

#### 1. Design

Inspired by protein logic gates[3], to avoid self-cyclization of individual MSP proteins, we added different Catcher/Tag sequences at both ends of MSP respectively. Only when the three sequences are simultaneously connected can the multi-polymerized MSP be constructed, as shown in the figure.

![](https://static.igem.wiki/teams/5301/engineer/7.png)

Figure 7: Protein Logic Gate - 3 Input AND Gate[3]

![](https://static.igem.wiki/teams/5301/engineer/eng-8new.png)

Figure 8: Conceptual Diagram of Multi-polymerized MSP

#### 2. Build

We added different series of Tag/Catcher sequences on both ends of MSP, and added split-mCherry sequences at the SpyTag/Catcher junctions to characterize protein interactions. Based on this, we constructed the initial multi-polymerized MSP sequence.

![](https://static.igem.wiki/teams/5301/engineer/9.png)

Figure 9: Schematic Diagram of the Initial Multi-polymerized MSP Sequence, (a) Fragment 1 - SpyCatcher-mCherry[1-10]-MSP-SnoopTag, (b) Fragment 2 - SnoopCatcher-MSP-SdyTag, (c) Fragment 3 - SdyCatcher-MSP-mCherry[11]-SpyTag

#### 3. Test

We used AlphaFold 2 to model and validate the above constructs. The results showed that cross-reactivity between SnoopTag and mCherry[1-10], and between SdyCatcher and mCherry[11], might lead to self-cyclization of monomeric MSP, while Fragment 2 of multi-polymerized MSP, which did not contain the mCherry sequence, formed the correct linear non-cyclized conformation.

![](https://static.igem.wiki/teams/5301/result/atu15.png)

Figure 10: Alpha Fold 2 Predicted Models of the Test Sequences, (a) Fragment 1 - SpyCatcher-mCherry[1-10]-MSP-SnoopTag, (b) Fragment 2 - SnoopCatcher-MSP-SdyTag, (c) Fragment 3 - SdyCatcher-MSP-mCherry[11]-SpyTag

#### 4. Learn

The results indicated that while the initial design of MSPs prevented self-cyclization to some extent, there were unexpected cross-reactions that made it difficult to assemble multi-polymerized MSP.

Based on this, we modified the linker design of the first and third proteins in the multi-polymerized MSP to ensure that MSPs could not self-assemble into cyclic structures. We also modeled and simulated the conformations of the two modified MSP proteins to verify that each part of the multi-polymerized MSP had the correct tertiary structure. (Refer to the [Results page](https://2024.igem.wiki/bnu-china/results) for modeling results.)

### Cycle 5 Designing Plasmids for Protein Expression Induction

#### 1. Design

After designing the protein sequences, we synthesized plasmids through GENEWIZ and transformed them into BL21(DE3) for protein expression. Our goal was to obtain high-concentration MSP and then mix and incubate the three components to construct multi-polymerized MSP.

#### 2. Build

Activated punctured bacteria were cultured and induced with 0.8 mM IPTG at OD600 0.6-0.8, then cultured at 16°C with shaking for 16 hours. After centrifugation, lysis, and nickel column purification, size exclusion chromatography further purified the protein to obtain high-purity MSP.

#### 3. Test

The induced expression of SCSdC-mCh[1-10] was relatively successful, with a large number of target protein bands visible on the SDS-PAGE results after nickel column purification. Purer protein was also collected after SEC purification. However, the results for SnCSdT and SnTST-mCh[11] were not ideal.

#### 4. Learn

We speculated that during the protein purification process, SnCSdT and SnTST-mCh[11] were degraded by proteases. We decided to add protease inhibitors to the buffer and re-extract the proteins.

### Cycle 6 Optimizing Expression Conditions and Constructing Multi-polymerized MSP

#### 1. Design

We planned to add protease inhibitors to the buffers involved in the purification process of SnCSdT and SnTST-mCh[11] and re-purify the proteins after exploring the optimal induction conditions, and finally attempted to connect the three MSPs to construct multi-polymerized MSP.

#### 2. Build

We added serine protease inhibitor AEBSF to the buffers during purification. After observing no significant improvement in the results, we speculated that other types of protease inhibitors might be causing MSP degradation and switched to using a protease inhibitor cocktail. We mixed three types of MSP in a 1:1:1 ratio, incubated them at room temperature in the dark, and validated using SDS-PAGE.

#### 3. Test

We did not observe any obvious target bands (i.e., multi-polymerized MSP formed by the cyclization of the three MSPs) on the SDS-PAGE, indicating that the three MSPs were not successfully connected.

#### 4. Learn

These results indicated that there were issues during the connection of multi-polymerized MSP. We further speculated that this might be due to insufficient protein concentration or inappropriate incubation conditions.

### Cycle 7 Optimizing Connection Methods and Successfully Constructing Multi-polymerized MSP

#### 1. Design

We hoped to increase the concentration of the three proteins by changing the mixing time, thereby enhancing the efficiency of protein connection to counteract the low sensitivity of SDS-PAGE.

#### 2. Build

In a new round of protein purification, we attempted the in-vitro construction of multi-polymerized MSP using two methods:

Firstly, we mixed three proteins after resuspension, and incubated in the dark at 4°C for a period of time. Then, we used an ultrafiltration tube to concentrate the protein mixture after incubation to increase the protein concentration.

Secondly, we used the original method but reduced the volume of the buffer used to elute the nickel column to achieve higher concentrations of each protein component.

#### 3. Test

We verified the results using SDS-PAGE. Compared to the individual components of the trimer, the mixture of the three proteins showed two new bands at around 100 and 180 kDa. We speculated that these might be trimers or dimers formed by the connection of MSP monomers.

To further verify the construction of the trimer, we observed the protein mixture that had been incubated for a period of time using a fluorescence inverted microscope. As shown in the figure, we observed significant mCherry red fluorescence under green excitation light.

![](https://static.igem.wiki/teams/5301/engineer/11.png)

Figure 11: Fluorescence inverted microscope image taken with a mobile phone.

#### 4. Learn

The display of red fluorescence indicated the possible successful construction of the trimer. However, we could not accurately determine whether SnCSdT was connected to the other two proteins, which may require further characterization.

## Membrane Protein Polymerization

### Cycle 1: Purification of sGFP1-10 and sGFP11 Proteins

#### 1. Design

Our inspiration comes from the split-GFP self-assembly technology, which provides a concept for constructing membrane protein dimers through the in vitro association of sGFP1-10 and sGFP11. However, due to the difficulty of directly linking membrane proteins to sGFP, we further adopted the streptavidin-biotin system. We decided to use the Escherichia coli BL21 (DE3) strain to express fusion proteins of streptavidin with sGFP (i.e., sGFP1-10 tether and sGFP11 tether) and purchased membrane proteins with an Avi tag, which can be biotinylated using a kit.

![](https://static.igem.wiki/teams/5301/engineer/23.png)

Figure 12: Constructed Plasmids.Figure A shows the plasmid map of pET-22b-mSA-sGFP1-10,and Figure B shows the plasmid map of pET-22b-mSA-sGFP11.

#### 2. Build

We reviewed the literature and, based on the experiences of previous iGEMers (mainly from the 2023 BNU-China data), established an experimental plan for expressing and purifying sGFP1-10/sGFP11 tethers. Firstly, a simple examination of the induction conditions for protein expression was conducted by setting the IPTG induction concentrations at 0.5mM and 1mM, respectively, and inducing for 16 hours at 16°C. Next, we purified the protein using nickel affinity chromatography, taking advantage of the His-tag on the fusion protein.

#### 3. Test

**1.Protein Crude Extraction:**

The SDS-PAGE results show that the induction of sGFP1-10 tether expression was relatively successful and consistent with the experimental results from 2023 BNU-China. The protein expression level was relatively high at an IPTG concentration of 1 mM; therefore, in this round of experiments, we maintained the IPTG induction concentration at 1 mM.

![](https://static.igem.wiki/teams/5301/engineer/24.png)

Figure 13: SDS-PAGE Analysis of Crude Extraction of sGFP1-10 Tether Protein

**2.Ni-NTA Purification:**

After gradient imidazole elution, SDS-PAGE analysis showed that the molecular weight of sGFP1-10 tether was 39.2 kDa, and that of sGFP11 tether was 17.0 kDa. Observing the gel, there were no distinct bands at the corresponding molecular weights, indicating that the purification efficiency of sGFP1-10/11 tethers from the supernatant was poor, and it could not be determined whether the protein was present.

![](https://static.igem.wiki/teams/5301/engineer/25.png)

Figure 14: SDS-PAGE Analysis of Purification of sGFP1-10/11 Tether Protein from Supernatant

**3.Molecular Sieve Selection Purification:**

To determine whether the target protein was present in the supernatant and to obtain a higher purity of the target protein, we performed further purification using gel filtration chromatography (molecular sieve). The protein peak profile during purification is shown below. It can be observed that there are many background peaks, and the target protein peak cannot be identified. Subsequent SDS-PAGE experiments also confirmed this, indicating that the purification of the protein from the supernatant was unsuccessful.

![](https://static.igem.wiki/teams/5301/engineer/26.png)

Figure 15: Gel Filtration Chromatography Result

#### 4. Learn

By analyzing the above experimental results and referencing relevant literature and the experiences of 2023 BNU-China, we believe that the main reason for the unsuccessful protein purification is that mSA in sGFP1-10/11 tethers causes a large amount of protein to exist as inclusion bodies in the lysate precipitate. Additionally, we conducted further analysis and reflection on the experimental conditions, and if necessary, we can revisit the IPTG induction conditions to obtain soluble protein. Therefore, next, we first validated Hypothesis 1 by reviewing literature on the extraction of inclusion body proteins.

### Cycle 2: Purification of sGFP1-10 and sGFP11 Inclusion Body Proteins

#### 1. Design

When expressing exogenous proteins in bacteria, the protein products often aggregate into inactive solid particles known as inclusion bodies. While the primary structure of inclusion body products is completely correct, their stereoconformation is incorrect, leading to misfolding and lack of biological activity. Inclusion body proteins are usually dissolved after being washed, followed by subsequent purification operations. Since dissolution often causes protein denaturation, refolding is necessary after purification to proceed with membrane protein dimer construction.

To obtain high-purity inclusion bodies, we washed the bodies along with some disrupted cell membranes and membrane proteins using low concentrations of denaturing agents and detergents. In the experiment, we used urea as a denaturant for dissolution. The refolding of proteins is very complex, and the refolding yield of inclusion body proteins is usually low, not exceeding 20%. Different proteins have distinct structures and functions, leading to variability in their refolding processes and differing levels of difficulty. Specific refolding methods for the target proteins should be designed and screened based on fundamental principles and preliminary experiments. Due to time constraints, we referenced literature and adopted refolding conditions similar to those in reference.

#### 2. Build

Without changing the cultivation conditions of the bacteria and the protein expression induction conditions, we performed protein purification operations, collecting the precipitate after centrifugation of the lysate.

The precipitate obtained from bacterial lysis and centrifugation is washed sequentially with Inclusion Body Buffer I and II. The precipitate is then dissolved in Inclusion Body Solubilization Buffer, followed by incubation and centrifugation. The supernatant is collected to obtain the inclusion body extract, which is subjected to nickel column affinity chromatography. After protein purification, SDS-PAGE analysis is performed to verify whether the inclusion body protein has been successfully purified. For the successfully purified inclusion body protein, renaturation is carried out according to the specific experimental protocol provided.

#### 3. Test

The SDS-PAGE analysis results of the purified inclusion body proteins are shown in the figure below, with distinct bands at the corresponding molecular weights, indicating successful extraction of the inclusion body proteins. Furthermore, the protein concentration and purity in some of the elution fractions meet the requirements; therefore, we will skip the molecular sieve purification and proceed directly to protein refolding.

![](https://static.igem.wiki/teams/5301/engineer/27.png)

Figure 16: SDS-PAGE Analysis of Purification of Inclusion Body Proteins

After protein refolding and concentration, we also performed SDS-PAGE electrophoresis for verification, but no corresponding bands were observed.

To ensure the accuracy of the experimental results, we concurrently conducted an ELISA experiment to test the activity and concentration of the refolded protein. However, the absorbance values of the protein refolding samples were all relatively low, indicating that the concentration of active sGFP1-10/11 tether in the protein refolding samples was low, which was consistent with the results of the SDS-PAGE.

![](https://static.igem.wiki/teams/5301/engineer/28.png)

Figure 17: ELISA Results of Refolding of Inclusion Body Proteins
Row A, wells 1-9 contain a gradient of mSA standard solutions, wells 10 and 11 are 0.5% BSA negative controls; Row B, wells 1-10 contain a gradient of mSA standard solutions, wells 11 and 12 are 0.5% BSA negative controls; Row C, wells 1 and 4 contain sGFP1-10 tether protein refolding samples, wells 2 and 5 contain sGFP11 tether protein refolding samples.

#### 4. Learn

In this experiment, although we initially succeeded in extracting the target protein, we did not detect satisfactory concentration and activity after the subsequent refolding treatment. Reflecting on our experimental process and results, we accurately grasped the denaturing extraction of protein inclusion bodies and successfully extracted denatured proteins, but we underestimated the difficulty of protein refolding and did not thoroughly explore the refolding conditions for sGFP1-10/11 tethers.

Through subsequent discussions with Professor Yang Dong and Professor Wang Zhanxin, and considering the existing conditions and the challenges of protein refolding, we decided to re-explore the conditions for purifying soluble proteins. Through this cycle, we realized that our communication with the professors during the experiment was lacking, and our background research was insufficient, which hindered the progress of the experiment.

### Cycle3 Exploration of Soluble Protein Purification and Dimer Construction

#### 1. Design

We rethought how to obtain active target proteins. Reducing growth rates can help express proteins and consequently decrease the likelihood of inclusion body formation. Therefore, we considered directly altering the culture conditions to lower growth or expression rates, at the cost of potentially decreasing the total protein yield.

We first adjusted the IPTG working concentrations to create a gradient of 0 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM, inducing at 16°C for 16 hours before proceeding with subsequent protein purification and activity assays. Additionally, we analyzed the possibility that the low protein concentration obtained from the supernatant purification led to the absence of target protein bands in the SDS-PAGE analysis. Therefore, we will attempt to purify soluble proteins from the supernatant again using low-temperature induction with 1 mM IPTG and concentrate the purified protein in hopes of obtaining active protein.

#### 2. Build

1.Set IPTG gradient concentrations for induced expression and perform SDS-PAGE analysis on the crude extract.

2.Purify the supernatant obtained from low-temperature induction with 1 mM IPTG, validate it with SDS-PAGE and ELISA experiments, and upon obtaining active protein, attempt to connect sGFP1-10/11 tethers.

#### 3. Test

**1.Results of IPTG Gradient Induction**

The results showed that the optimal protein expression for sGFP1-10 tether was achieved at a concentration of 0.75mM, while for sGFP11 tether, the optimal protein expression was achieved at a concentration of 0.5mM.

**2.Purification and Concentration of Soluble Proteins**

![](https://static.igem.wiki/teams/5301/engineer/29.png)

Figure 18: SDS-PAGE Analysis of Concentrated sGFP1-10 Protein

We observed a corresponding band for the target protein, but the band was faint, indicating that the purification of the supernatant proteins was successful, although the concentration of the purified protein was low. We incubated sGFP1-10/11 tethers at 4°C in the dark and observed whether they emitted fluorescence using a fluorescence microscope.

![](https://static.igem.wiki/teams/5301/engineer/30.png)

Figure 19: Fluorescence microscopy was used to observe the mixed incubated samples (4×10)
 A and C represent the background fluorescence, and B and D are the observation results after the corresponding samples were added. By comparing these results, the corresponding fluorescence signals are obtained to eliminate false positives.

#### 4. Learn

We have obtained the target protein from the purified supernatant and observed fluorescence after mixed incubation. Next, we will further purify the protein under an IPTG concentration gradient to determine the optimal IPTG induction concentration for producing soluble proteins. Additionally, we will continue with protein purification to increase protein concentration and explore the optimal conditions for dimer formation by adjusting the temperature, time, and mixing ratios during incubation.

Currently, we are purifying the proteins separately before combining them; however, we will also attempt to mix and lyse both engineered bacteria for purification, investigating whether sGFP1-10 tether and sGFP11 tether can directly associate during the lysis process to obtain dimer proteins directly.

## Fabrication and characterization of monomer MSP nanodiscs

### Cycle1 The manufacturing process of nanodiscs

#### 1. Design

We tried to use three kinds of purified MSP proteins with SpyTag and SpyCatcher to self-cyclide to construct nanodiscs with different particle sizes to meet the needs of our experimental design. According to the experimental conditions mentioned in the references, protein and lipid were co-incubated in corresponding proportions. In order to explore the optimal conditions, we constantly adjusted and improved protein-lipid ratio on the basis of literature, and tried to add detergent DDM[4].

#### 2. Build

In the preparation of the spNW15 nanodiscs, we made a gradient attempt on the protein-lipid ratio between 1：100 and 1：110, with a control group with or without the addition of detergent. For the production of the spMSP1D1 nanodiscs, we constantly modify the protein-lipid ratio from 1:60 to 1:200, and strive to achieve the optimal conditions. And we constructed spMSP1D1 nanodiscs using the protein-to-lipid ratio of 1:60 as reported in the literature. For the construction of the spNW50 nanodiscs, we attempted to construct nanodiscs using the protein-to-lipid ratio of 1:600 as described in the literature.

#### 3. Test

For a series of trial samples, we conducted Native-PAGE test and inally confirmed the best protein-lipid ratio according to the test results.

![](https://static.igem.wiki/teams/5301/engineer/12.png)

Figure 20: Results of Native-PAGE analysis. The Figure shows the nanodiscs strips successfully produced by spNW15 (Figure 12a), spMSP1D1(Figure 12b) and spNW50(Figure 12c) and their protein lipid ratios.

According to the results of Native-PAGE, we found that spNW15 nanodisc strips are similar to those in the literature, and are banded and discrete, while spMSP1D1 and spNW50 are narrow strips that are inconsistent with the literature. At the same time, the same strip has different shapes and positions in different Native-PAGEs[5].

#### 4. Learn

According to the results, we believe that in the process of Native-PAGE characterization, the mobility of target bands is related to the isoelectric point of proteins, and the lipid charge may affect the characterization result after the addition of lipids. To solve the problem, we decided to visualize other representations.

### Cycle2: Characterization of nanodiscs

#### 1. Design

DLS can measure the particle size of our nanodiscs with high throughput and check whether it conforms to the literature records. Through negative stain electron microscopy, we can intuitively see whether we have constructed the disk structure of the nanodisc, and the particle size can be quantified according to the scale. The samples obtained in the last cycle were examined by DLS and negative stain electron microscopy.

According to the experimental design of modeling and the gradient experiment, we analyzed the main effect factors to make the subsequent construction and characterization of the nanodiscs more directional and save the number of tests.

#### 2. Build

The gradient manipulation was carried out under the experimental conditions of lipid type, protein-lipid ratio, culture temperature and culture time, and the gradient results were characterized by negative stain electron microscopy and DLS[(See more--Model)](https://2024.igem.wiki/bnu-china/model).

#### 3. Test

We successfully observed and quantified the samples using negative stain electron microscopy.

We not only actually saw the disk-like structure of the three kinds of nanodiscs through negative stain electron microscopy (Figure 21a, 21b, 21c), but even saw the side of the nanodiscs (Figure 21d).For the spMSP1D1 nanodiscs incorporating the Low-Density Lipoprotein Receptor(LDL-R) ，and we can saw the image of successfully containing membrane proteins in the nanodiscs (Figure 22a).This was consistent with the images of nanodiscs containing membrane proteins in the literature (Figure 22b)[6].

The negative stain electron microscopic quantization results of spMSP1D1 (Figure 23a) and spNW15 (Figure 23b) were also consistent with the literature records, while the negative stain electron microscopic quantization results of spNW50 were the same as those of DLS (Figure 23c). Finally, the box diagram (Figure 23d) was drawn according to the particle sizes of the three nanodiscs. It can be seen that we produced three nanodiscs with different particle sizes.

A small number of negative stain electron microscope results showed that the nanodiscs clustered (Figure 24a) or formed flower-like structures (Figure 24b). However, the results of DLS (Figure 25) differ greatly from the theoretical values and the quantified values of negative stain electron microscopy.

![](https://static.igem.wiki/teams/5301/engineer/13.png)

Figure 21: Negative stain electron microscope images of spNW15 (a), spMSP1D1 (b) and spNW50 (c) nanodiscs showed clear disk-like structures. The side structure of the nanodisc can be seen from (d).

![](https://static.igem.wiki/teams/5301/engineer/14.png)

Figure 22：Negative stain electron microscope images of spMSP1D1 nanodiscs containing LDL-R(a), images of nanodiscs with membrane proteins in the literature as seen under negative staining electron microscopy(b).

![](https://static.igem.wiki/teams/5301/engineer/15.png)

Figure 23: Diameter size distribution of spMSP1D1 nanodiscs (a) and spNW15 nanodiscs (b) under stain electron microscopy, negative stain electron microscope images of spNW50 nanodiscs(c), box diagram of spMSP1D1 nanodiscs, spNW15 nanodiscs, spNW50 nanodiscs under negative staining electron microscopy(d).

![](https://static.igem.wiki/teams/5301/engineer/16.png)

Figure 24: Stain electron microscopy image of clustered nanodiscs(a), and the flower-like structures of spNW50 nanodiscs under stain electron microscopy(b).

![](https://static.igem.wiki/teams/5301/engineer/17.png)

Figure 25: According to the statistical results of DLS data measurement, the theoretical particle sizes of spNW15(a) and spMSP1D1(b) are about 10-20nm, and there is a large gap between the measured values and the theoretical values.

#### 4. Learn

Since the hydraulic diameter of nanoparticles in solution is measured by DLS, and the agglomeration of nanodiscs with small particle size is easy, the data measured by electron microscopy will be larger than that measured by electron microscopy. The flower-like nanodiscs may be due to the fact that the amount of lipid added is less than that of protein, and the MSP is not fully separated.

Future outlook: We can still make further improvements to the initial successful nanodiscs samples, including increasing the dilution of the samples to reduce aggregation between the nanodiscs, and appropriately increasing the protein-lipid ratio to prevent the formation of flower-like nanodiscs. In the future, we hope to further improve the manufacture, characterization and application of nanodiscs.

## Functional Verification

### Research

As a project in the infectious disease track, we cannot experiment with real viruses, which requires higher biosafety levels.

We aim to develop an in vitro simulation system for "Infection-Blocking-Curing" process to preliminarily validate the antiviral design of nanodiscs while providing a convenient and safe solution for future high-risk virus-related research simulations. This requires us to creatively apply relevant system knowledge for design and engineering implementation.

### Cycle1 Sibling Nanodiscs with Liposome Fluorescence Leakage Experiments

#### 1. Design

We designed a liposome in vitro "infection-blocking" system to observe the destructive effect of nanodiscs on viruses, considered direct validating the lysate.

We discovered a type of SNARE protein-based membrane fusion perforation system, which resembles the membrane fusion mechanism of HIV infecting CD4+ T cells[7,8]. Research has constructed a nanodisc-liposome interaction system through SNARE protein assembly, quantitatively demonstrating significant advantages of large nanodiscs[7] over small ones, inspiring our design of a liposome pseudovirus system.

![](https://static.igem.wiki/teams/5301/engineer/18.png)

Figure 26. Schematic Diagram of the Interaction Mechanism Between Nanodiscs and Liposomes.This inspires us to use liposomes as a substitute for viruses to interact with nanodiscs, in order to characterize the antiviral effects of the nanodiscs.

Additionally, assuming the system is effective, the liposome pseudovirus not only ensures high safety and a clean biological background but also allows highly engineered synthetic liposomes to serve as variable simulation virus platforms, achieving precise and controllable quantitative data.

1. We designed Sibling Nanodiscs: a virus-simulating disc and an antiviral nanodisc. Using the stable membrane proteins of nanodiscs and their membrane fusion-triggering characteristics, we selected suitable viral envelope proteins and corresponding host receptor membrane proteins, stabilizing and solubilizing them with nanodiscs.
2. We chose pH-responsive fluorescent dyes to characterize liposome rupture or leakage, constructing HPTS-loaded small unilamellar vesicles (SUVs) to monitor content leakage in response to the pH gradient inside and outside synthetic liposomes.
3. Utilizing a calcium-responsive artificial fusion transfer system (nano-CRAFT) and employing typical ultracentrifugation in a sucrose gradient to fuse special viral simulation discs with liposomes, loading envelope proteins onto the surface of synthetic liposomes[8].

![](https://static.igem.wiki/teams/5301/engineer/19.png)

Figure 27: CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs[10]

4. 4.Finally, we mixed the antiviral nanodiscs with positive liposome "viruses," allowing the recognition and conformational change between membrane proteins to leverage the intrinsic infection capability of the viral envelope proteins, simulating "infection-blocking" in vitro.

#### 2. Build

1. Use the Avanti extruder to filter HEPES dye-blank liposomes.
2. Prepare Sibling Nanodiscs, optimizing lipid type and ratio for high lipid fluidity, switching to MSP.
3. Establish quantifiable measures of success: quantitative fluorescence leakage experiments.

#### 3. Test

1. Source, domains, feasibility, and purification of CD4 protein.As a membrane protein, CD4 was designed as a full-length membrane protein to ensure the functionality of nanodiscs in viral envelope removal and to guarantee correct glycosylation conformations, with limited existing research on this. Most studies focus on the extracellular domain of CD4, leaving us with a lack of theoretical support[7].
2. The purification of MSP1E3D1 protein is quite challenging. Compared to spontaneously cyclized proteins like spNW1D1, this protein's water transport domain is more exposed. Existing inorganic nanodisc schemes use amphipathic styrene maleic acid (SMA) to stabilize nanodiscs, which have been verified to effectively transport membrane proteins and fuse cell membranes[7].
3. Fluorescence interference issues. In the overall design, membrane proteins may be linked to GFP fluorescence, and MSP may be linked to mCherry fluorescence. If liposomes also contain fluorescent groups, the fluorescence in the system becomes chaotic. If the excitation spectra of several fluorescent dyes are close, simultaneous excitation by a laser can lead to fluorescence cross-talk.
4. The active infection capability is questioned, and the system's convincing power in validating viruses is not very high.

#### 4. Learn

Due to the weak persuasiveness of abiotic systems and the difficulty in obtaining specific membrane proteins in large quantities, we are considering the use of biological physiological functions, specifically secreted protein-lipid complexes known as virus-like particles, to simulate and characterize viruses in vitro.

We reviewed literature and materials on existing engineered systems of virus-like particles, which have good examples in drug delivery, vaccine development, gene engineering, etc. Examples include Adeno-associated Virus (AAV), Pseudoviruses, and the VSV-G system[8,9].

In the next iteration, we will attempt a new approach using the Mammalian retrovirus-like protein PEG10 system.

### Cycle2 SEND and Fluorescent Reporting System

#### 1. Design

We designed a new in vitro "infection-blocking" system to directly verify the blocking effect of nanodiscs on viral infection. We utilized the SEND system to simulate the virus.

To avoid unnecessary complexity in the experiment and to ensure compatibility with human cells, we used the 293FT cell line as the target for simulated viral infection, reflecting real-world conditions where host cells are susceptible to viral invasion and serve as containers for virus production.

The fluorescence reporting system used in this study was conducted in a system with cellular activity, and the in vitro simulation of viruses had stronger persuasiveness, more mature experiments, and shorter experimental cycles.

#### 2. Build

Mammalian cells co-expressed three transient plasmids: capsid, cargo RNA, and fusogen[10], and the final construction of cargo RNA plasmids are shown as follows:

![](https://static.igem.wiki/teams/5301/engineer/20.png)

Figure 28: Plasmid profile of cargoRNA (human)

We cultured the HEK293FT cell line, which is particularly suitable for generating lentiviral constructs using the Invitrogen ViraPower Lentiviral Expression System[10].

#### 3. Test

First, after approximately 48 hours post-transduction, we collected the supernatant from the mammalian cells. SDS-PAGE analysis of the supernatant showed successful secretion of VSV-G and PEG10 proteins, indicating partial success in plasmid transduction. (figure 21a)

![](https://static.igem.wiki/teams/5301/engineer/21.png)

Figur 29: (a) SDS-PAGE analysis of the supernatant. From left to right: marker, transfected cell supernatant (containing VLP) diluted 2-fold, 5-fold, 10-fold, and 20-fold, culture medium, culture medium diluted 10-fold, untransfected cell supernatant, and untransfected cell supernatant diluted 10-fold . (b) Culture medium contrast. Left is untransfected cell and right is transfected cell. Right one is greener. (c) Apply fluorescent microscopy to detect strong intracellular fluorescence. (d) Fluorescent microscopy result of single cell.

Secondly, we visually observed the culture medium color and employed fluorescent microscopy to detect strong intracellular fluorescence, confirming the proper assembly and infectivity of the VLP system. (figure 21b.21c) Furthermore, we diluted the cells and observed the distribution of fluorescence in individual cells. The fluorescence existed within the cells, which is consistent with our expected results, further confirming that we have produced VLP. (figure 21d)

Finally, we infected normal cells with the supernatant, setting up control and gradient experiments to establish quantifiable measurement standards.

We conducted a control infection experiment on HEK293 FT cells and obtained preliminary fluorescence images and data that varied over time.

![](https://static.igem.wiki/teams/5301/engineer/22.png)

Figure 30. Fluorescence of HEK293 FT cells in control infection experiment over time

#### 4. Learn

In the final control infection experiment, due to time constraints, more fluorescence intensity data could not be obtained, and visual counting of fluorescent cells may introduce errors. In the future, we plan to conduct more rigorous validation, such as fixed observation scenarios and obtaining fluorescence intensity values to reflect infection rates. In addition, we hope to directly confirm the expression of PEG10 VLP extracellular vesicles through Western Blot and electron microscopy, and verify the blocking function of nanodisks on VLPs from a microscopic perspective using methods such as dynamic light scattering (DLS).

For more results, please refer to the [results page](https://2024.igem.wiki/bnu-china/results).

## References

[1] Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7

[2] Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7

[3] Zibo Chen et al. ,De novo design of protein logic gates.Science368,78-84(2020).DOI:10.1126/science.aay2790

[4] Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7

[5] Benedicto, V. G. L., Haguar, Z., Abdulhasan, A., & Narayanaswami, V. (2024). Apolipoprotein E3 Containing Nanodiscs as Vehicles for Transport and Targeted Delivery of Flavonoid Luteolin. ACS omega, 9(2), 2988–2999. https://doi.org/10.1021/acsomega.3c09120

[6] Nasr, M., Baptista, D., Strauss, M. et al. Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14, 49–52 (2017). https://doi.org/10.1038/nmeth.4079

[7] Smirnova, I.A., Ädelroth, P. & Brzezinski, P. Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents. Sci Rep 8, 14950 (2018). https://doi.org/10.1038/s41598-018-33208-1

[8] From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Grimm D, Kay MA. Curr Gene Ther. 2003 Aug;3(4):281-304. PubMed.

[9] Hwang, BY., Schaffer, D. Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors. Gene Ther 20, 807–815 (2013). https://doi.org/10.1038/gt.2013.1

[10] Luigi Naldini et al., In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector.Science272,263-267(1996).DOI:10.1126/science.272.5259.263

{% 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 %}

[Construction of Mono-MSP](#0)

[1 MSP Plasmids Construction](#0-0)

[2 Cultures expansion and inducible expression](#0-1)

[3 Protein expression and purification](#0-2)

[Construction of Multi-Polymerized MSP](#1)

[1 Plasmid Construction](#1-0)

[2 Induced Protein Expression](#1-1)

[3 Protein Extraction and Purification](#1-2)

[4 Structure and Function Validation](#1-3)

[Membrane Protein Polymerization](#2)

[1 Construction and Verification of Plasmid Vectors](#2-0)

[2 Expression Induction and Protein Purification](#2-1)

[3 Protein Activity Analysis](#2-2)

[4 BiFC (Biomolecular Fluorescence Complementation)](#2-3)

[5 Binding of Biotinylated Membrane Proteins](#2-4)

[Assembly of Nanodiscs](#3)

[1 spNW15](#3-0)

[2 spMSP1D1](#3-1)

[3 spNW50](#3-2)

[Functional Verification](#4)

[1 Construction of the three plasmids](#4-0)

[2 HEK 293FT cell culture](#4-1)

[3 VLPs obtainment after plasmid transfection](#4-2)

[4 Cells infected by VLPs](#4-3)

[5 Further Experiments](#4-4)

## Part1 Construction of Mono-MSP

### 1 MSP Plasmids Construction

In order to successfully construct nanodiscs, we decided to use the Membrane Scaffold Proteins (MSPs) that have been reported in the literature for initial trials and validation. By reviewing the literature, we decided to first use four types of monomeric MSPs, namely spNW50, spMSP1D1, spNW15, and MSP1E3D1, to manufacture nanodiscs.

We first synthesized the following plasmids at GENEWIZ, and the company returned the plasmids to us via transformation.

Then, we isolated single colonies using the streak plate method.

Lastly, through PCR method, we confirmed that the target genes had been obtained.

![](https://static.igem.wiki/teams/5301/result/figure-1.png)

Figure 1: Final plasmid map of spNW15(a), spMSP1D1(b), MSP1E3D1(c), spNW50(d).

![](https://static.igem.wiki/teams/5301/result/atu2.png)

Figure 2: The PCR results of spNW15(a), spMSP1D1(b), MSP1E3D1(c), spNW50(d). On the far left is the marker. The other lanes are different single colonies. The red box indicates the target band.

### 2 Cultures expansion and inducible expression

We performed amplification and cultivation of the obtained target colonies separately, and induced with IPTG when the OD600 value reached 0.6-0.8.

Table 1: Conditions of expanded cultures.

| Protein Name | Seed Fluid Volume: Medium Volume | Kanamycin Concentration (μg/mL) | Culture Temperature (℃) | Speed (rpm) | Culture Time (h) | Final OD600 Value |
| --- | --- | --- | --- | --- | --- | --- |
| spNW15 | 1:100 | 50 | 37 | 180 | 3-4 | 0.6-0.8 |
| spMSP1D1 | 1:100 | 50 | 37 | 200 | 3-4 | 0.7 |
| spNW50 | 1:100 | 50 | 37 | 180 | 3-4 | 0.6-0.8 |
| MSP1E3D1 | 1:100 | 50 | 37 | 180 | 2.5-3 | 0.6-0.8 |

The concentration of IPTG and the induction time can both have a certain impact on protein expression levels. We conducted a brief exploration of the induction concentration and time for spNW50 and MSP1E3D1. We added different amounts of IPTG solution to the bacterial solutions, set the concentration gradient of the final concentration of IPTG, and selected the most appropriate concentration of IPTG. The most suitable IPTG concentration was screened by SDS-PAGE gel electrophoresis.

Table 2: IPTG gradient induction results of spNW50. SDS-PAGE lanes with different IPTG concentrations at the same induction time (10h) were analyzed (“+” and “-“ were used to distinguish the amount of protein at 245KDa, "+" meant the amount of protein was normal, and "-" meant no protein).

| IPTG concentration (mM) | Amount of protein |
| --- | --- |
| 0 | - |
| 0.2 | + |
| 0.4 | + |
| 0.6 | + |
| 0.8 | + |

For spNW50, it was found that for the same induction time (10h), there was little difference in protein amount under different IPTG concentration(Figure 3c).

Table 3: IPTG gradient induction results of MSP1E3D1. SDS-PAGE lanes with different IPTG concentrations were analyzed (“+” and “-“ are used to distinguish the amount of target protein, "++" means more protein, "+" means average protein, and "-" means no protein).

| IPTG concentration (mM) | Amount of protein |
| --- | --- |
| 0 | - |
| 0.1 | + |
| 0.2 | + |
| 0.3 | - |
| 0.4 | - |
| 0.5 | + |
| 0.6 | + |
| 0.7 | + |
| 0.8 | ++ |
| 0.9 | + |

According to the electrophoretic results (Figure 3d), MSP1E3D1 bands were most obvious when the IPTG concentration was 0.8mM, and the IPTG concentration of 0.8mM was selected.

What's more, for spNW50, considering that induction time may have a certain effect on protein expression, we set two time gradients to explore the optimal induction time.

Table 4: Time gradient induction results ofspNW50. SDS-PAGE lanes with the same IPTG concentration and different induction times were analyzed (“+” is used to distinguish the amount of protein at 245KDa, "+" means the amount of protein is average, and "++" means the amount of protein is large).

| IPTG concentration (mM) | Induction time (h) | Amount of protein |
| --- | --- | --- |
| 0.2 | 10 | + |
| 30 | + |
| 0.4 | 10 | + |
| 30 | ++ |

It was found that for the same IPTG concentration, there was little difference in protein spNW50 amount under different induction time (Figure 3c).

We used shaking tables for inducible expression under the induction conditions shown in the table below (Table 5). SDS-PAGE was used to analyze whether the induction was successful (Figure 3)

Table 5: Conditions for inducible expression

| Protein name | IPTG concentration (mM) | Culture temperature (℃) | Speed (rpm) | Culture time (h) |
| --- | --- | --- | --- | --- |
| spNW15 | 0.2 | 16 | 180 | 16 |
| spMSP1D1 | 0.2 | 16 | 180 | 16 |
| spNW50 | 0.2 | 16 | 180 | 16 |
| MSP1E3D1 | 0.8 | 28 | 150 | 16 |

![](https://static.igem.wiki/teams/5301/result/atu3.png)

Figure 3: Analysis result of crude extraction of IPTG-induced protein spNW15(a), spMSP1D1(b), spNW50(c), MSP1E3D1(d) on SDS-PAGE, with marker on the far left and protein on the right. The red box indicates the target band.

### 3 Protein expression and purification

The bacteria were broken up in the metal bath, and the crude protein extract was obtained. SDS-PAGE analysis was performed to test whether the induced expressions were successful.

#### 3.1 spNW15

The theoretical size of spNW15 was 40.3kDa, and the crude protein extract after induction had obvious bands in the 40kDa attachment, which suggested that the expression of spNW15 was successfully induced (Figure 4a).

The bacteria were ultrasonically broken in Buffer A, and the proteins were refined by elution with nickel bead affinity chromatography and imidazole with gradient concentration. We conducted SDS-PAGE analysis to check the purification effect (Figure 4b). It can be seen that a large number of target proteins were purified in 300mM imidazole eluant and 500mM imidazole eluant, but the purity was not high and there were some impurity bands, which may be caused by the dimerization or polymerization of spNW15, and further purification was required through molecular sieve.

In order to obtain higher protein purity, we purified the protein using molecular sieve chromatography, and obtained spNW15 protein with higher purity through SDS-PAGE analysis (Figure 4c).From the results of SDS-PAGE, it can be seen that SEC was effective, and our spNW15 was purified. Subsequently, ultrafiltration tubes were used to concentrate and measure the concentration, so as to prepare the nanodiscs.

![](https://static.igem.wiki/teams/5301/result/atu4.png)

Figure 4: SDS-PAGE analysis of the expression results of protein spNW50. Lanes 1 was marker, the other lanes were crude extract(a), the extraction results(b), molecular sieve effluent(c).

#### 3.2 spMSP1D1

The theoretical molecular weight of spNW50 is 123.9 kDa. Compared with the liquid phase of crude protein extracts without induction, there is a distinct band obtained in the crude protein extract after induction, indicating that the induction expression of spNW50 was successful (Figure 6a).

The bacteria were ultrasonically broken in Buffer A, and the proteins were refined by elution with nickel bead affinity chromatography and imidazole with gradient concentration. SDS-PAGE analysis was performed to check the purification effect (Figure 5b). It can be seen that a large number of target proteins were purified from 100mM imidazole and 300mM imidazole elution, but the purity was not high and there were some impurity bands, which may be caused by dimerization or polymerization of spMSP1D1. Further purification by molecular sieve was required.

In order to obtain higher purity proteins, molecular sieve chromatography was used to purify proteins. After SDS-PAGE analysis (Figure 5c), we obtained high purity spMSP1D1 and suspected spMSP1D1 dimer proteins, which were then concentrated and measured by ultrafiltration tube for subsequent preparation of nanodiscs.

What's more, we further used anti-His antibodies and confirmed the successful purification of spMSP1D1 protein through Western Blot (Figure 5d). Additionally, it has been determined that what was previously suspected to be spMSP1D1 dimers may not be dimers but rather non-specific bands.

![](https://static.igem.wiki/teams/5301/result/atu5.png)

Figure 5:(a)SDS-PAGE analysis of the product results of protein spMSP1D1. Lanes 1 was marker, the other lanes were crude extract, (b)the extraction results,(c) molecular sieve effluent(d) Western blot result image.

#### 3.3 spNW50

The theoretical size of spNW50 was 123.9kDa. Compared with the liquid phase of uninduced crude protein extraction, the induced crude protein extract showed more obvious bands in the range of 135-180kDa, which suggested that the induced expression of spNW50 was successful (Figure 6a).

A cracking buffer containing 0.02%Triton x 100 was used and ultrasonically broken, followed by nickel strain elution for fine protein purification. SDS-PAGE analysis was performed again to verify the purification effect (Figure b). Obvious bands can be seen, and there are few impurity bands. So it is considered that we have purified relatively pure spNW50.

The protein concentration was determined using the Bradford method (see protocol for details) to facilitate subsequent fabrication of the nanodiscs.

![](https://static.igem.wiki/teams/5301/result/atu6.png)

Figure 6: SDS-PAGE analysis of the product results of protein spNW50. Lanes 1 was marker, the other lanes were crude extract(a), the extraction results(b), molecular sieve effluent(d). Figure c standard curve determined by Bradford method for determination of protein concentration. The ordinate is OD value of protein solution at 595nm, and the abscess is protein concentration (mg/mL).

#### 3.4 MPS1E3D1

The theoretical size of MSP1E3D1 was 32.6kDa. Compared with the liquid phase of uninduced crude protein extraction, the induced crude protein extract showed more obvious bands in the range of 25kDa to 35kDa, suggesting that MSP1E3D1 was successfully induced. Moreover, it was observed that the MSP bands were more obvious when the IPTG concentration was 0.8mM, so the optimal induced concentration of IPTG was 0.8mM (Figure 7a).

A cracking buffer containing 0.02%Triton x 100 was used and ultrasonically broken, and then nickel strain eluted to refine the protein. SDS-PAGE analysis was performed again to verify the purification effect (Figure 7b). Obvious bands were visible at 25-35kDa in the lane of the sample eluant, and there were few impurity bands, so it was considered that we purified relatively pure MSP1E3D1.

MSP1E3D1 was further purified by molecular sieve (Figure 7c). The purification effect was tested by SDS-PAGE analysis (Figure 7d). A shallower band can be seen at 25-35kDa in the lane of Peak 1, presumed to be purified MSP1E3D1, but at a lower concentration.

MSP1E3D1 purified by molecular sieve was concentrated by ultrafiltration tube, and then the purification success was verified by SDS-PAGE (Figure 7e). There was a relatively obvious band at 25-35kDa of the swim lane of MSP1E3D1, which was presumed to be the concentrated MSP1E3D1, indicating successful purification.

![](https://static.igem.wiki/teams/5301/result/atu7.png)

Figure 7: SDS-PAGE analysis of the product results of protein MSP1E3D1. Lanes 1 was marker, the other lanes were crude extract(a), the extraction results(b), molecular sieve effluent(d), passing through molecular sieve after concentration(e). Figure c was the results of protein MSP1E3D1 passing through molecular sieve showed six peaks.

Ultimately, Bradford method (Figure 6c) was used to determine the protein concentration of the above four proteins (see protocol for details) to facilitate subsequent fabrication of the nanodiscs.

## Part2 Construction of Multi-Polymerized MSP

We aim to utilize different linkers (Spy/Sdy/Snoop) to connect Mono-MSPs to form multi-polymerized MSP, thus creating larger nanodiscs. Larger nanodiscs are advantageous for accommodating more receptor proteins and can increase the probability of nanodisc-virus binding in vivo. Compared to directly producing longer MSP proteins, this method effectively alleviates the metabolic stress on the bacteria. Additionally, through the fluorescence emitted by mCherry, we can effectively validate our idea.

![](https://static.igem.wiki/teams/5301/result/figure-8-fix.png)

Figure 8: Schematic Diagram of Multi-Polymerized MSP Cyclization

### 1 Plasmid Construction

To produce polymeric MSPs, we constructed plasmids to synthesize three MSPs separately, in order to achieve controllable cyclization of MSP and controllability of the nanodisc particle size. In the experiments of the previous section, we purified MSP proteins of different lengths to construct nanodiscs of varying sizes. However, the purification of proteins with a large molecular weight (such as spNW50) presented certain difficulties, thus further illustrating the necessity of our newly designed method for constructing larger nanodiscs (Multi-polymerized MSP).

We obtained the gene sequence of spNW15 from NCBI and integrated the sequences of the three linkers (Spy/Sdy/Snoop) in a specific order at its N-terminus and C-terminus to form three MSPs that can be connected to each other. Based on the different linkers fused at both ends of the MSPs, we sequentially name these fusion proteins used to generate multi-polymerized MSP as SCSdC-mCh[1-10], SnCSdT, and SnTST-mCh[11]. We added 5' (NcoI) and 3' (XhoI) to the ends of the genes (BBa\_K5301005, BBa\_K5301006, BBa\_K5301007) through GENEWIZ, cloning them into the vector pET-28a(+) (Kanamycin) to construct three recombinant plasmids, which were then introduced into BL21 (DE3). Subsequently, we picked multiple single colonies from the plate for colony PCR to check if the plasmids were successfully introduced into the host bacteria. We selected colonies with the desired bands for the next step of induced expression.

![](https://static.igem.wiki/teams/5301/result/figure-9.png)

Figure 9: Plasmid Map of Multi-Polymerized MSP

![](https://static.igem.wiki/teams/5301/result/figure-10.png)

Figure 10: Colony PCR Results of Host Bacteria Introduced with pET28a\_SnTST-mCh[11], All Four Colonies Successfully Obtained the Desired Band, Indicating Successful Plasmid Transformation

### 2 Induced Protein Expression

We selected suitable single colonies and cultured them overnight in 20 mL LB (Kanamycin). The overnight culture was then added to fresh 200 mL LB (Kanamycin) at a 1:100 ratio and incubated at 37°C for approximately 3 hours (slightly less than 3 hours, depending on the concentration of the activated bacterial liquid). When the culture reached an OD600 of 0.6-0.8, we induced protein expression with IPTG and continued incubation at 16°C for 16 hours. For the SCSdC-mCh[1-10] and SnCSdT, we chose a final IPTG concentration of 0.8 mM. After observing poor expression of the SnTST- mCh[11] in the multi-polymerized MSP, we set an IPTG concentration gradient to find the optimal IPTG concentration for protein expression. The bacterial culture was then centrifuged, and the pellet was subjected to SDS-PAGE to compare protein expression levels under different IPTG concentrations, identifying the optimal induction concentration as 1 mM.

Table 6: IPTG Concentration Gradient and Corresponding Volume of 200 mM IPTG Solution Added to 200 mL LB (Kanamycin)

| Number | IPTG Concentration (mM) | IPTG Solution Volume (μL) |
| --- | --- | --- |
| 1 | 0.2 | 47.61 |
| 2 | 0.5 | 119.03 |
| 3 | 0.8 | 186.83 |
| 4 | 1 | 238.05 |

![](https://static.igem.wiki/teams/5301/result/figure-11.png)

Figure 11: Expression of the SnTST- mCh[11] Under Different IPTG Concentrations. The Target Protein Size is 33.6 kDa. Expression of the Target Protein Was Observed at All Four IPTG Concentrations, with the Highest Expression at 1 mM IPTG.

### 3 Protein Extraction and Purification

#### 3.1 Protein Purification

After determining the optimal induction conditions for the three proteins, we scaled up the culture. We incubated the strains producing SCSdC-mCh[1-10], SnCSdT, and SnTST- mCh[11] at 16°C with IPTG concentrations of 0.8 mM, 0.8 mM, and 1 mM, respectively, for 16 hours, thus obtaining a large amount of target protein.

In the plasmid design, we included a His-tag in the target proteins to facilitate the use of nickel affinity chromatography for obtaining high-purity target proteins. As shown in the figure, the use of 300 mM and 500 mM imidazole successfully eluted a substantial amount of SCSdC-mCh[1-10] and SnCSdT

Due to the potential interference of endogenous E. coli proteins and possible dimerization or degradation of MSP proteins with SDS-PAGE, we did not verify the successful purification of SnTST- mCh[11] through SDS-PAGE. We further used anti-His antibodies and confirmed the successful purification of SnTST- mCh[11] through Western Blot.

![](https://static.igem.wiki/teams/5301/result/atu12.png)

Figure 12: Purification of multi-polymerized MSP. (a) SDS-PAGE analysis of purified SCSdC-mCh[1-10]. The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa. (b) SDS-PAGE analysis of purified SnCSdT. The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SnCSdT is 42.1 kDa. (c) Western Blot analysis of purified SnTST- mCh[11]. The molecular weight of SnTST- mCh[11] is 33.6 kDa.

Due to the nickel column purification results not meeting the expected protein purity, we further separated and obtained high-purity SCSdC-mCh[1-10] through SEC, as shown in the figure.

![](https://static.igem.wiki/teams/5301/result/figure-13.png)

Figure 13: High-purity SCSdC-mCh[1-10] obtained through further separation by SEC. (a) SEC absorption peak chromatogram of the purification results of SCSdC-mCh[1-10]. A total of 6 absorption peaks were obtained before the salt peak (absorption peak at 104 mL), with elution volumes of 42 mL, 56 mL, 62 mL, 73 mL, 80 mL, and 86 mL, respectively. (b) SDS-PAGE analysis of the SEC results for SCSdC-mCh[1-10]. Based on the molecular weight data of SCSdC-mCh[1-10], absorption peaks 1 and 2 correspond to SCSdC-mCh[1-10].

#### 3.2 Protein Concentration Determination

To facilitate the subsequent connection of multi-polymerized MSPs and the construction of large cyclic nanodiscs, we used the Bradford method to plot a standard curve and measured the concentration of SCSdC-mCh[1-10] protein to be 0.228 mg/ml.

![](https://static.igem.wiki/teams/5301/result/figure-14.jpeg)

Figure 14: Standard curve plotted using the Bradford method to measure protein concentration. The measured OD value of the protein sample is 0.351, corresponding to a concentration of 0.228 mg/ml.

### 4 Structure and Function Validation

When constructing multi-polymerized MSP, We predicted and analyzed the structure of three fragments and the overall structure of the multi-polymerized MSP using AlphaFold2, to confirm that the structure of the fusion protein is rational.

![](https://static.igem.wiki/teams/5301/result/atu15.png)

Figure 15: Alpha Fold 2 prediction model of three fragments of multi-polymerized MSP,(a) SCSdC-mCh[1-10] (b) SnCSdT (c) SnTST-mCh[11]

![](https://static.igem.wiki/teams/5301/result/figure-add.gif)

Figure 16: Alpha Fold 2 prediction model of the overall structure of multi-polymerized MSP

By controlling the reaction conditions, we successfully connected the three segments of MSP proteins.
To characterize the connection of the large cyclic MSP, we used SDS-PAGE to verify the molecular weight of the multi-polymerized MSP and observed the fluorescence of mCherry under a fluorescent inverted microscope to characterize the successful cyclization of the MSPs. Although the SDS-PAGE results are not clear (for specific content, please refer to the engineering section), clear fluorescence can be seen under the fluorescence microscope (Figure 17).

![](https://static.igem.wiki/teams/5301/result/figure-17-add.png)

Figure 17: The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.

In future experiments, we hope to further characterize multi-polymerized MSP to confirm its successful polymerization. Due to time constraints, we have only used Mono-MSP to produce nanodiscs and validate the Nanodisguiser function thus far. In upcoming experiments, we aim to use multi-polymerized MSP to manufacture nanodiscs and insert membrane proteins to better achieve the functionality of Nanodisguiser.

## Part3 Membrane Protein Polymerization

### 1 Construction and Verification of Plasmid Vectors

#### 1.1 Construction of Plasmid Vectors

To obtain Monomeric Streptavidin (mSA) fused with split-GFP, we designed two plasmids: pET-22b-mSA-sGFP1-10 and pET-22b-mSA-sGFP11, where mSA-sGFP1-10(or sGFP1-10 tether
[BBa\_K5301017](https://parts.igem.org/Part%3ABBa_K5301017)) and mSA-sGFP11 (or sGFP11 tether [BBa\_K5301018](https://parts.igem.org/Part%3ABBa_K5301018)) are our target fragments. To enhance protein expression, the sequence was optimized for codon usage.

![](https://static.igem.wiki/teams/5301/result/tu15.png)

Figure 18: Constructed Plasmids. Panel A shows the plasmid map of pET-22b-mSA-sGFP1-10, and Panel B shows the plasmid map of pET-22b-mSA-sGFP11.

#### 1.2 PCR Verification

After isolating single colonies on solid media, different colonies were picked, and colony PCR was performed to verify whether the plasmid was successfully transformed.

![](https://static.igem.wiki/teams/5301/result/tu16.png)

Figure 19: PCR Results of sGFP1-10 Tether, Trans2K DNA Marker.

The target fragment ([BBa\_K5301017](https://parts.igem.org/Part%3ABBa_K5301017)) length is 1071 bp, and all colonies successfully obtained the target fragment.

![](https://static.igem.wiki/teams/5301/result/tu17.png)

Figure 20: PCR Results of sGFP11 Tether, Trans2K DNA Marker.

The target fragment ([BBa\_K5301018](https://parts.igem.org/Part%3ABBa_K5301018)) length is 477 bp, and colonies 1, 2, 3, 5, and 6 successfully obtained the target fragment.

Colonies with successfully transformed plasmids were inoculated into 20 mL of LB liquid medium for single strain culture.

### 2 Expression Induction and Protein Purification

#### 2.1 Screening of Expression Induction Conditions

The presence of mSA in sGFP1-10 and sGFP11 tethers often leads to the formation of inclusion bodies, increasing purification difficulty. After reviewing the literature, we found that low-temperature expression can reduce inclusion body formation, so we determined 16°C as the induction temperature. To achieve more efficient expression of sGFP1-10/sGFP11 tethers, we set up a gradient of IPTG concentrations: 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM. The results indicated that protein expression of sGFP1-10 tether reached its optimum at 0.75 mM, while sGFP11 tether reached its optimum at 0.5 mM.

![](https://static.igem.wiki/teams/5301/result/tu18.png)

Figure 21: SDS-PAGE Analysis of sGFP1-10 Tether Protein Extraction Using IPTG Concentration Gradient, 10-180 kDa RealBand Pre-stained Protein Marker.

![](https://static.igem.wiki/teams/5301/result/tu19.png)

Figure 22: SDS-PAGE Analysis of sGFP11 Tether Protein Extraction Using IPTG Concentration Gradient, 10-180 kDa RealBand Pre-stained Protein Marker.

#### 2.2 Protein Purification

We used 1 mM IPTG to induce the strain for expressing the target protein and extracted the protein after inducing for 16 hours at 16°C. During the plasmid construction process, we incorporated a His-tag into the target protein to facilitate purification using nickel column affinity chromatography based on His-tag-specific binding.
In this section, we explored the conditions repeatedly, which will be described in detail in the [engineering section](https://2024.igem.wiki/bnu-china/engineering). Here, we present some of our successful results.

The molecular weight of sGFP1-10 tether is 39.2 kDa, while that of sGFP11 tether is 17.0 kDa. After purifying the supernatant obtained from the lysate of the bacterial culture, we found that the amount of soluble protein was low, and the protein existed in inclusion bodies in the precipitate. Therefore, we simultaneously purified the supernatant and inclusion body proteins, sequentially using elution buffers containing imidazole at concentrations of 100 mM and 150 mM to elute the proteins. The SDS-PAGE results, as shown in the figures, indicated that sGFP1-10 tether was eluted more effectively with the 150 mM elution buffer, while sGFP11 tether showed better elution with the 100 mM elution buffer. Gel analysis of the nickel beads revealed that a significant amount of sGFP11 tether was not eluted.

![](https://static.igem.wiki/teams/5301/result/tu21.png)

Figure 23: SDS-PAGE Analysis of sGFP1-10/sGFP11 Tether Protein Purification. Protein was expressed on a large scale using 1 mM IPTG induction at 16°C, followed by purification using nickel affinity chromatography. Molecular weight comparison was performed using the 10-180 kDa RealBand Pre-stained Protein Marker. Sample lanes are indicated above the image.

To improve the elution of proteins from the nickel beads, we attempted to increase the imidazole concentration, sequentially using elution buffers containing imidazole at concentrations of 100 mM, 250 mM, and 500 mM.It can be observed that increasing the imidazole concentration results in a larger amount of eluted protein. However, a higher imidazole concentration is not necessarily better; the elution buffer with 250mM imidazole eluted the highest amount of protein.

![](https://static.igem.wiki/teams/5301/result/tu22.png)

Figure 24: Results of Protein Purification After Changing Imidazole Concentration Gradient.

#### 2.3 Protein Concentration

We determined the protein concentration using the Bradford method, a dye-binding assay. Coomassie Brilliant Blue is red in its free state, with a maximum absorbance at 488 nm; upon binding to protein, it changes to blue, and the protein-dye complex has maximum absorbance at 595 nm. The absorbance value is proportional to the protein content, making it suitable for quantitative protein analysis. The binding of protein to Coomassie Brilliant Blue reaches equilibrium in about 2 minutes, and the reaction completes rapidly; the complex remains stable at room temperature for up to 1 hour.

**1. NanoDrop**

First, we prepared a gradient standard of BSA and prepared protein samples, using NanoDrop to determine the standard curve and measure protein concentrations. The results are as follows. The R² value of the standard curve for this measurement was 0.928, indicating a good fit. The measured concentrations were 81.540 μg/mL for sGFP1-10 tether and 81.540 μg/mL for sGFP11 tether.

![](https://static.igem.wiki/teams/5301/result/tu23.png)

Figure 25: Results of Protein Concentration Measurement Using NanoDrop.

**2. Microplate Reader**

We prepared a gradient standard BSA solution and sample, using a microplate reader to determine the standard curve and measure protein concentrations (note: this batch of protein is different from that measured by NanoDrop). The gradient standard BSA solution was added to wells B1 to B11 of a 96-well plate, while the protein samples were added to wells C1 to C6, D1 to D8, and E1 to E8. The BSA standard curve is shown in Figure 11, and the measured protein concentrations are presented in Table 1.

![](https://static.igem.wiki/teams/5301/result/tu24.png)

Figure 26: Results from the Microplate Reader. Specific samples are shown in Table 7.

![](https://static.igem.wiki/teams/5301/result/tu25.png)

Figure 27: BSA Standard Curve.

Table 7: Protein Concentration Results

|  |  | The concentration of imidazole used for elution | Absorbance | Concentration (mg/mL) |
| --- | --- | --- | --- | --- |
| sGFP1-10 tether | 50mM | C1 | 0.241 | 0.024 |
| 100mM | C3 | 0.256 | 0.037 |
| D1 | 0.322 | 0.092 |
| D2 | 0.377 | 0.138 |
| E1 | 0.279 | 0.056 |
| E2 | 0.352 | 0.117 |
| 150mM | D3 | 0.336 | 0.104 |
| D4 | 0.301 | 0.074 |
| E3 | 0.462 | 0.209 |
| E4 | 0.384 | 0.144 |
| 250mM | C5 | 0.303 | 0.076 |
| sGFP11 tether | 50mM | C2 | 0.238 | 0.022 |
| 100mM | C4 | 0.25 | 0.032 |
| D5 | 0.346 | 0.112 |
| D6 | 0.304 | 0.077 |
| E5 | 0.376 | 0.137 |
| E6 | 0.377 | 0.138 |
| 150mM | D7 | 0.251 | 0.033 |
| D8 | 0.27 | 0.048 |
| E7 | 0.28 | 0.057 |
| E8 | 0.33 | 0.099 |
| 250mM | C6 | 0.296 | 0.070 |

### 3 Protein Activity Analysis

In the process of purifying inclusion body proteins, we used a high concentration of urea for denaturation and solubilization. To ensure that streptavidin protein and split-GFP retain biological activity in subsequent conjugation experiments, we refolded the proteins. To determine the effectiveness of refolding, we measured the activity and concentration of the sGFP1-10 / sGFP11 tether proteins, using a method based on the ELISA assay.

Specifically, this experiment utilized the strong affinity between streptavidin and biotin, using HRP (horseradish peroxidase) as the labeled enzyme and biotin as the linking molecule. The enzymatic activity of HRP causes a color change in the substrate TMB, and the absorbance was measured using a microplate reader to assess the activity of streptavidin.

![](https://static.igem.wiki/teams/5301/result/tu26.png)

Figure 28: ELISA Results
Figure 12 ELISA Test Results;
Row A, wells 1-9 contain gradient concentrations of mSA standard solution, and wells 10 and 11 serve as negative controls with 0.5% BSA.
Row B, wells 1-10 contain gradient concentrations of mSA standard solution, and wells 11 and 12 are negative controls with 0.5% BSA.
Row C, wells 1 and 4 contain renatured samples of sGFP1-10 tether protein, and wells 2 and 5 contain renatured samples of sGFP11 tether protein.
Row D, well 1 contains a purified sample of sGFP1-10 tether protein, and well 2 contains a purified sample of sGFP11 tether protein.
Rows E and F, well 1 in both rows contains a purified sample of sGFP11 tether inclusion bodies, and well 2 in both rows contains a purified sample of sGFP1-10 tether inclusion bodies.

Due to some experimental errors, we were unable to obtain the mSA standard curve. Therefore, it is not possible to obtain specific protein activity data. But we have judged the protein activity through direct observations. By comparing the absorbance values with those of the mSA standards and the 0.5% BSA negative controls, it can be observed that the protein activity obtained from the inclusion body purification is very low. Even after the renaturation process, the activity did not improve. In contrast, the protein purified directly from the supernatant exhibits some activity and can be used for subsequent ligation experiments.

### 4 Biomolecular Fluorescence Complementation(BiFC)

We first investigated the incubation conditions for the binding of sGFP1-10/sGFP11 tether by mixing solutions of sGFP1-10 tether protein and sGFP11 tether protein in various volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. After incubation at 4°C, we observed whether fluorescence appeared.

![](https://static.igem.wiki/teams/5301/result/tu28.png)

Figure 29: Results of Fluorescence Observation(4\*10).
Fluorescence microscopy observation of the mixed incubation samples. Images A and C show background fluorescence, while images B and D display the results after the corresponding samples were added.

We observed fluorescence in some of the mixed incubation samples, indicating that the sGFP1-10 tether and sGFP11 tether successfully bound. Next, we will change the concentration ratio of sGFP1-10/11 tether, incubation time, etc. to explore the optimal conditions for their binding. Meanwhile, we will analyze the possible reason for the weak fluorescence due to low protein concentration, so we will also continue to optimize the method of purifying sGFP1-10/11 tether protein.

### 5 Binding of Biotinylated Membrane Proteins

In subsequent experiments, we will proceed with the purification and activity validation of sGFP1-10/11 tether, and simultaneously conduct BiFC (Biomolecular Fluorescence Complementation) experiments. We will utilize GFP handles to purify the successfully bound sGFP1-10/sGFP11 tether dimers. Once sufficient dimers are obtained, we will biotinylate the membrane proteins purchased with avi-tags and link them through the streptavidin-biotin system, ultimately constructing membrane protein dimers.

## Part4 Assembly of Nanodiscs

### 1 spNW15

#### 1.1 Native-page:

We prepared nanodiscs with different lipid-to-protein ratios, and immediately performed Native-PAGE characterization after the preparation was completed. Compared with lane 6, lane 4 (1:100 spNW15) had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful (Figure 30).

![](https://static.igem.wiki/teams/5301/result/figure-33.png)

Figure 30:from left to right:marker、1:100spNW15、1:110spNW15、spNW15

#### 1.2 Negative Stain Electron Microscope(EM)

We prepared negative stain electron microscope samples using the nanodiscs from lane 4 (1:100 spNW15) in Native-PAGE and performed negative stain electron microscopy imaging. The results are shown below (Figure 31). In the image, we can see that the black circular objects are the nanodiscs. Their diameter is approximately 15 nm, which is close to the theoretical value.

![](https://static.igem.wiki/teams/5301/result/figure-34.png)

Figure 31: (a, b) The negative stain EM image of spNW15 nanodiscs shows that the black circular objects in the image are the nanodiscs. (c) By analyzing the quantization results of electron microscope images, their diameter is approximately 15 nm, which is close to the theoretical value. (d) Because of the small size of the nanodiscs, the error of DLS results is large.

### 2 spMSP1D1

#### 2.1 Native-PAGE

We prepared nanodiscs with different lipid-to-protein ratios, and immediately performed Native-PAGE characterization after the preparation was completed. Compared with lane 1, other lanes had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful (Figure 32a).

Next, we will prepare spMSP1D1 nanodiscs incorporating the membrane protein LDL-R and perform preliminary validation using Native-PAGE (Figure 32b). We prepared nanodiscs with a LDL-R:spMSP1D1:DOPC ratio of 2:5:60 and 1:2:130. First, the membrane protein is mixed with DOPC and incubated on ice for 1 hour to form lipid-detergent micelles. Then, spMSP1D1 protein is added to the protein-lipid mixture and incubated for 10 minutes. Fresh Bio-beads are then added to adsorb the detergent from the mixture, inducing the self-assembly of nanodiscs. The mixture is gently incubated at 4°C for 2 hours before being supplemented with fresh Bio-beads and continuing to incubate overnight at 4°C with gentle shaking.

![](https://static.igem.wiki/teams/5301/result/figure-35.png)

Figure 32: (a) The first lane is a control for spMSP1D1 protein, and lanes two to four are nanodiscs with protein-to-lipid ratios of 1:60, 1:90, 1:100 and 1:110, respectively. (b) The result of Native-PAGE. The last lane is spMSP1D1 nanodiscs incorporating membrane.

#### 2.2 negative stainEM (negative stain electron microscope)

We prepared negative stainEM samples using the spMSP1D1 nanodiscs and performed negative stain electron microscopy imaging.
The results are shown below (Figure 33a).
In the image, we can see that the black circular objects are the nanodiscs.
Their diameter is approximately 6-8 nm, which is close to the theoretical value of 11nm (Figure 33c).
Surprisingly, we also observed the nanodiscs from the side (Figure 33b).
We also used negative stain EM to observe the nanodiscs incorporating LDL-R (Figure 33e).
In the images, some black dots can be seen on the nanodiscs, which we believe are the membrane proteins.

![](https://static.igem.wiki/teams/5301/result/figure-36.png)

Figure 33:(a) The negative stain electron microscope image of spMSP1D1 nanodiscs shows that the black circular objects in the image are the nanodiscs. (b) We observed the nanodiscs from the side. (c) According to the quantitative statistics of electron microscope results, the particle sizes of our nanodiscs are close to the value in the literature. (d) Because of the small size of the nanodiscs, the error of DLS results is large. (e) TEM results of nanodiscs incorporating membrane proteins.

### 3 spNW50

#### 3.1 Native-PAGE

The lipid-to-protein ratios had been used of 1:600,
and immediately performed Native-PAGE characterization after the preparation was completed.
Compared with lane and 5, lane 2 and 3 had a significant difference,
which is similar to the band shape in the literature.
We preliminary considered that it is successful.(figure 34)

![](https://static.igem.wiki/teams/5301/result/figure-37.png)

Figure 34:100mM nanodisc failed, 300mM nanodisc molecular weight has a certain difference.

#### 3.2 DLS (Dynamic Light Scattering) &negative stainEM (negative stain electron microscope)

We prepared DLS samples using the nanodiscs from lane 3 in Native-PAGE and performed the DLS.
The result showed that, the diameter of nanodiscs was around 300nm (figure 35d.)

Then we prepared negative stain electron microscope samples using the spNW50 nanodiscs and
performed negative stain electron microscopy imaging.
The results are shown below (Figure 35abc).
In the image, we can see that the black circular objects are the nanodiscs.
Their diameter is approximately 300 nm, which is close to the DLS result.

![](https://static.igem.wiki/teams/5301/result/atu35.png)

Figure 35:(a, b, c) The negative stain electron microscope image of spMSP1D1 nanodiscs shows that the black circular objects in the image are the nanodiscs. (d) The frequency distribution histogram of particle size measured by DLS shows that the peak particle size is around 200-300 nm.

![](https://static.igem.wiki/teams/5301/result/figure-39.png)

Figure 36:Three monomer MSPS, pMSP1D1 (a), spNW15 (b) and spNW50 (c), have successfully produced nanodisks with different particle sizes. The difference in particle size (d) can be highlighted by the box plot.

## Part5 Functional Verification

### 1 Construction of the three plasmids

In the part of functional verification, we established a new SEND system to simulate the process of virus invading cells via membrane fusion. The novel SEND system demands three plasmids: capsid, cargoRNA, and fusogen. The capsid and fusogen plasmids we utilized were sourced from the commercial offerings of Miaoling Biotechnology. We transferred the pre - made plasmid products into E.coli DH5α for amplification and extraction.

![](https://static.igem.wiki/teams/5301/result/figure-30.png)

Figure 37: Capsid and fusogen plasmid

A. Plasmid profile of capsid; B. Plasmid profile of fusogen

Regarding the cargoRNA plasmid, we joined the commercial cargo backbone to the superfolderGFP fragment through homologous recombination. The superfolderGFP fragment was obtained from an official plasmid donated by iGEM. For the effective function of cargoRNA, the reporter gene (superfolderGFP) needs to be connected on both sides of PEG105'UTR and PEG103'UTR; thus, we opted for seamless cloning of the plasmid. Firstly, we amplified and extracted the cargo backbone plasmid and superfolderGFP plasmid. Subsequently, we obtained a linearized cargo backbone (carrying PEG10 UTR sequences on both sides) by reverse PCR. Meanwhile, the superfolderGFP fragment sequence was procured from the plasmid by PCR.
To obtain the two pure linear PCR products, we employed agarose gel for slice recycling. Finally, using an infusion kit, we constructed the recombinant cargoRNA plasmid.
To affirm the validity of the plasmid, we carried out fragment sequencing. All the experimental data indicated that the recombinant cargoRNA plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5301/result/figure-38-change.png)

Figure 38: Construction of cargoRNA plasmid

A. Plasmid profile of commercial cargo backbone;

B. Plasmid profile of superfolder GFP;

C. E.coli transferred by the sfGFP plasmid which fluoresces green under UV light;

D. Electrophoresis results of sfGFP gene fragment;

E. Electrophoresis results of cargo backbone gene fragment;

F. cargoRNA plasmid fragment sequencing.

### 2 HEK 293FT cell culture

The cells used in the verification experiment were HEK 293FT cells from Shanghai Binsui Biotechnology.
HEK 293FT cells are adherent cells with a polygonal epithelial-like appearance. Through microscopic observation during multiple passages, the cell status and viability were confirmed.

![](https://static.igem.wiki/teams/5301/result/figure-32.png)

Figure 39: HEK 293FT cell

### 3 VLPs obtainment after plasmid transfection

To confirm the successful transfection of the three plasmids and the effective expression of VLP, we conducted SDS - PAGE verification.
The results of SDS-PAGE showed that the cells produced proteins similar in size to PEG10 and VSV-G. However, protein bands also appeared at the same positions in the control medium and in the supernatant of normal cells. (Fig 40B.)
To further verify the successful transfection of the plasmids, fluorescent monitoring of the cells was performed. We observed that 24 hours after transfection, the cells emitted green fluorescence. (Fig 40A.) And these results suggest that the transfected cells may have expressed exosomes containing PEG10 VLPs.

![](https://static.igem.wiki/teams/5301/result/figure-40-add.png)

Figure 40: Cells expressed exosomes containing PEG10 VLPs

A. After transfection of the three plasmids, cells emit green fluorescence (red circles);

B. SDS-PAGE of supernatant (VLP: supernatant suspected to contain VLP; Medium: cell culture-medium; Cell supernatant: supernatant of untransfected cells)

### 4 Cells infected by VLPs

In order to confirm that PEG10 VLP exosomes are capable of infecting target cells and delivering mRNA, and to verify the blocking effect of nanodiscs on VLP, we performed a controlled infection experiment on HEK293 FT cells. The infusions added to the cells of the experimental group, positive control group and negative control group were successively: VLP supernatant containing nanodiscs, VLP supernatant alone, and cell culture medium containing neither VLP nor nanodiscs. After adding the corresponding solution, fluorescence monitoring was performed on the cells at an interval of 30 minutes. In the observation of the positive control group, we found that the internal cells did emit green fluorescence. This indicates that the cells acquired the mRNA of the GFP protein and expressed it. The only possible source of this mRNA is the supernatant we added. This result confirms that the novel SEND system can produce PEG10 VLP exosomes capable of effectively transporting mRNA, leading to its expression in target cells.

![](https://static.igem.wiki/teams/5301/result/figure-41-add.png)

Figure 41: After being infected by supernatant suspected to contain PEG10 VLP exosomes, the interior of the cells fluoresced green.

Furthermore, fluorescence observations showed that the cells with only culture medium did not express fluorescence. And on the whole, cells treated with VLPs alone expressed more GFP protein compared to those that also received nanodiscs. (Figure 42) This result partially validates the blocking effect of nanodiscs on the pseudovirus VLPs. It is speculated that the nanodisc has a blocking effect on membrane fusion virus.

![](https://static.igem.wiki/teams/5301/result/figure-42-add.png)

Figure 42: Fluorescence of HEK293 FT cells in control infection experiment over time

### 5 Further Experiments

In the final control infection experiment, we were unable to obtain further fluorescence intensity data due to time constraints. Counting fluorescent cells by eye can introduce certain errors. Additionally, the monitoring of fluorescence levels during the experiment may have been influenced by the selection of the microscopic field of view. Our conclusions may be affected by these factors, so we plan to conduct more rigorous validations in future experiments, such as fixing the observation field and obtaining fluorescence intensity values to reflect infection rates. For conclusions based on indirect evidence, we aim to design more direct experiments. For example, we plan to use Western Blot and electron microscopy to directly confirm the expression of PEG10 VLP exosomes. Regarding the verification of the blocking function of nanodiscs on VLPs, we hope to use methods like DLS to provide a more microscopic perspective on the membrane fusion process.

{% endblock %}