iGEM 2023 — Team nudt-china

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Notebook

[2022](#section1)

[2023](#section2)

November 2022

Organizing campus promotions for iGEM 2023.

December 2022

Holding a biology contest to select team members for NUDT-CHINA 2023.

January 2023

Establishment of team NUDT-CHINA 2023. Team member chose their interesting role to play according to their interests, including Wet Lab, Human Practice, Art&Design.

February 2023

Conducting a literature review on industries associated with obesity.

Conducting a questionnaire survey among over 2,000 participants.

March 2023

Brainstorming for 2023 team project.

Learning the molecular biology and basic experimental techniques.

Laboratory safety training.

April 2023

Determining the initial direction of the 2023 iGEM project, considering to choose UCP1 as our fundamental weight-losing protein to deliver. However, the specific method to deliver remained undecided.

May 2023

Determining the direction of using PVCs as delivery tool. Til here, project ARROW was born.

Conducting preliminary Proof of Concept design and plasmid design.

Conducting an interview with Professor Zhang. Our dedication to the project became stronger.

June 2023

Collection and study of relevant literature regarding the application of our research project.

Development of the 1st Generation ARROW Head protein.

Preliminary Safety Form and Project Description.

Start modeling for UCP1 functionality.

 Offline meeting of the Wet Lab group for literature reading, reporting the progress of the project and arranging the follow-up tasks.

July 2023

Team uniform & Project logo & Team 10th Anniversary Logo design.

Online meetings of the experimental group for literature reading and plasmid design.

Confirming PVC specificity with PVCs that tail fibers were modified with DARPin and gain positive results.

Conducting AlphaFold2 Multimer V3-based structure prediction for reprogrammed PVC.

Start constructing 25K PVC plasmid with normal molecular strategy.

Conducting AlphaFold2-based structure prediction for ARROW Head structure.

Interview with drug quality monitoring expert Xiao. Inspired with his advices on ARROW further implementation.

August 2023

Coming up with the 2nd Generation ARROW Head protein, changing the UCP1 to the C-terminus of the fusion protein.

Production of the Promotion Video.

Creation of team giveaways and mascots.

Offline meeting of the wet lab group for further design and engineering.

Commencement of production for the “Gene Editing” Science Popularization Picture Book.

September 2023

Team Roster Confirmed.

Attending National Science Popularization Day Series Events.

Purifying PVCs (loaded with 2nd Generation ARROW Head protein) with different E.coli culture condition.

Interview with master Zhou. Inspired with effective Golden Gate assembly method.

Conduct 2nd Generation ARROW Head delivery experiment to Hela Cells.

Interview with expert from drug company Xiao. Informing us with detailed possible further application of our project.

October 2023

Team family photo.

Interview with synthetic biology experts Zhou about 3rd ARROW Head Constuction.

WIKI Construction.

Production of Presentation Video.

November 2023

Participation of Grand Jamboree.

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Engineering

[Introduction](#section1)

[Fat-burning Payload Module](#section2)

[Adipose-targeting PVC Module](#section3)

[Reference](#section4)

# Introduction

##### The engineering of a mitochondrial uncoupling payload protein and the reprogramming of PVCs to target adipocytes are two critical steps in the development of ARROW, our weight-loss drug (see the detailed design of ARROR in the [**Description**](https://2023.igem.wiki/nudt-china/description) and [**Design**](https://2023.igem.wiki/nudt-china/design) pages). Through multiple design-build-test-learn cycles, we have successfully engineered the fat-burning payload, a UCP1-based fusion protein that can be effectively expressed in bacteria and packaged into PVCs while maintaining its ability to enhance cell metabolism upon delivery into target cells. Simultaneously, we have been working on constructing an adipocyte-targeting PVC coat by modifying its tail fibers. In this section on engineering success, we will go through the engineering cycles we went through to develop the fat-burning payload and adipocyte-targeting tail fibers. (Figure 1).

![overview](https://static.igem.wiki/teams/4960/wiki/design/engineering/new/1.jpg)

**Figure 1. An Adipocyte-targeting UCP1-delivering PVC Particle.**

![top2](https://static.igem.wiki/teams/4960/wiki/design/engineering/top22.png)

# Engineering of the Fat-burning Payload Module

![overview](https://static.igem.wiki/teams/4960/wiki/design/1234.jpg)

**Figure 2. An Schematic Diagram of the Fat-burning Payload Module Engineering.**

### The First Engineering Cycle

### Design:

##### In order to efficiently package UCP1 into PVCs, a Pdp1NTD protein domain must be fused to its N-terminus. Additionally, we decided to fuse an EGFP tag onto UCP1 to improve solubility and provide a visible tag. Hence, it would be crucial to test whether this gene fusion affects the subcellular localization and function of UCP1. It has been reported that a C-terminus fusion of EGFP does not affect the mitochondrial localization and function of UCP1 (Bates et al., 2020). Hence, we first designed the Pdp1NTD-UCP1-EGFP construct (Fig. 3a) and validated its localization and function in HEK-293T cells.

### Build and Test:

##### To assess the function of the fusion protein, we transfected HEK-293T cells with pNC087, which encodes a PCMV promoter-driven Pdp1NTD-UCP1-EGFP protein expression cassette. Cells were imaged at 48 h post transcription to identify the cellular localization of the fusion protein. Cellular glucose consumption was then evaluated by measuring the remaining glucose levels in the cell culture medium. Results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was localized all over the cytoplasm and nucleus (Fig. 3b). Consistent with the failed mitochondrial localization, glucose levels in the pNC087-transfected cells showed no significant difference compared to the control group transfected with pcDNA3.1(+) vector only (Fig. 3c). Interestingly, Alphafold2-based prediction of Pdp1NTD-UCP1-EGFP structure revealed an unexpected interaction between the Pdp1NTD domain and the UCP1 domain, which might be the cause of the UCP1 function loss in this design (Fig. 3d).

![overview](https://static.igem.wiki/teams/4960/wiki/new-12-8/engineering2.jpg)

**Figure 3. The design and characterization of the 1st Generation Fat-burning payload.**
(a) Design of the 1st Generation of Fat Burning Payload Fusion Protein (Pdp1NTD-UCP1-EGFP). (b) Localization of Pdp1NTD-UCP1-EGFP payload protein in HEK-293T cells under wide-field microscopy. HEK-293T cells were transfected with pNC087 (PCMV-Pdp1NTD-UCP1-EGFP) and imaged at 48 h post transfection, scale bar: 100 μm. Data are representative image of 3 independent experiments. (c) Bar Chart Illustrating Glucose Consumption in pNC087 Transfected HEK-293T Cells. Glucose concentration in the cell culture medium concentration was measured 48 h post transfection; data shows mean±SD, n=3 independent experiments. (d) AlphaFold2-based Structural Prediction of Pdp1NTD-UCP1-EGFP Fusion Protein.

### Learn:

##### According to the structural prediction, the failure of the Pdp1NTD-UCP1-EGFP construct was probably due to the undesired interaction between the Pdp1NTD and UCP1, which affects the localization and function of the protein. This problem could be corrected by altering the linker length or changing the overall protein design.

# The Second Engineering Cycle

### Design:

##### Based on the knowledge gained from the initial engineering cycle, we then tried multiple design options with AlphaFold2. Interestingly, structural prediction showed that by simply swapping the UCP1 and EGFP domain, the Pdp1NTD-EGFP-UCP1 configuration could effectively reduce the interaction between Pdp1NTD and EGFP (Fig. 4a). We then tested this construct in HEK-293T cells.

### Build and Test:

##### Similar to the first engineer cycle, we transfected HEK-293T cells with pNC088, a Pdp1NTD-EGFP-UCP1 expressing plasmid, and evaluated the cellular localization and function of the fusion protein at 48 h post-transfection. As expected, both wide-field fluorescent imaging (Fig. 4b) and live-cell confocal imaging (Fig. 4c) showed a highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria (labeled by MTS-mcherry). Moreover, cells transfected with pNC088 showed a significantly higher glucose consumption compared to the control cells transfected with pcDNA3.1(+) vector (Fig. 4d), suggesting a significantly improved energy consumption in these cells.

![overview](https://static.igem.wiki/teams/4960/wiki/new-12-8/engineering3.jpg)

**Figure 4. The design and characterization of the 2nd Generation Fat-burning payload.**
(a) AlphaFold2-assisted Prediction of the Structure of 2nd Generation of Payload Fusion Protein. (b, c) Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells. For wide-field microscopy in b, cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1). For confocal images in c, cells were co-transfected with MTS-mcherry and PNC088. Photos were taken 48 h post transfection, scale bar: 100 μm for wide-field microscopy and 10 μm for confocal microscopy. Data are representative images of 3 independent experiments. (d) Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC088 or pcDNA3.1(+). Glucose concentration in the cell culture medium was measured 48 h after transfection; data shows mean±SD, n=3 independent experiments.

##### With the Pdp1NTD-EGFP-UCP1 construct as a promising payload, we then proceeded to investigate whether it can be successfully expressed in E. coli and assembled into PVCs. Therefore, we electroporated our payload plasmid pNC093 (expressing Pdp1NTD-EGFP-UCP1) together with an EGFR-targeting pPVC vector (pNC090, similar to the pAWP78-PVCpnf\_pvc13-E01DARPin plasmid reported by Kreitz et al.) into E.coli to generate the PVCEGFP−UCP1EGFR-targeting  particle (Fig. 5a). The particle then purified by ultracentrifuge. The expression of Pdp1NTD-EGFP-UCP1 protein was then validated by SDS-PAGE (Fig. 5b). Importantly, TEM imaging of the purified PVC fraction showed the existence of syringe-like structures similar to the previously published paper (Fig. 5c), suggesting the Pdp1NTD-EGFP-UCP1 payload could be efficiently packed into PVCs. Additionally, by incubating these PVC particles with HEK-293T cells transfected with either EGFR-expressing plasmid or empty vector control, we demonstrated these PVCEGFP−UCP1EGFR-targeting  particles could selectively enhance the energy expenditure in EGFR-expressing cells (Fig. 5d).

![overview](https://static.igem.wiki/teams/4960/wiki/new-12-8/engineeringimg5.png)

Figure 5. Delivery of the Fat Burning Payload through PVCEGFP−UCP1EGFR-targeting  Particles to HEK-293T Cells. (a) Schematic representation of the construction of PVCEGFP−UCP1EGFR-targeting  particles. (b, c) Charactrization of the assembled PVCEGFP−UCP1EGFR-targeting  particles by SDS-PAGE (b) and negative-strain TEM (c). Scale bars, 100nm. (d) Charactrization of cellular metabolism in PVCEGFP−UCP1EGFR-targeting  treated HEK-293T cells transfected with either pNC089 or pcDNA3.1(+) plasmids. Glucose concentration in the cell culture medium was measured 48 h after PVCEGFP−UCP1EGFR-targeting  administration; data shows mean±SD, n=3 independent experiments.

### Learn:

##### Through the aforementioned two cycles, we have successfully generated a UCP1-based payload protein that works as we expected. Once overexpressed in mammalian cells, it is correctly located in mitochondria and is able to enhance cellular energy expenditure. It can also be effectively expressed in E. coli and loaded into PVCs. Additionally, PVCs carrying these payload proteins could effectively enhance cell metabolism in a target-dependent manner.

# Engineering of the adipose-targeting PVC Module

![overview](https://static.igem.wiki/teams/4960/wiki/design/456.jpg)

**Figure 6. Schematic diagram of the adipose-targeting PVC coat module engineering**

![](https://static.igem.wiki/teams/4960/wiki/subpage2/bkpic3.png)

# The First Engineering Cycle

### Design:

##### To facilitate the targeted delivery of UCP1 into white adipose tissue using PVC, our design involved modifying the tail fiber protein of PVC (PVC13). We achieved this by attaching a previously described 9-mer peptide that specifically targets adipose tissue (Kolonin et al., 2004). In order to ensure optimal exposure of the targeting peptide on the tail fiber, we used protein structure prediction deep learning model AlphaFold2, to examine the trimeric structure of PVC13(as shown in Figure 7a) via AlphaFold2. By considering different types of linkers to connect PVC and the 9-mer peptide (as depicted in Figure 4a), we identified three potential candidates for our initial trials: 1GS, 3GS, and 1EAAAK. These candidates were chosen based on their ability to individually and effectively expose the 9-mer peptide. Our initial cloning strategy employed traditional infusion cloning methods, utilizing SphI and SalI enzymes for the double enzyme digestion of the pNC090 vector (PAWP78-PVCpnf pvc13-E01DARPin). We then performed PCR amplification for the subsequent fragments, fNC177 and fNC178, which were specifically designed for the insertion of targeting peptide and linker sequences. To maximize the abundance of infusion products, these two fragments were overlapped during the cloning process.

### Build and Test:

![overview](https://static.igem.wiki/teams/4960/wiki/new-12-8/engineering4.jpg)

Figure 7. Design and Construction of Adipose-targeting Tail Fiber Proteins. (a) Alphafold2-based design of adipose-targeting peptide-presenting tail fibers. (b) Agarose gel electrophoresis image showing the digestion of pNC090 (PAWP78-PVCpnf pvc13-E01DARPin) with SphI and SalI restriction enzymes. (c) Overlapping PCR Analysis of fNC177 and fNC178.

##### During the experiments, we encountered challenges with our plasmid constructs. Specifically, when digesting pNC090 with SphI and SalI restriction enzymes, we observed a band around 6 kb, resulting in a lower than expected concentration after gel purification(Figure 7b). Additionally, during the overlap PCR of inserting fragments fNC177 and fNC178, the desired band at approximately 6 kb did not appear, despite our effort to optimize reaction conditions (Figure 7c).

# The Second Engineering Cycle

### Design and Future Work

##### During our interview with Mr.Deyang Zhou, renowned in the field of mammalian cell synthetic biology, we discussed the challenges encountered in cloning the 25 Kbp plasmid. To streamline the plasmid construction process, he suggested exploring the utilization of the Golden Gate Assembly method. Surprisingly, upon devising a specific cloning strategy for each plasmid, we observed a significant reduction in workload. In particular, we designed a novel PVC 13 part (BBa\_K4960024) to facilitate subsequent cloning of the targeting sequence into the pPVC plasmid. The engineered model plasmid, pNC092 (pAWP78-PVCpnf\_pvc13-2\*BsaI), features this PVC13 part and can serve as a template for future teams seeking to modify PVCs (Figure 8). Unfortunately, due to time constraints, we could not complete the construction of the intended plasmids before the WIKI FREEZE deadline. However, we are currently undergoing construction using the Golden Gate Assembly method. Once these constructs are finalized, we will promptly proceed with testing the fat-burning payload on 3T3-L1 cells, enabling us to continue the development of the adipose-targeting PVC coat Module.

![overview](https://static.igem.wiki/teams/4960/wiki/design/engineering/img7.png)

Figure 8. Schematic Diagram of Golden Gate Assembly Strategy for New pvc13-based pPVC Constructs

##### The aforementioned engineering process outlines the comprehensive efforts involved in building, testing, and optimizing the UCP1-delivering adipocyte-targeting PVC particle, ARROW, from scratch. This intricate process can be categorized into two key modules: the fat-burning payload module and the adipocyte-targeting PVC coat module. Thorough characterization of the fat-burning payload has undeniably demonstrated its efficacy. For more detailed insights into our future developments, we encourage you to refer to our Implementation and Integrated Practices pages.

# Reference

##### Bates, R., Huang, W., & Cao, L. (2020). Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Mol Ther Methods Clin Dev, 19, 236-249. https://doi.org/10.1016/j.omtm.2020.09.009 Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by targeted ablation of adipose tissue. Nat Med, 10(6), 625-632. https://doi.org/10.1038/nm1048

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Experiments

[Cell Part Protocol](#section1)

[Molecular Part Protocol](#section2)

# Cell Part Protocol

In Vitro PVC Delivery Experiments

Protocol for Cell Culture and Cubculture

Protocol for Cell Transfection and Stimulation

Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis

# Molecular Part Protocol

Agarose Gel Electrophoresis

Double Enzyme DNA Digestion& Agarose Gel Electrophoresis

Electroporation

Gel Purification

Infusion

Preparation of Electrocompetent Bacterial Cells

Overlap PCR & Agarose Gel Electrophoresis

PCR & Agarose Gel Electrophoresis – Colony PCR

PCR & Agarose Gel Electrophoresis

Plasmid Conservation

Plasmid Extraction

Protocol for LB Medium Preparation

PVC Purification

Transformation

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# Engineered Photorhabdus Virulence Cassette (PVC) particles could specifically deliver payload proteins into target mammalian cells

To demonstrate that PVCs could serve as a viable target-specific protein-delivering system, we
first engineered Escherichia coli to produce PVCs from P. asymbiotica ATCC 43949 (PVCpnf) using
a method similar to the one published by Kreitz et al.(Kreitz et al., 2023). Similar to their
design, we split the PVC system into separate structural/accessory plasmid (pPVC) and
payload/regulatory plasmid (pPayload). To evaluate the target-specific protein delivery function
of engineered PVCs, we first synthesized a pPVC plasmid carrying E01DARPin (pNC090, similar to
the pAWP78-PVCpnf\_pvc13-E01DARPin plasmid reported by Kreitz et al.) that specifically targets
EGFR and a pPayload plasmid carrying Cre recombinase (pNC091, similar to the
pBR322-Pdp1NTD-Cre-HiBiT plasmid reported by Kreitz et al.). Active PVCs were then purified from
the lysate of *E. coli* cells electroporated with pNC090 and pNC091 via ultracentrifuge **(Figure
1a)**. Negative-stain transmission electron microscopy resembled canonical PVC structures
similar
to the ones reported by Kreitz et al. **(Figure 1b)**, suggesting a successful assembly of
PVCs.

![fig1](https://static.igem.wiki/teams/4960/wiki/new-12-8/resultsfig1.jpg)

**Figure 1. Validation of PVC Particles Specificity Delivery of Payload into Mammalian
Cells.****(a)** Schematic representation of the Construction of Engineered PVC particles. PVCs were
purified
from *E.coli* electroporated with pNC090 (pPVC with EGFR targeting E01DARPin) and pNC091
(pPayload
with Cre) via ultracentrifuge. **(b)** Negative-strain TEM of the purified Cre-loading, EGFR-Targeting PVCs. Scale bars:
600 nm.
**(c)** PVC-dependent activation of gene expression in mammalian cells. Left: Schematic
diagram of
mammalian cell gene expression induced by Cre-delivering PVCs. Right: HEK-293T cells transfected
with either pcDNA3.1(+) plasmids or pLZ362 (PCMV-LoxP-STOP-LoxP-sNluc) were treated
with
Cre-carrying, EGFR-Targeting PVCs. sNluc production in the culture medium was measured 72 h
after PVC administration; data shows mean±SD, n=3 independent experiments.

To evaluate the protein-delivering function of PVCs, we incubated these EGFR-targeting,
Cre-delivering PVCs (

*PVC*CreEGFR\_targeting

                   )
with HEK-293T cells co-transfected with pLZ362
(PCMV-LoxP-STOP-LoxP-sNluc, a gift from Lihang Zhang, Westlake University) and either
pNC089
(PCMV-EGFR) or pcDNA3.1(+) plasmids. Secreted Nanoluc (sNluc) levels in the culture
medium were
then evaluated at 72 h post PVCs treatment. Results showed a significant increase in sNluc
levels in EGFR-expressing cells treated with PVCs, while the cells without EGFR expression
showed low sNluc levels **(Figure 1c)**. These results demonstrated that engineered PVCs
could
specifically deliver payload proteins into target mammalian cells, thereby serving as a possible
delivery mechanism for our purposes.

# Structural-informed engineer of a UCP1-based Payload Protein

In PVCpnf, the N-terminus domain of Pdp1 (Pdp1NTD) is critical for the efficient loading of payload
proteins into the PVCs. Hence, to efficiently load mitochondrial uncoupler proteins (UCP1 in our
case) into the PVCs, an N-terminus Pdp1NTD domain must be supplemented. In addition, we also decided
to fuse an EGFP tag to provide a potential visualization signal and improve the solubility of UCP1
in the E. coli expression system.

Since the UCP1-EGFP construct has been previously reported
in
transgenic mice (Bates et al., 2020), we started by constructing a Pdp1NTD-UCP1-EGFP construct as an
initial test **(Figure 2a)**. To validate the protein function in the best possible scenario, we
transfected the HEK-293T cells with pNC087, a mammalian expression vector carrying the
PCMV-Pdp1NTD-UCP1-EGFP cassette. Cells were imaged at 48 h post transfection to validate
the
subcellular location of the Pdp1NTD-UCP1-EGFP fusion protein. Unfortunately, results showed that
instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was localized all over the
cytoplasm and nucleus **(Figure 2a)**, suggesting the interaction between UCP1 and key chaperone
proteins enabling its mitochondria translocation was compromised by the protein fusion. Moreover, by
evaluating the remaining glucose level in the culture medium, we analyzed the glucose consumption of
the cells transfected with either pNC087 or pcDNA3.1(+) control plasmid, which, in a way,
represented the level of cellular energy expenditure. Consistent with the failed mitochondrial
localization, glucose levels in the pNC087-transfected cells showed no significant difference
compared to the control group transfected with pcDNA3.1(+) vector only **(Figure 2b)**.

To
understand how the fusion of Pdp1NTD and EGFP affected the function of UCP1, we performed structural
prediction using AlphaFold2. Interestingly, we observed an unexpected interaction between the
Pdp1NTD domain and UCP1 (**Figure 2c**, red box), which could possibly change the local protein
structure and affect the translocation and function of UCP1. With the same protocol, we predicted
the structure of a few design options and found that we could simply solve this problem by swapping
UCP1 and EGFP **(Figure 2d)**.

To test this option, we then constructed pNC088, a
mammalian cell expression vector carrying the PCMV-Pdp1NTD-EGFP-UCP1 cassette. As
expected, both
wide-field fluorescent imaging **(Figure 2e)** and live-cell confocal imaging **(Figure 2f)**
showed highly
specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria markers (MTS-mcherry,
**Figure
2f**). Moreover, cells transfected with pNC088 showed a significantly higher glucose
consumption
compared to the control cells transfected with pcDNA3.1(+) vector **(Figure 2g)**, suggesting a
significantly improved energy consumption in these cells.

![fig2](https://static.igem.wiki/teams/4960/wiki/new-12-8/resultsfig22222.jpg)

**Figure 2. Functionality of UCP1-based Payload Protein in HEK-293T Cells.
(a)** Localization of UCP1-based payload protein Pdp1NTD-UCP1-EGFP in HEK-293T cells under
wide-field
microscopy. HEK-293T cells were transfected with pNC087 Pdp1NTD-UCP1-EGFP and imaged 48 h post
transfection, scale bar: 600 μm. Data are representative image of 3 independent experiments
**(b)** Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC087
or
pcDNA3.1(+). Glucose concentration in the cell culture medium concentration was measured 48 h post
transfection; data shows mean±SD, n=3 independent experiments.
**(c)** AlphaFold2 prediction of Pdp1NTD-UCP1-EGFP protein structure. The unexpected interaction
between
SepC and UCP1 is labeled in a red box.**(d)** AlphaFold2 prediction of Pdp1NTD-EGFP -UCP1 protein structure.**(e, f)** Localization
Pdp1NTD-EGFP-UCP1 in HEK-293T cells.For wide-field microscopy in **e**, cells were transfected
with
pNC088 (PCMV-Pdp1NTD-EGFP-UCP1).For confocal images in **f**, cells were
co-transfected
with
MTS-mcherry and PNC088. Photos were taken 48 h post transfection, scale bar: 100μm for wide-field
microscopy and 10 μm for confocal microscopy. Data are representative images of 3 independent
experiments.**(g)** Charactrization of cellular metabolism in HEK-293T cells transfected with
either
pNC088 or pcDNA3.1(+). Glucose concentration in the cell culture medium was measured 48 h after
transfection; data shows mean±SD, n=3 independent experiments.

# PVC-mediated delivery of the Pdp1NTD-EGFP-UCP1 effectively altered the energy expenditure in mammalian cells in a target-dependent manner.

With the PCMV-Pdp1NTD-EGFP-UCP1 construct as a promising payload, we then proceeded to
investigate
whether it can be successfully expressed in E. coli and loaded into PVCs. For such matter, we
constructed pNC093, a pPayload plasmid carrying Pdp1NTD-EGFP-UCP1 payload. By electroporating E.
coli cells with both pNC093 and a pPVC plasmid carrying E01DARPin (pNC090), we could then get a

*PVC* EGFR\_UCP1EGFR\_targeting

particle that specifically targets EGFR-expressing cells and
delivers Pdp1NTD-EGFP-UCP1 protein **(Figure 3a)**.

To validate whether Pdp1NTD-EGFP-UCP1
protein could be correctly expressed, we performed SDS-PAGE analysis on purified PVCs, which showed
a strong and clear band on approximately 69 kDa, which resembles Pdp1NTD-EGFP-UCP1
**(Figure3b)**. In the meantime, negative-stain transmission electron microscopy on purified PVCs
**(Figure 3c)**
showed similar structures to the Cre-carrying PVCs in Figure 1b, suggesting that the
Pdp1NTD-EGFP-UCP1
protein could be correctly loaded into the PVCs. Additionally, by incubating these PVC particles
with HEK-293T cells transfected with either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids.
we
demonstrated these

*PVC*EGFR\_UCP1EGFR\_targeting

particles could selectively enhance the energy
expenditure in EGFR-expressing cells **(Figure 3d)**. Altogether, our findings demonstrate a
successful
engineer of a PVC-based strategy to boost cellular energy expenditure by specifically deliver UCP1
into target cells.

![fig3](https://static.igem.wiki/teams/4960/wiki/new-12-8/resultsfig3.jpg)

**Figure 3. Delivery of the Fat Burning Payload through  *PVC*EGFR\_UCP1EGFR\_targeting

Particles
to HEK-293T Cells.(a)** Schematic representation of the construction of
 *PVC* EGFP\_UCP1EGFR\_targeting

particles.**(b, c)** Charactrization of the assembled  *PVC*EGFP\_UCP1EGFR\_targeting

particles by
SDS-PAGE**(b)** and negative-strain TEM**(c)**. Scale bars, 100nm.**(d)** Charactrization
of
cellular
metabolism in  *PVC* EGFP\_UCP1EGFR\_targeting

treated HEK-293T cells transfected with either pNC089
or pcDNA3.1(+) plasmids. Glucose concentration in the cell culture medium was measured 48 h after
 *PVC* EGFP\_UCP1EGFR\_targeting

administration; data shows mean±SD, n=3 independent
experiments.

# Structure-informed design of adipose-targeting PVC Tail Fiber Protein

To enable the PVC-based delivery of UCP1 into white adipose tissue, we then decided to equip the
tail fiber protein of PVC (PVC13) with an adipose tissue-targeting 9-mer peptide developed by
Kolonin et al. in 2004 (Kolonin et al., 2004). To ensure the efficient exposure of the targeting
peptide on the tail fiber, we predicted the structure of PVC13 trimers inserted with adipose tissue
targeting peptide flanked by different linkers **(Figure 4a)**. We were unable to peruse
validating
these constructs due to the limited time before the jamboree and the practical difficulties in
working with such a huge plasmid (~25kb), while we did make some effort in simplifying the cloning
process by designing a new PVC13 part which allows subsequent Golden-gate-based cloning of the
targeting sequence into the pPVC plasmid (**Figure 4b**, see also the second part of our [**Engineering
page**](engineering) for more information).

![fig4](https://static.igem.wiki/teams/4960/wiki/new-12-8/resultsgolden.jpg)

**Figure 4. AlphaFold2-guided Design of Adipose Cell-targeting PVC Coat Expressing Plasmids.
(a)** AlphaFold2 based prediction of engineered PVC tail fiber trimer structure. Structure of
adipose-targeting CKGGRAKDC peptide-presenting PVC tail fiber with indicated linkers were
shown.**(b)**
Schematic diagram of the newly-designed pvc13 part allowing Golden Gate cloning of targeted sequence
into the pPVC plasmid.

# Reference

* Bates, R., Huang, W., & Cao, L. (2020). Adipose Tissue: An Emerging Target for Adeno-associated
  Viral Vectors. Mol Ther Methods Clin Dev, 19, 236-249. https://doi.org/10.1016/j.omtm.2020.09.009
* Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by
  targeted ablation of adipose tissue. Nat Med, 10(6), 625-632. <https://doi.org/10.1038/nm1048>
* Kreitz, J., Friedrich, M. J., Guru, A., Lash, B., Saito, M., Macrae, R. K., & Zhang, F. (2023).
  Programmable protein delivery with a bacterial contractile injection system. Nature, 616(7956),
  357-364. <https://doi.org/10.1038/s41586-023-05870-7>

![top3](https://static.igem.wiki/teams/4960/wiki/design/result/top33.png)

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