iGEM 2020 — Team iBowu-China

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# Team:iBowu-China/Notebook

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## Notebook

####

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# Team:iBowu-China/Engineering

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## Engineering Success

####

### Overview

In our magneto-hyperthermia-based lung cancer therapy, solutions were designed and modified.

• DNA of magnetic protein with a high expression promoter was first designed to be delivered by the liposome to the cancer cells. The magnetic protein can spontaneously form crystals after recombinantly expressed with inka-box (iBox) and PAK4cat.

• Regarding cancer cell targeting, the surface of the liposome was modified with the ligand specific to epidermal growth factor receptor (GE11), which is highly expressed in cancer cell, and PEG for immune escape.

• Moreover, the expressed and iron-loaded magnetic protein was delivered by ADC (Antibody Drug Conjugation), which is usually used as the drug specific delivery.

• Also, the magnetocaloric effect can be control by the area of the localized alternating magnetic field.

Currently, we have successfully expressed the magnetic protein crystal and completed purification. The magnetic properties were also confirmed by magnetic force microscopy. (
[See statistics and further explanation in ‘Results’.](https://2020.igem.org/Team%3AiBowu-China/Proof_Of_Concept#Results-MFM)) Moreover, we uploaded eight parts regarding magnetic protein crystal, GE11, and the lung-tissue-specific promoter.
([See detailed explanation in ‘Parts Overview".](https://2020.igem.org/Team%3AiBowu-China/Parts_Overview#Overview))

### Research:

Magnetic hyperthermia is advantageous in that the heating source can be located directly at the target region. Local hyperthermia only addresses small areas, such as the tumor per se. Therefore, the utilization of magnetic materials right to tumor areas is expected to improve the therapeutic efficiency. Non-small cell lung carcinoma (NSCLC) is a clinically common type of epithelial cancer, yet is not sensitive to chemotherapy or radiotherapy. Because of this, magnetic hyperthermia has been considered a promising tool.

### Hypothesis:

We postulate that an engineering protein could be assembled for magnetic sensing and generate substantial magnetocaloric effect. With these features, this protein is an alternative material for magnetic hyperthermia. Moreover, it should be targeted to the tumor cells for the specific therapy.

### Design:

Our project aims to increase cancer-killing efficacy by inducing magneto-hyperthermia on the magnetic protein crystals (MPCs). The DNA sequence that encodes the magnetic protein is designed to be delivered into the tumor cells through liposomes. DNA is selected as the delivered material instead of mRNA because it is a more stable form of genetic information and can therefore guarantee a successful expression. The designed vector also includes a lung Tissue-Specific Promoter (TSP), SFTBP Promoter, which is a high expression promoter in lung cancer cells (Figure 1). With the SFTBP promoter, the magnetic protein will be highly expressed in lung cancer cells and less-expressed in other healthy cells. Beside ftn-PAK4cat, GFP-PAK4cat is designed to monitor the progress of crystallization.

![](https://static.igem.org/mediawiki/2020/8/89/T--iBowu-China--genetic_circuits.png)

**Figure 1.** Schematic of ftn-PAK4cat and GFP-PAK4cat plasmid. GFP and ferritin are linked with inkabox-PAK4cat respectively. Both are promoted by tumor specific promoters, ensuring targeting accuracy.

### Build:

#### Core Component - Magnetic Protein Crystal

Magnetic protein crystal is an engineered protein crystal that contains more than 10 million ferritin subunits. This protein crystalizes spontaneously after expression in mammalian cells and is able to load iron. Crystallization of protein attributes to the conformational changes of the inka-PAK4 complex. PAK4 is a metazoan-specific kinase within the family of p-21 activated kinases. When expressed with its inhibitor Inka1, the catalytic domain of PAK4 binds with Inka1 and furthers the formation of a rod-like crystal. Its structure is illustrated in Figure 2. We engineered ferritin into the hollow channel in inka-PAK4 crystal by expressing them together. With ferritin, the crystal is capable of mineralizing substantial amounts of iron. By applying an external alternating magnetic field, the iron generates heat and causes death of tumor cells through the intense heat. Furthermore, its characteristic of magneto taxis allows another layer of insurance of targeting since the magnetic field can orient the crystal to the tumor tissues.

![](https://static.igem.org/mediawiki/2020/6/6f/T--iBowu-China--Ferritin-iBox-Pak4cat-structure.png)

**Figure 2.** Structure of Ft-PAK4 Cat protein: showing how ferritin fits into the hollow channel of the crystal.

#### Delivery System — Phospholipid Capsule

Nucleic acids can be easily decomposed in the biological environment. Thus, liposomal capsule is designed to ensure successful delivery of the nanoparticles. Due to its identical chemical composition with the cellular membrane, liposome guarantees biocompatibility and biosafety. Under the dual targeting of the internal lung-specific promoter and the external alternating magnetic field, the liposomes containing the DNA can fuse with the cancer cells' membranes and deliver the cargo through endocytosis. In the cytoplasm, the phospholipid membrane is disintegrated, and DNAs are released to form proteins through transcription and translation, as illustrated below in Figure 3.

![](https://static.igem.org/mediawiki/2020/d/da/T--iBowu-China--pdd5.jpeg)

**Figure 3.** Lipid-mediated transfection in mammalian cells. DNAs are artificially enclosed by the liposome and will be spontaneously released after entering the tumor cell.

### Modification — Improved Targeting and Safety

At present, there are still some problems with liposomes. In our design, the liposome is modified with epidermal growth factor (EGF) GE11 and polyethylene glycol (PEG) to achieve active targeting and to prevent detection and destruction by immune cells, respectively.

#### Targeting

**Active Targeting:**
EGFR ligand:
Epidermal growth factor receptor (EGFR) displays tyrosine kinase activity which is essential for DNA synthesis and mitosis. This transmembrane protein is overexpressed in a variety of cancer cell types including lung cancer.

GE11 is used to target EGFR. It is a polypeptide chain which has an affinity toward EGFR and is able to form a steady conjugation with lysosome (Figure 4). The integration of GE11 ligand and EGFR induces structural change of the receptor and further promotes endocytosis. Polypeptide demonstrate a low immunogenic potential and can be easily synthesized. Therefore, GE11 shows great characteristics of an active targeting.

![](https://static.igem.org/mediawiki/2020/e/e2/T--iBowu-China--Ge11.jpeg)

**Figure 4.** Chemical structure of GE11

**Lung Tissue-Specific Promoter:**
SFTBP promoter is incorporated in the vector to avoid the expression of engineered DNA outside lung tissue. SFTBP gene codes for pulmonary surfactant protein B, which is an important protein for preventing the collapse of alveoli after exhalation. Due to the property of only promoting expression in lung cells, the use of SFTBP promoter ensures the nanoparticles act solely on lung tissues.

#### Safety

**PEG-laytion:**
When the nanoparticles enter the biological environment, it will be rapidly covered by a large number of biomolecules including proteins. This NP-protein interaction provides the nanoparticles bio-identity which can be detected by the macrophages. PEG-laytion prevents uptake by macrophages and elongates circulation time, enabling the generation of enough heat to kill the tumor cells. The chemical structure of polyethylene glycol shown in Figure 5.

![](https://static.igem.org/mediawiki/2020/d/d0/T--iBowu-China--Chemical_structure_of_a_unit_of_polythene_glycol.png)

**Figure 5.** Chemical structure of a unit of polyethylene glycol. Polyethylene glycol is a macromolecule with a number of identical monomers. The figure shows only one unit of it.

In general, our magnetic protein crystal encapsulated in liposome delivery system was modified with targeting ligand GE11 and immune escaping molecule PEG (Figure 6).

![](https://static.igem.org/mediawiki/2020/1/11/T--iBowu-China--EngFig6.jpeg)

**Figure 6.** Schematic of the plasmid containing liposome, modified with PEG and GE11 peptide.

### Test:

Despite the challenges and restrictions of COVID-19, we have completed several stages of our experiments. We have successfully expressed and isolated magnetic protein *in vitro*. But results have indicated that loading iron into the magnetic protein is difficult to achieve *in vivo*. Further research is required to validate this approach.

### Learn:

To solve the problem of iron loading and improve targeting in late practical application, we sought a better delivery systems. We studied relevant research papers and consulted numerous experts in the fields related our project, the main topics of discussion being active targeting and delivery strategies. They have recommended a few alternatives to GE11: HER2 (Human Epidermal Growth Factor Receptor 2), CD19, and CD20. Other delivery methods were also mentioned: ADC (Antibody Drug Conjugation), Soy Protein, and Albumin.

### Improve:

Based on the suggestions mentioned above, we have decided to express magnetic protein crystals *in vitro*, load iron into the expressed crystals, then deliver the crystals to the tumor cells (Figure 7). This approach would address the issue of not being able to load iron *in vivo*.

![](https://static.igem.org/mediawiki/2020/c/c0/T--iBowu-China--EngFig7.jpeg)

**Figure 7.** Schematic of the magnetic protein crystal containing liposome, modified with PEG and GE11 peptide.

In addition, we developed another system of delivering the magnetic protein crystals to the tumor cells--through antibody drug conjugate (ADC). ADC is biopharmaceutical drug composed of humanized monoclonal antibody conjugated with drug particles by chemical linkers. Through ADC conjugation, we are able to link the humanized anti-TROP2 monoclonal antibody to the magnetic protein crystal domain (Figure 8). We use the domain because it is easier to transport than the crystals due to its smaller size. The protein domain is smaller yet still fully functional. Therefore, we have successfully designed an ADC nanoparticle. However, due to time constraints, we will use this as part of our experimental plan for the future.

![](https://static.igem.org/mediawiki/2020/9/98/T--iBowu-China--ADC.png)

**Figure 8.** ADC-based magnetic protein delivery: the magnetic protein is conjugated with a specific antibody, which is capable of targeting tumor-associated antigen, by a chemical linker.

---

---

### References

[1] Margana, R. K. , & Boggaram, V. . (1997). Functional analysis of surfactant protein b (sp-b) promoter. sp1, sp3, ttf-1, and hnf-3alpha transcription factors are necessary for lung cell-specific activation of sp-b gene transcription. Journal of Biological Chemistry, 272(5), 3083-3090.

[2] Li, T. L. , Wang, Z. , You, H. , Ong, Q. , & Cui, B. . (2019). Engineering a genetically encoded magnetic protein crystal. Nano Letters, 19(10).

[3] Baskaran, Y. , Ang, K. C. , Anekal, P. V. , Chan, W. L. , & Robinson, R. C. . (2015). An in cellulo-derived structure of pak4 in complex with its inhibitor inka1. Nature Communications, 6, 8681.

[4] Genta, I. , Chiesa, E. , Colzani, B. , Modena, T. , Conti, B. , & Dorati, R. . (2017). Ge11 peptide as an active targeting agent in antitumor therapy: a minireview. Pharmaceutics, 10(1).

[5] Bardia, A. , Mayer, I. A. , Diamond, J. R. , Moroose, R. L. , Isakoff, S. J. , & Starodub, A. N. , et al. (2017). Efficacy and safety of anti-trop-2 antibody drug conjugate Sacituzumab govitecan (immu-132) in heavily pretreated patients with metastatic triple-negative breast cancer. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology, 35(19), JCO2016708297.

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# Team:iBowu-China/Experiments

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Document

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## Experiment

#### Protocol

iBowu-China has compiled an account of everything that has happened in the lab over the summer. We are excited to show you how busy we have been in iGEM 2020! We believe that sharing our notes in labs will convey the passion and dedication of our team members, as we continuously push our limits to achieve high-quality results.

### 1. Preparation of Liquid LB Solution for E. Coli Competent Cell culture Petri Dishes

#### Preparation of Agarose Gel

| Ingredients | 100ml | 250ml |
| --- | --- | --- |
| NaCl | 1g | 2.5g |
| Tryptone | 1g | 2.5g |
| Yeast | 0.5g | 1.25g |
| Agarose | 1.5g | 3.75g |
| Kana Antibiotic | 100μl | 250μl |

1. 1.Solid LB solution prepared
2. 2.Carefully pour the solution into clean petri dishes in clean benches
3. 3.Allow the solution to dry over 30 minutes
4. 4.Seal the petri dishes with tape- 5.Store in 4 ̊C fridge, upside down to prevent accumulation of moisture

### 2. Gel Electrophoresis

| Ingredients | 1× | 1.5× |
| --- | --- | --- |
| LE Agarose | 0.3g | 0.45g |
| TAE | 30ml | 30ml |
| Yeast | 0.5g | 1.25g |
| M5 Gelred (nucleic acid dye) | 2μl | 2μl |

1. 1.Weigh the LE Agarose with an electronic balance.
2. 2.Pour respective mass of agarose into the conical flask.
3. 3.Pour respective volume of TAW into the conical flask.
4. 4.Heat the solution with microwave for 1 minute.
5. 5.If the agarose is completely dissolved, proceed to step 6. If the agarose is not completely dissolved, repeat step 4.
6. 6.After cooling, add respective volume of nucleic acid dye to the solution.
7. 7.Set up the gel mold.
8. 8.Gently pour the gel inside the mold, making sure the liquid is evenly spread.
9. 9.Insert the comb of the selected specification into the positioning slot of the glue making rack.
10. 10.After the gel has solidified (30-60min), gently pull out the comb, take the gel out and put it in the electrophoresis tank.

**Loading DNA samples**
Use a pipette to slowly add the DNA sample vertically to the loading hole until below the opening.

**Gel**

1. 1.Emerge the gel in the TAE buffer, making sure the liquid covers all the wells.
2. 2.Make sure the wells are placed toward the cathode.
3. 3.Pipette 20% volume of 6x Loading buffer into the samples, blend with vortex.
4. 4.Always put a marker in the leftmost lane.
5. 5.Add sample that is well mixed with loading buffer into the gel, record the order on the lab note.
6. 6.Electrophoresis should last for 30min, 80V, 220microamps.
7. 7.If Marker appears too short, run the gel for an additional 30 minutes with close monitoring.
8. 8.After electrophoresis, obtain an image with the gel inspection machine and smart phone.

### 3. Plasmid Extraction (Omega E.Z.N.A. Plasmid DNA Mini Kid I)

1. 1.Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
2. 2.Centrifuge at 10,000 x g for 1 minute at room temperature
3. 3.Decant or aspirate and discard the culture media
4. 4.Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields
5. 5.Transfer suspension into a new 1.5 mL microcentrifuge tube
6. 6.Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minutes incubation may be necessary.
7. 7.Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate form.
8. 8.Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step
9. 9.Prepare the vacuum manifold according to manufacturer’s instructions
10. 10.Connect the HiBind® DNA Mini Column to the vacuum manifold
11. 11.Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column
12. 12.Turn on the vacuum source to draw the sample through the column
13. 13.Turn off the vacuum
14. 14.Add 500 μL HBC Buffer.
15. 15.Turn on the vacuum source to draw the buffer through the column
16. 16.Turn off the vacuum
17. 17.Add 700 μL DNA Wash Buffer.
18. 18.Turn on the vacuum source to draw the buffer through the column
19. 19.Turn off the vacuum
20. 20.Repeat Steps 17-19 for a second DNA Wash Buffer wash step
21. 21.Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube
22. 22.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
23. 23.Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube
24. 24.Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
25. 25.Let sit at room temperature for 1 minute.
26. 26.Centrifuge at maximum speed for 1 minute.
27. 27.Store DNA at -20°C

### 4. Gel Extraction

1. 1.Carefully excise the DNA fragment of interest using scalpel from gel. Minimize the size of the gel slice by removing extra agarose.
2. 2.Weighing the volume of the gel slice in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1g/mL.
3. 3.Add 1 volume XP2 Binding Buffer. (A gel slice of mass 0.3g will have a volume of 0.3mL)
4. 4.Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
5. 5.Insert a HiBind® DNA Mini Column in a 2mL Collection Tube.
6. 6.Add 700μL DNA/agarose solution from step 5 to the HiBind® DNA Mini Column.
7. 7.Centrifuge at 10,000×g for 1 minute at room temperature.
8. 8.Discard the filtrate and reuse collection tube.
9. 9.Repeat Steps 6-8 until all of the sample had been transferred to the column.
10. 10.Add 300μL XP2 Binding Buffer.
11. 11.Centrifuge at maximum speed (≥13,000xg) for 1 minute at room temperature.
12. 12.Discard the filtrate and reuse collection tube.
13. 13.Add 700μL SPW Buffer.
14. 14.Centrifuge at maximum speed for 1 minute at room temperature.
15. 15.Discard the filtrate and reuse collection tube.
16. 16.Repeat Steps 13-15 for a second SPW Buffer wash step.
17. 17.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
18. 18.Transfer the HiBind® DNA Mini Column to a clean 1.5mL microcentrifuge tube.
19. 19.Add 25μL Elution Buffer or deionized water directly to the center of the column membrane.
20. 20.Set at room temperature for 2 minutes
21. 21.Centrifuge at maximum speed for 1 minute.
22. 22.Store DNA at -20℃.

### 5. Polymerase Chain Reaction

1. 1.Set up a PCR system.
2. 2.Place in PCR machine and set the program as needed.
3. 3.30min before PCR ends, make agarose gel for DNA recycling.
4. 4.Add 6x Loading Buffer into each system.
5. 5.The first lane of the left-hand side should always be the Marker.

**PCR System(50μL)**

| Ingredients | Volume |
| --- | --- |
| 5\*Prime Star Buffer | 10 μl |
| dNTP | 4μl |
| Forward Primer | 1.5μl |
| Reverse Primer | 1.5μl |
| DNA template | 1μl |
| Prime Star Polymerase | 1μl |
| ddH2O | 31μl |

### **PCR System(50μL)**

|  |  |  |
| --- | --- | --- |
| Pre-Denaturing | 98°C, 2 min | 1 cycle |
| Denaturing | 98°C, 15s | 35 cycles |
| Annealing | 55°C, 15s | 35 cycles |
| Extending | 68°C, 6min | 35 cycles |
| Further Extending | 68°C, 5 min | 1 cycle |

### 6.Gibson Assembly

1. 1.Design plasmid and order primers..
2. 2.Generate DNA segments by PCR.
3. 3.Run PCR product on an agarose gel to check for size and yield. If there are significant amounts of undesired product, gel purify DNA segments.
4. 4.Combine segments in Gibson Assembly Reaction.

#### Gibson Assembly System

|  |  |  |
| --- | --- | --- |
| Ingredients | 20μL | 10μL |
| 2xGB mix | 2μL | 1μL |
| Linearized vector | 2μL | 1μL |
| PCR segment | 2μL | 1μL |
| water | 6μL | 3μL |

1. \*2-3 Fragment Assembly
2. 5.Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts.
3. 6.Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
4. 7.Transform the DNA into bacteria and screen for the correct plasmid product by Restriction Digest.

### 7.Transformation of Plasmid DNA into Competent E. coli

1. 1.Take 200μl of competent cell suspension from the -70℃ refrigerator and thaw it on ice for 1-2 min.
2. 2.Add plasmid DNA solution (content not more than 50ng, volume not more than 10μl), shake gently, and place on ice for 30 minutes.
3. 3.Heat shock in a 42℃ water bath for 90 seconds, and then quickly put it on ice for 2 minutes, without shaking the bacterial solution during the whole process.
4. 4.Add 200μl LB liquid medium (without antibiotics) to the tube, mix well and culture with shaking at 37°C (225 rpm) for 1 hour to restore the normal growth of bacteria and express the antibiotic resistance gene encoded by the plasmid.
5. 5.Shake the above bacterial liquid and spread it on an antibiotic-containing LB agar plate, and place it face up for half an hour. After the bacterial liquid is completely absorbed by the medium, invert the culture at 37°C for 12-16 hours.

### 8.HEK293T Cell Culture and Transfection

1. HEK293T cells are maintained in high-glucose DEME (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific) with 1% P/S (Penicillin-Streptomycin, Thermo Fisher Scientific) and 10% FBS (fetal bovine serum, Thermo Fisher Scientific). Before seeding, the cells are cultured on flask. Then, cells are plated at 50,000 cells per well in a 6-well plate. After overnight incubation, cells are transfected by using Lipofectamine 2000 (Thermo Fisher Scientific).

### 9.Protein Crystals Purification

1. Pipetted out medium from wells, and rinsed once with sterile water. Added lysis buffer(1% NP-40 in 0.1M HEPES at pH 7.4)directly to the cell culture well and scraped cells off the surfaces. Pipetted the resulting cells into labeled eppendorf tubes and placed on a rocker (KS250 basic, Ika labortechnik Staufen)at room temperature for 40 minutes. After rocking, centrifuged at 2000 rpm for 1 minute on a benchtop centrifuge(Micro Star 12, VWR). Discarded supernatant and resuspended pellet in 0.1M HEPES buffer. Check crystal yield by pipetting a few microliters of suspension onto a coverslip and observing under the microscope.

### 10.Iron Loading

1. For iron loading, added fresh prepared FeSO4 solution (50 mM, 100X) to a 100 μL aliquot of the crystal suspension, incubated at room temperature for 2 hours. The final suspension changed color to pale yellow. To verify iron loading, freshly prepared the Prussian Blue (PB) stain (Sigma-Aldrich), pipetted a 10 µL droplet of the iron-loaded crystals onto a glass slide or coverslip and added 1 µL of the PB stain. The droplet can be immediately checked by brightfield optical microscopy.

### 11.MFM

1. Magnetic force microscopy (MFM) measurements were performed by a Bruker Dimension Icon (Santa Barbara, CA, USA). Before the MFM experiment, normal atomic force microscopy (AFM) experiment in tapping mode was carried out on the samples to reveal the accurate surface topography. The probe used for imaging the surface topography was a non-magnetic probe (Olympus). After the topographic measurement, lifted the cantilever up at a certain distance above the surface of the sample in the second pass to measure the magnetic force between the probe and the sample. Magnetic measurement was conducted using a magnetic MESP probe (Bruker) with the tip radius of ~ 35 nm, the normal spring constant of ~ 2.8 N/m, and the resonance frequency of ~ 75 kHz. Magnetic data was recorded by changes in the amplitude and phase of the probe oscillation.

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# Team:iBowu-China/Results

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Document

### Results

####

We have engineered Ft-PAK4, a genetical protein crystal containing ferritin that grows inside mammalian cells.
We have isolated and characterized isolated Ft-PAK4 crystals and Wt-PAK4 crystals.
We have detected magnetic forces when PAK4 crystals loaded with iron in vitro. These protein crystals are attracted to external magnetic field and move toward magnets.
We have presented regimens in order to realize the magnetic control of genetic targeting in vivo in the future therapy.

### 1.Ft-PAK4 protein crystals grow inside mammalian cells

####

Previous study has identified Inka1 as a potent inhibitor of PAK4, which contains two copies of the kinase inhibitory domain and these small regions of themselves can support PAK4cat crystal formation in cells [18]. When inkabox complexes with PAK4cat, conformational changes cause the complex to spontaneously crystallize, producing long rod-shaped crystals [1].
We generated the iron storing construct driven by promoter CMV, by fusing a ferritin to the N-terminal inkabox portion of the inkabox-PAK4cat plasmid, which we call Ft-PAK4. HEK293 cells, human embryonic kidney cells 293, a specific cell line originally derived from human embryonic kidney cells grown in tissue culture, are commonly used in cell biology research, because of their reliable growth and propensity for transfection. HEK293T cells derived from the HEK293 cell line expressing a mutant version of the SV40 large T antigen. Due to they can continue to express the SV40 antigen, the cells are often used in transfection experiments with high transfection efficiency.
According to Baskaran et al., HEK293T cells transfected with the control inka-PAK4 plasmid grow with a single needle-like crystal. In our experiment, we can observe that Ft-PAK4 protein expression usually begins in 24 hours after transfection, but this only happens if a small amount of the control inka-PAK4 plasmid co-transfected, and their growth lasts around 48 to 72 hours.
To make the crystals more easily visible, we also structured the Wt-PAK4 construct, which replace ferritin with green fluorescence protein (GFP). With the same results, the first crystals appeared within 24 hours after transfection and continued to grow up to 72 hours after transfection. We observed that intracellular crystals were morphologically different, that is, the Ft-PAK4 crystals grew slower and shorter but thicker than Wt-PAK4 crystals.

![](https://static.igem.org/mediawiki/2020/1/11/T--iBowu-China--result1.png)

Figure 1 a) Wt-PAK4 and b) Ft-PAK4 expression results in intracellular crystals

### 2.Characterize isolated Ft-PAK4 crystals

####

We harvested all crystals in 72 hours after transfection. To characterize isolated crystals, we used a lysis buffer which can break the cell membrane and crystals were subsequently released. And then, the crystals were separated by slow centrifugation, allowing most of the crystals to precipitate. After the supernatant discarded, the crystal-containing pellet was resuspended in buffer containing 0.1 M HEPES.
We checked crystal yield by pipetting a few microliters of suspension onto a coverslip and observing under the microscope. Qualitatively, the Ft-PAK4 crystals tended to be a longer and thicker structure than the Wt-PAK4 crystals. It seemed a little bit different from the situation when expressed in cells. It may be caused by the ferritin-in-core structure. These results imply that ferritins enclosed by PAK4 improve the stubbornness of the crystals, while the Wt-PAK4 crystals are much more fragile when being scraped, centrifuged and suspended.

### 3.Isolated Ft-PAK4 crystals load iron in vitro

####

![](https://static.igem.org/mediawiki/2020/a/a6/T--iBowu-China--result2.png)

Figure 2 Crystals isolated from cells. a) Wt-PAK4 crystals, b) Ft-PAK4 crystals.

To test whether the ferritin subunit in the isolated PAK4 crystals could store iron, we carried out the following experiment. Ferritins can catalyze the oxidation of Fe2+ to Fe3+ [28]. Based on it, we added fresh FeSO4 solution to isolated crystals to wash out free Fe2+, and the final suspension changed color to pale yellow. To verify iron loading, the crystals were assayed with Prussian blue (PB) staining for the presence of mineralized Fe3+. The highly colored PB can proved the presence of iron.
As shown in figure 3.5, the Ft-PAK4 crystals turned a deep blue color in the center while the Wt-PAK4 crystals without ferritin appeared slightly blue on the edges. This result confirms that ferritins are necessary for the Ft-PAK4 crystals to load iron.

![](https://static.igem.org/mediawiki/2020/8/8c/T--iBowu-China--result3.png)

Figure 3 Prussian Blue (PB) staining rusults: a) Wt-PAK4 crystal, b) Ft-PAK4 crystals.

### 4.Ft-PAK4 crystals mineralized iron generate magnetic forces

####

Magnetic force microscopy (MFM) measurements were performed by a Bruker Dimension Icon (Santa Barbara, CA, USA). Before the MFM experiment, a normal atomic force microscopy (AFM) experiment in tapping mode was carried out on the samples to reveal the accurate surface topography. The probe used for imaging the surface topography was a non-magnetic probe (Olympus). After topographic measurement, lifted the cantilever up at a certain distance above the surface of the sample in the second pass to measure the magnetic force between the probe and the sample. Magnetic measurement was conducted using a magnetic MESP probe (Bruker) with the tip radius of ~ 35 nm, the normal spring constant of ~ 2.8 N/m, and the resonance frequency of ~ 75 kHz. Magnetic data was recorded by changes in the amplitude and phase of the probe oscillation.
The Ft-PAK4 crystals, after loading irons, indeed has magnetic response signals according to the results shown in Figure 3.5. The magnetic response signal strength increases as the tip-to-sample distance decreases. The results show that these protein crystals can be attracted by the applied magnetic field and move towards the magnet.

![](https://static.igem.org/mediawiki/2020/6/6e/T--iBowu-China--result4.png)

Figure 4 Magnetic response of Ft-Fe crystal. (A) and (B) The typical magnetic response of two crystals under different lift height. (C) The magnetic response of standard sample under different lift height. (D) The frequency shift as the function of lift height of the two crystals, the inset shows the frequency shift as the function of lift height of the standard sample. The scale bars in A, B, C are 2 µm.

### 5.Conclusion and perspective

####

We have shown that the Ft-PAK4 protein could assemble for magnetic sensing and generate substantial magnetic forces. This protein is genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. We postulate that this well-designed Magnetic Protein Crystal (MPC) could be modified into a more targeted tool. By applying these internal proteins into magnetic hyperthermia therapy, the heating doses can be deposited using minimal control and be selectively localized within the tumor area.
Based on the current research progress, the Ft-PAK4 system cannot realize the magnetic control of genetic targeting in vivo. The following work is to solve iron-load problem through other targeted carriers, and regulate the expression of Ft-PAK4 by optimizing the specific expression promoter.

#### Targeting problem

To solve the problem of targeting in application, we present two therapy regimens: through a modified liposome delivery system or conjugated with antibodies. Amphiphilic lipid molecules could be self-assembled into liposomes and act as drug carriers. Through encapsulating MPC in liposomes, magnetic liposomes offer multiple functions for biomedical applications. On the other hand, antibody-drug conjugates (ADCs) allow an antibody to be conjugated to MPC via a linker which is delivered to its unique target. For example, many studies have shown that Trop2 may be a more effective target for NSCLC.

#### Specific expression problem

We choose a tissue-specific expression of promoter to replace original promoter CMV.

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