iGEM 2020 — Team ZJU-China

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# Team:ZJU-China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:ZJU-China/Engineering

Engineering

![](https://static.igem.org/mediawiki/2020/6/6b/T--ZJU-China--wiki_loading.gif)

* [Overview](#overview)
* [Research](#research)
* [Imagine & Design](#imagine)
* [Build](#build)
* [Testing](#testing)
* [Actions under COVID-19](#action)

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* [Home](https://2020.igem.org/Team%3AZJU-China)
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* Project
  + [Description](https://2020.igem.org/Team%3AZJU-China/Description)
  + [Background](https://2020.igem.org/Team%3AZJU-China/Background)
  + [Design](https://2020.igem.org/Team%3AZJU-China/Design)
  + [Magnetosome](https://2020.igem.org/Team%3AZJU-China/Magnetosome)
  + [Experiments](https://2020.igem.org/Team%3AZJU-China/Experiments)
  + [Results](https://2020.igem.org/Team%3AZJU-China/Results)
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* Model
  + [Model](https://2020.igem.org/Team%3AZJU-China/Model)
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  + [Proof of Concept](https://2020.igem.org/Team%3AZJU-China/Proof_Of_Concept)
* Parts
  + [Contribution](https://2020.igem.org/Team%3AZJU-China/Contribution)
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* Human
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  + [Safety](https://2020.igem.org/Team%3AZJU-China/Safety)
  + [Implementation](https://2020.igem.org/Team%3AZJU-China/Implementation)
  + [Entrepreneurship](https://2020.igem.org/Team%3AZJU-China/Entrepreneurship)

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* Home
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* Project
  + [Description](https://2020.igem.org/Team%3AZJU-China/Description)
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* Model
  + [Model](https://2020.igem.org/Team%3AZJU-China/Model)
  + [Engineering](https://2020.igem.org/Team%3AZJU-China/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AZJU-China/Proof_Of_Concept)
* Parts
  + [Contribution](https://2020.igem.org/Team%3AZJU-China/Contribution)
  + [Improve](https://2020.igem.org/Team%3AZJU-China/Improve)
  + [New Parts](https://2020.igem.org/Team%3AZJU-China/Newparts)
* Human
  + [Human Practices](https://2020.igem.org/Team%3AZJU-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AZJU-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AZJU-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AZJU-China/Partnership)
  + [Safety](https://2020.igem.org/Team%3AZJU-China/Safety)
  + [Implementation](https://2020.igem.org/Team%3AZJU-China/Implementation)
  + [Entrepreneurship](https://2020.igem.org/Team%3AZJU-China/Entrepreneurship)

# Engineering

## Overview

**Our project is typically problem-based. And each step to support engineering success follows the design cycle as iGEM suggests, which starts from researching, and is followed
with imagination, designing, building, testing, learning, and improving, and continues with a re-research again. Thankfully, with the effort made by every member of ZJU-2020, we
constructed two pivotal parts of our project to demonstrate our design was workable by both wet experiments and modeling analysis. And the whole process of our engineering is
as below.**

## Research

As we mentioned in the background of our project, there are obvious disadvantages of present contrast agents for MRI of not only breast cancer but all kinds. We wanted to make
improvements in the specificity of contrast agents, and we understood clearly that we must create a modified contrast agent to meet the standard of specificity. Adding
targeting molecule on gadolinium, which is now widely used for MRI was first proposed but quickly dismissed for the toxicity of gadolinium. And then we started to search if
there is a biocompatible magnetic material for MRI, and we finally found a biological one, which is called magnetosome as our product.

For the proper choice for targeting a molecule, the antibody was aforethought. We also studied other kinds of molecules that targeted HER2 cells such as peptides which tended to
the acidic environment of tumors; nucleic acid aptamers that bind HER2 specifically and so on. For the convenience of design and building a standardized platform in the future, we
finally chose a single-chain variable fragment (scFv) as the targeting molecule for HER2.

## Imagine & Design

By studying the protocols of the transgene on MSR-1, we can obtain the surface-modified magnetosomes by transferring the recombinant plasmid containing the fusion protein of our wanted protein and mamC
(membrane protein covers the most area of the surface of magnetosomes) into magnetotactic bacteria, using biparental conjugation. So at first, we designed to link scFv directly to mamC to functionalized the magnetosomes.

However, no research showed such a design like us. Instead, they preferred to choose a third one as an anchor for the molecule that they wanted to link to mamC in case that the
magnetosomes would be unfunctional for direct linkage. Refer to those researches, we decided on ZZ, the Fc-binding domain of an immunoglobulin-binding protein from the cell
wall of *Staphylococcus aureus* as the anchor and add Fc to scFv for fusion with mamC-ZZ (Figure 1).

For more detailed information about our design, you can visit the [Design](https://2020.igem.org/Team%3AZJU-China/Design) page of our wikis.

![](https://static.igem.org/mediawiki/2020/thumb/5/54/T--ZJU-China--Engineering_fig1.png/800px-T--ZJU-China--Engineering_fig1.png)

###### Figure 1. (A) Map of mamC-ZZ vector; (B) Map of scFv-Fc vector.

## Build

The building process is described as the experiment part of our wikis, we constructed mamC-ZZ and scFv-Fc proteins as we designed according to recent studies. And we submitted
them to the iGEM Registry following the BioBricks assembly standard.

For more detailed information about our parts, we strongly recommend you visit the corresponding parts pages on the iGEM BioBricks Registry.

## Testing

In the wet experiments, we first confirmed the sequences of the two fusion proteins we designed by amplifying them from PCR (Figure 2). And the sequencing results showed no
difference with the maps of the two vectors.

For more detailed information about our experiments, you can visit the [Results](https://2020.igem.org/Team%3AZJU-China/Results) and [Model](https://2020.igem.org/Team%3AZJU-China/Model%20style%3D) part of our wikis.

![](https://static.igem.org/mediawiki/2020/4/40/T--ZJU-China--Engineering_fig2.png)

###### Figure 2. (A) scFv-Fc amplified from pET19b; (B) mamC-ZZ amplified from pGEX-2TK.

Then, we transferred pET19b\_scFv-ZZ and pGEX-2TK\_mamC-ZZ into *E.coli* SHuffle® and BL21 (DE3) to express the fusion proteins respectively and obtained the
purified proteins successfully (Figure 3).

![](https://static.igem.org/mediawiki/2020/a/ac/T--ZJU-China--Engineering_fig3.png)

###### Figure 3. (A) Western blotting results of immunoprecipitation of scFv-Fc; (B)&(C) Western-blot results of GST mamC-ZZ under different inducing IPTG (B) concentration and inducing time (C).

To validate the interaction between mamC-ZZ and scFv-Fc, co-immunoprecipitation was carried out and the results turned out that the two fusion proteins bound together
specifically (Figure 4).

![](https://static.igem.org/mediawiki/2020/9/9e/T--ZJU-China--Engineering_fig4.jpg)

###### Figure 4. Western blot results of mamC-ZZ and scFv which introduce different primary antibody.

In order to prove that scFv-Fc fusion protein can specifically target HER2-positive breast cancer cells, we have demonstrated the specificity and effectiveness of this targeting by flow cytometry (Figure 5).

![](https://static.igem.org/mediawiki/2020/c/ce/T--ZJU-China--Results_fig10_newone.jpg)

###### Figure 5. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.

As MSR-1 was microaerophilic, we added resazurin into the liquid medium to see the growth of cells by the change of the color. The results showed that the color of the liquid medium which was added into MSR-1 was obviously different from that without the addition of MSR-1 (Figure 6). To further prove that the magnetotactic bacteria were truly cultured, bacterial PCR was carried out using the primers designed from the 16srDNA of *Magnetospirillum magneticum* and we used *E.coli* and *Agrobacterium* as control. The results showed that the magnetotactic bacteria were indeed grown (Figure 7).

![](https://static.igem.org/mediawiki/2020/9/95/T--ZJU-China--MSR-1.jpeg)

###### Figure 6. Liquid medium cultured MSR-1 (red test tubes) contrast to the blank control ( blue test tubes).

![](https://static.igem.org/mediawiki/2020/1/19/T--ZJU-China--bacterial_PCR_of_MSR-1.jpeg)

###### Figure 7. Results of the bacterial PCR.

In the modeling analysis, we mainly focused on three aspects of our product, including the yield of scFv-Fc and magnetosomes in their expression chassis, the combination and
disaggregation of these two proteins and the interaction of HER2 and the modified magnetosomes *in vivo*.

In the first part, we constructed two deterministic models to compute the production of scFv-Fc and modified magnetosomes. And the results showed that the expression of these two
proteins can be precisely and highly activated after the addition of IPTG (Figure 8,9).

![](https://static.igem.org/mediawiki/2020/1/1e/T--ZJU-China--wiki_model_fig1b_new.png)

###### Figure 8. Dynamics of target protein. Horizontal axis shows the length of time. Vertical axis demonstrates the amount of protein (scFv-Fc) within the system.

![](https://static.igem.org/mediawiki/2020/5/5d/T--ZJU-China--wiki_model_fig2b_new.png)

###### Figure 9. Dynamics of target protein. Horizontal axis shows the length of time. Vertical axis demonstrates the amount of protein (mamC-ZZ) within the system.

In the second part, we constructed a deterministic model to determine the combination and disaggregation of scFv-Fc and modified magnetosomes *in vitro* and it showed that they bound rapidly in a very short time, ensuring that the feasibility of auto-assembling *in vitro* (Figure 10).

![](https://static.igem.org/mediawiki/2020/e/ef/T--ZJU-China--wiki_model_fig3b_new.png)

###### Figure 10. Combination of scFv-Fc and modified magnetosomes (the blue line refers to the combination product of scFv-Fc and modified magnetosomes, and the orange line refers to pure magnetosomes).

In the last part, we constructed a model about modified magnetosomes *in vivo* to describe the combination and degradation with HER2 and the complex of our modified magnetosomes and scFv-Fc. It turned out that the complex can bind HER2 rapidly while degraded in a relatively long period of time (Figure 11,12), which makes it potential for rapid detection and long-term treatment if we develop it into a drug delivery vector.

![](https://static.igem.org/mediawiki/2020/c/cb/T--ZJU-China--wiki_model_fig6a_new.png)

###### Figure 11. Magnetosome binding in a short time.

![](https://static.igem.org/mediawiki/2020/4/44/T--ZJU-China--wiki_model_fig6b_new.png)

###### Figure 12. Metabolism of magnetosomes in the body for a long time.

For more detailed information about our modeling, you can visit the [Model](https://2020.igem.org/Team%3AZJU-China/Model) page of our wikis.

## Actions under COVID-19

Due to the spread of the COVID-19 virus, the majority of our lab work has been delayed. For example, because MSR-1 was not a common strain like *E.coli*, we need to purchase it from abroad to obtain quality strains. Besides that, the cultural condition had a tough standard which should be taught by experienced technicians. Though we connect a professor studying the culture of magnetotactic bacteria, yet teaching the methods simply through the internet is basically useless for there being so many details culturing this microaerobic bacteria. However, for making some progress in the area of contrast agents, we still tried our best to carry out relative experiments to demonstrate our design and listed the plans for the following experiments. For the detailed content of the future work, we set a wiki page specialized for it.

[aannnaaaaannnnnnnnnnnnn](http://www.zju.edu.cn/)

aannnnnnnnn

![](https://static.igem.org/mediawiki/2020/e/ea/T--ZJU-China--wiki_gifff.gif)

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# Team:ZJU-China/Experiments

Experiments

![](https://static.igem.org/mediawiki/2020/6/6b/T--ZJU-China--wiki_loading.gif)

* [Cloning
  Expression Purification](#Cloning)
* [Optimum Expression of
  Protein](#about)
* [Expression Results
  Verification](#services)
* [Cell Experiment](#showcase)
* [Bacterial PCR of MSR-1](#bacteria_pcr_of_msr-1)
* [References](#references)

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* [Home](https://2020.igem.org/Team%3AZJU-China)
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* Project
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* Parts
  + [Contribution](https://2020.igem.org/Team%3AZJU-China/Contribution)
  + [Improve](https://2020.igem.org/Team%3AZJU-China/Improve)
  + [New Parts](https://2020.igem.org/Team%3AZJU-China/Newparts)
* Human
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  + [Safety](https://2020.igem.org/Team%3AZJU-China/Safety)
  + [Implementation](https://2020.igem.org/Team%3AZJU-China/Implementation)
  + [Entrepreneurship](https://2020.igem.org/Team%3AZJU-China/Entrepreneurship)

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* Home
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* Project
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  + [Background](https://2020.igem.org/Team%3AZJU-China/Background)
  + [Design](https://2020.igem.org/Team%3AZJU-China/Design)
  + [Magnetosome](https://2020.igem.org/Team%3AZJU-China/Magnetosome)
  + [Experiments](https://2020.igem.org/Team%3AZJU-China/Experiments)
  + [Results](https://2020.igem.org/Team%3AZJU-China/Results)
  + [Future](https://2020.igem.org/Team%3AZJU-China/Future)
* Model
  + [Model](https://2020.igem.org/Team%3AZJU-China/Model)
  + [Engineering](https://2020.igem.org/Team%3AZJU-China/Engineering)
  + [Proof of Concept](https://2020.igem.org/Team%3AZJU-China/Proof_Of_Concept)
* Parts
  + [Contribution](https://2020.igem.org/Team%3AZJU-China/Contribution)
  + [Improve](https://2020.igem.org/Team%3AZJU-China/Improve)
  + [New Parts](https://2020.igem.org/Team%3AZJU-China/Newparts)
* Human
  + [Human Practices](https://2020.igem.org/Team%3AZJU-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AZJU-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AZJU-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AZJU-China/Partnership)
  + [Safety](https://2020.igem.org/Team%3AZJU-China/Safety)
  + [Implementation](https://2020.igem.org/Team%3AZJU-China/Implementation)
  + [Entrepreneurship](https://2020.igem.org/Team%3AZJU-China/Entrepreneurship)

# Experiments

## Cloning

For cloning of the fusion of **single-chain variable fragment (scFv)** and **fragment crystallizable
(Fc)**,
the amino acid sequence of anti-HER2 scFv described by Ahmadzadeh, M.[[1]](#references) and the CH2 & CH3 region of immunoglobulin heavy constant
gamma 1,
which is the constant region of immunoglobulin heavy chains [(UniProtKB-P01857)](https://www.uniprot.org/uniprot/P01857),
was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1
was used as the linker between scFv and Fc,
a FLAG tag was fused to the C-terminus as well. Furthermore, we employed codon optimization to
maximize the expression of a functional protein in *Escherichia coli*.
The gene was synthesized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned
into gene expression vector pET19b, introduced a 10× histidine tag at the N-terminus.

![](https://static.igem.org/mediawiki/2020/5/56/T--ZJU-China--wiki_experiment_fig1.png)

###### Figure 1. Vector of scFv-Fc, pET19b.

For cloning of the fusion of mamC [(GeneBank: AVM72836.1)](https://www.ncbi.nlm.nih.gov/protein/AVM72836.1)
and ZZ [(GeneBank:M74186.1 )](https://www.ncbi.nlm.nih.gov/nuccore/M74186.1), the gene was synthesized and
codon optimized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene
expression vector pGEX-2TK,
introduced a GST tag at the N-terminus.

![](https://static.igem.org/mediawiki/2020/8/87/T--ZJU-China--wiki_experiment_fig2.png)

###### Figure 2. Vector of mamC\_ZZ, pGEX-2TK.

## Expression of scFv-Fc

After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pET19b was transformed into
*Escherichia coli* SHuffle®T7 Express Cells. The cells were grown in LB medium
at 37°C until the
OD600 reached 0.6, at which time protein expression was induced by adding filter sterilized
isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then incubated at 30°C for an
additional
time to expression. Afterwards, the cells were harvested by centrifugation at 4000 rpm for 12
min.
The cell pellet was resuspended in lysis buffer (50 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.1% CA630,
10%
glycerol and 5 mM β-mercaptoethanol) and the cells were lysed by sonication. The cell extraction
was
clarified by centrifugation at 10000 rpm for 15 min.

## Expression of mamC-ZZ

After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was
transformed into
BL21 (DE3). The process of growth, induction and harvest was the same as that of scFv-Fc. The
cell pellet team
was resuspended in lysis buffer (50 mM Tris-HCl pH8.0 5 mM EDTA and 1.5 mM NaCl,
phenylmethylsulfonyl fluoride).
Add lysozyme (Solarbio, Beijing, CN) to a final concentration of 500 μg/mL, incubating for 30
min at room
temperature and centrifuging at 12000 rpm for 30 seconds. Discard the supernatant to remove the
periplasm
protein. Add RIPA IV (Sangon, Shanghai, CN) and incubate on ice for 10 min, vibrating the tubes
for several
times during the incubation. The cell extract was clarified by centrifugation at 10000 rpm for
10 min.

## Purification of scFv-Fc

The protein was purified by immunoprecipitation (IP) using anti‐DYKDDDDK G1 affinity
resin (GenScript, Nanjing, CN). Resuspend the resin to form a uniform slurry and transfer
40 μL of the slurry into a 1.5 mL vial. Add 500 μL TBS into the vial and gently mix the
resin, centrifuge at 6000 rcf for 30 seconds, and remove supernatant carefully. Repeat
this step for three times. Add 1 mL supernatant of the cell lysate to the washed resin.
Mix by end‐to‐end rotation on a tube rotator overnight at 4°C. To remove the nonspecific
binding segment, centrifuge at 6000 rcf for 30 seconds, carefully remove supernatant and add
500 μL TBS to wash the resin three times. Remove as much supernatant as possible without
disturbing the resin, add 120 μL of acid elution buffer (0.1 M glycine HCl, pH 3.5) into
the washed resin and use a wide bore pipette tip to gently resuspend the resin. Incubate
at room temperature for 5 minutes, mix gently by tapping the tube once or twice during the
incubation period. After incubation, centrifuge at 6000 rcf for 30 seconds. Carefully transfer
supernatant into a new vial containing 5 μL 1 M Tris, pH 9.0 for further application.

## Optimum Concentration of IPTG for Expressing

The transformed cells were grown until the OD600 reached 0.6, at which time
protein expression was induced by a gradient from 0 to 2 mM of IPTG. The cells
were then incubated at 30°C for an additional 24 h.

## Optimum Induction Time for Expressing

The transformed cells were grown until the OD600 reached 0.6, at which time
protein expression was induced by 2 mM IPTG. The cells were then incubated at
30°C for a gradient from 0 to 24 h. For negative control, none inducer was
introduced.

## Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS–PAGE)

The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer (TIANGEN, Beijing, CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5%
stacking gel, 8% separating gel). Gels were run at 110V for 15vmin and then at 160V
for ~50 min in running buffer.

## Coomassie Staining

Following SDS–PAGE, gels was stained with Coomassie brilliant blue (0.1% Coomassie
brilliant blue R-250, 25% isopropanol, 10% acetic acid) for 2 h at room temperature.
Destain in destaining buffer (5% ethanol, 10% acetic acid) overnight.

## Western Blotting of Proteins

Following SDS–PAGE, samples were transferred to nitrocellulose membrane (Merck) by
semi-dry transfer (Bio-Rad). Membranes were blocked 1 h at room temperature in 5%
(w/v) nonfat milk in TBST, probed with a rabbit anti-FLAG antibody (Abcam) overnight
at 4°C, washed with TBST, probed with a goat anti-rabbit HRP-conjugated antibody
(Sangon) for 1 h at room temperature, washed with TBST, and subsequently imaged using
ChemiDoc (Bio-Rad).

Particularly, the blocked membranes were directly probed with a rabbit
anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary
antibody.

## Co-immunoprecipitation

The supernatant of cell lysate of mamC-ZZ or purified mamC-ZZ was incubated with
purified FLAG-tagged scFv-Fc overnight at 4°C. Afterwards, the mixture was incubated
with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. The FLAG-tagged proteins scFv-Fc
and the interacted mamC-ZZ from the lysate were then immobilized on the resin, whereas
the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex
was eluted with acid elution buffer. The elution was then digested by thrombin to cut off
GST region. The product was then analyzed by SDS–PAGE and western blotting. For negative
control, only purified scFv-Fc was used as input.

## Cell Culture

MDA-MB-453 and MDA-MB-231 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium
(DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5%
CO2 at 37°C. All cells were passaged at 80% confluency.

## Targeted Cell Uptake Evaluation of scFv-Fc Using Flow Cytometry

MDA-MB-453 and MDA-MB-231 cells at 80% confluence were collected by centrifugation after
treatment using 0.5% trypsin-EDTA and resuspended in 50 μL PBS. Then, the cells were washed
thrice with PBS, at 400 g for 5 min. Next, the cells were incubated (4°C in dark) by scFv-Fc (or
goat anti-rabbit IgG H&L (HRP) as isotype control), rabbit anti-DDDDK tag (binds to
FLAG® tag sequence) antibody, goat anti-rabbit IgG H&L (Alexa Fluor® 488)
and Fixable Viability Dye eFluorTM 450 (eBiosciencTM), during each
incubation the cells were washed thrice with PBS, centrifuge at 400 rcf for 5 mins. In the end, the cells were
resuspended in 300 μL PBS, and the Alexa Fluor® 488 and Fixable Viability Dye
eFluoTM 450 fluorescence of the cells was immediately analyzed using a flow cytometer
(CytoFLEX, Beckman Coulter Inc., USA).

## Culture of Magnetotactic Bacteria

Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose
*Magnetospirillum gryphiswaldense*
strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation
Center(Taiwan, China).

The culture medium(BCRC medium no.611) of M. Gryphiswaldense MSR-1 is prepared as follows.

| Magntospirillum medium | |
| --- | --- |
| KH2PO4 | 0.68g |
| Na-thioglycolate | 0.05g |
| L(+)-Tartaric acid | 0.37g |
| Succinic acid | 0.37g |
| Trace elements solution | 5ml |
| Resazurin | 0.5mg |
| Distilled water | 1000ml |
| NaNO3 | 0.12g |
| Na-acetate | 0.05g |
| Vitamin solution | 10ml |
| Fe(III) quinate solution | Hyundai |
| Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N2 gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add. | |

| Trace elements solution | |
| --- | --- |
| Nitrilotriacetic acid | 1.5g |
| MgSO4·7H2O | 3g |
| MnSO4·2H2O | 0.5g |
| NaCl | 1g |
| CaCl2·2H2O | 0.1g |
| ZnSO4·7H2O | 0.18g |
| CuSO4·5H2O | 0.01g |
| Kal(SO4)2·12H2O | 0.02g |
| H33BO3 | 0.05g |
| Na2MoO4·2H2O | 0.01g |
| Distilled water | 1000mL |
| FeSO4·7H2O | 0.1g |
| CoSO4·7H2O | 0.18g |
| NiCl2·6H2O | 0.025g |
| Na2SeO3·5H2O | 0.3mg |
| Na2WO4·2H2O | 0.4mg |
| Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH). | |

| Vitamin solution | |
| --- | --- |
| Biotin | 2mg |
| Folic acid | 2mg |
| Pyridoxine-HCl | 10mg |
| Thiamine-HCl·2H20 | 5mg |
| Riboflavin | 5mg |
| Vitamin B12 | 0.1mg |
| p-Aminobenzoic acid | 5mg |
| Lipoic acid | 5mg |
| Distilled water | 1000mL |
| Nicotinic acid | 5mg |
| D-Ca-pantothenate | 5mg |
| Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N2 gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add. | |

| Ferric quinate solution, 0.01 M | |
| --- | --- |
| FeCl3·6H2O | 0.45g |
| Quinic acid | 0.19g |
| Distilled water | 100mL |
| Ferric quinate solution:Dissolve and autoclave at 121℃ for 15 min. | |

In the incubation period, aerobic environment is conducive to the growth of
*M.gryphiswaldense* strain
MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack
or
AnaeroPack was purchased from Mitsubishi Gas Chemical(Tokyo, Japan).

## Bacterial PCR of MSR-1

Because magnetotactic bacteria grew too slowly to form a visible colony, we tent to demonstrate that it was truly grown in the medium. The liquid medium was centrifuged at 12000 rpm for 10 minutes and the sediment was used for bacterial PCR using specific primers designed from the 16srDNA of *Magnetospirillum gryphiswaldense*, *E.coli* and *Agrobacterium* were used in the bacterial PCR as the negative control.

## References

[1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E. (2020). Anti-HER2
scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility
and Biological Activity. *Molecular biotechnology, 62*(1), 18–30.
https://doi.org/10.1007/s12033-019-00221-2

[aannnaaaaannnnnnnnnnnnn](http://www.zju.edu.cn/)

aannnnnnnnn

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# Team:ZJU-China/Results

Results

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* [Expression of scFv-Fc](#expression_of_scfv_fc)
* [Expression of mamC-ZZ](#expression_of_mamc_zz)
* [Protein Interaction](#protein_interaction)
* [Specifically Target HER2](#specifically_target_her2)
* [Magnetotactic Bacteria](#culture)
* [Future Work](#future_work)
* [References](#references)

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# Results

## Expression of scFv-Fc

If not specified, all expression chassis of scFv is SHuffle®. The supernatant and the pellet of cell lysate was analyzed by SDS–PAGE, corresponding to lane 1 and
lane 2, respectively. According to the Coomassie staining result below, we pointed out that scFv-Fc was presented in the correct weight and expression level was higher in
supernatant rather than pellet, indicating the solubility of this fusion protein.

![](https://static.igem.org/mediawiki/2020/9/9a/T--ZJU-China--Results_fig1.jpg)

###### **Figure 1. Coomassie staining results of scFv-Fc to investigate the solubility.**

Besides, scFv-Fc was also expressed in BL21 (DE3), which meant the absence of two disulfide bonds, leading to the incorrect folding of scFv. Therefore, the protein expressed in
BL21 (DE3) cannot interact with HER2 theoretically.

### Optimum Concentration of IPTG for Expression

We carried out a gradient experiment to investigate the optimum concentration of IPTG for expressing. The transformed cells were grown until OD600 reached 0.6, we introduced
the expression with IPTG, to a final concentration of 0.5, 1, and 2 mM, respectively. For negative control, IPTG was not introduced.

![](https://static.igem.org/mediawiki/2020/5/54/T--ZJU-China--Results_fig2.jpg)

###### **Figure 2. Western blotting results of scFv-Fc under different inducing concentration of IPTG.**

As Figure 2 shows above, the best concentration for expressing was 2 mM of IPTG. There was a small amount of expression without IPTG induction, probably due to promoter
leakage.
The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth.

### Optimum Induction Time for Expression

A gradient experiment was performed to make sure that the target protein was induced and to figure out the best time for expressing. When OD600 reached 0.6, 2 mM of IPTG was
induced. The expression time was 0, 1, 8, and 24 h, respectively. For negative control, none inducer was introduced.

![](https://static.igem.org/mediawiki/2020/b/bf/T--ZJU-China--Results_fig3.jpg)

###### **Figure 3. Western blotting results of scFv-Fc under different induction time.**

As Figure 3 shows above, the longer time for expressing, the higher expression level the cells reached. In the next work, the expression period was carried out under 2 mM of
IPTG
inducing and 24 h of additional incubating.

### Purification of the Proteins

We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section. The target protein appeared at 54kDa.
Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form a polymer, which was immobilized as well. At the same time, there was
protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction.

![](https://static.igem.org/mediawiki/2020/6/68/T--ZJU-China--Results_fig4.jpg)

###### **Figure 4. Western blotting results of immunoprecipitation of scFv-Fc.**

## Expression of mamC-ZZ

### Optimum Concentration of IPTG for Expression

A gradient experiment was conducted to investigate the optimum IPTG concentration for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6, we
induced the expression with IPTG, to a final concentration of 1, 2, and 3mM, respectively. For negative control, IPTG was not introduced.

![](https://static.igem.org/mediawiki/2020/thumb/e/eb/T--ZJU-China--Results_fig5.jpg/800px-T--ZJU-China--Results_fig5.jpg)

###### **Figure 5. Western blot results of GST mamC-ZZ under different inducing IPTG concentration.**

As Figure 5 is shown above, the best concentration for expressing was 2 mM of IPTG.

### Optimum Inducion Time for Expression

A gradient experiment was carried out to investigate the optimum inducing time for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6 and
induced the expression with IPTG. In a single experimental blocks, a group of gradient expressing time, which set from 1 h to 4 h, was tested.

![](https://static.igem.org/mediawiki/2020/thumb/c/cf/T--ZJU-China--Results_fig6.jpg/799px-T--ZJU-China--Results_fig6.jpg)

###### **Figure 6. Western blot results of GST mamC-ZZ under different induction time.**

### Purification of the Proteins

Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the
experiment section. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single, double and triple volume of GST mamC-ZZ solution, was
tested.

![](https://static.igem.org/mediawiki/2020/thumb/0/05/T--ZJU-China--Results_fig7.jpg/800px-T--ZJU-China--Results_fig7.jpg)

###### **Figure 7. Coomassie staining results of GST mamC-ZZ under different purification conditions**

As results are shown above, the efficiency of purification did not demonstrate a significant difference among different glutathione resin volumes which added in. In the
following work, we added equal glutathione resin as GST mamC-ZZ solution to purify the recombinant protein.

## Protein Interaction

### Immunoprecipitation

To prove that mamC-ZZ can combine with scFv-Fc, we conducted the immunoprecipitation experiments.

As for input, GST mamC-ZZ was incubated with purified FLAG scFv-Fc overnight at 4°C (Figure 8B). The left block was incubated with rabbit anti-FLAG (binds to FLAG®
tag sequence) antibody and goat anti-rabbit IgG H&L (HRP), and the right block was only incubated with goat anti-rabbit IgG H&L (HRP).

Due to the similar molecular weight of scFv-Fc and mamC-ZZ, we designed thrombin-digestion before western blotting. Thus, scFv-Fc will appear at about 54kDa, digested mamC-ZZ
will appear at about 26kDa by interacting with the secondary antibody directly. All the samples were digested by thrombin to cut off any potential GST region.

The mixture of GST mamC-ZZ and FLAG scFv-Fc was incubated with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. Figure 8A indicated the FLAG scFv-Fc and the interacted mamC-ZZ
was then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted. The right block was only
incubated with the secondary antibody, improving mamC-ZZ was pulled down by the interaction of scFv-Fc.

![](https://static.igem.org/mediawiki/2020/d/d1/T--ZJU-China--resultsfig8.png)

###### **Figure 8. Western blot results of mamC-ZZ and scFv which introduce different primary antibody.** (A) One block was incubated with rabbit anti-DDDDK tag antibody and goat anti-rabbit IgG H&L (HRP), and another block was only incubated with goat anti-rabbit IgG H&L (HRP), which can interact with mamC-ZZ by Fc region and show the specific position of mamC-ZZ. (B) Western blot results of input control block.

Furthermore, according to lane 1 and lane 2 (Figure 8A), purified proteins showed stronger interaction than unpurified proteins. However, it suggested an inspiring result,
which meant scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly.

## Specifically Target HER2

### Cultivation of Tumor Cells

We cultured two kinds of tumor cells which is MDA-MB-253 represented HER2 positive breast cancer and MDA-MB-231 represented HER2 negative breast cancer.

![](https://static.igem.org/mediawiki/2020/thumb/8/8d/T--ZJU-China--Results_cell_culture1.jpg/800px-T--ZJU-China--Results_cell_culture1.jpg)

###### **Figure 9. Images of MDA-MB-253 and MDA-MB-453 at different time after subculture.** (A) This image of MDA-MB-231 was taken one day after subculturing, the amount of cell is low; (B) This image of MDA-MB-231 was taken four days after subculturing, tumor cell was at good survival conditions and developed a high cell intensity; (C) This image of MDA-MB-453 in medium intensity was taken at six days after subculturing; (D) Ten days after subculturing, MDA-MB-453 showed a great cell intensity, and the morphology of some tumor cells has transformed account for long-time cultivation.

As images are shown above, both MDA-MB-453 and MDA-MB-231 was cultured successfully and prepared to attend further experiments steps.

### Flow Cytometry

MDA-MB-453 and MDA-MB-231 was both incubated with scFv-Fc on ice for 30 minutes, and stained with Fixable Viability Dye eFluor 450 and Alexa Fluor 488 for flow cytometry.

![](https://static.igem.org/mediawiki/2020/thumb/7/73/T--ZJU-China--Result_round_FCM.jpg/800px-T--ZJU-China--Result_round_FCM.jpg)

###### **Figure 10. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.**

As results are shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from the
negative
control group, which indicated that scFv-Fc could specifically bind with the certain target on MDA-MB-453 membrane (Figure 10A).

At the same time, we can also see that MDA-MB-231 has an obvious fluorescence peak after incubated by scFv-Fc (Figure 10B).

Then, the following analysis shows that although scFv-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks were significantly different. Obviously, the
high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted
to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Figure 10C). And the fluorescence of MDA-MB-453 was about 10 times higher than
MDA-MB-231’s, which was corresponding with the difference of HER2 expression level between HER2 positive and negative cells mentioned in existing studies[[1]](#references).

To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results
indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D).

## Culture of Magnetotactic Bacteria

### Microaerobic Culture

As magnetotactic bacteria needed microaerobic conditions to grow, we chose liquid medium rather than solid for the growth of bacteria. In order to show the change of oxygen
content in the culture medium visibly, we added resazurin into the medium. Resazurin is an indicator of dissolved oxygen. It turns blue when there is dissolved oxygen in the
solution and turns red when there is no oxygen. The results showed that the color of the liquid medium which were added into MSR-1 were obviously different from that without
the addition of MSR-1 (Figure 11), indicating that the dissolved oxygen may be consumed out by MSR-1.

![](https://static.igem.org/mediawiki/2020/9/95/T--ZJU-China--MSR-1.jpeg)

###### **Figure 11. Liquid medium cultured MSR-1 (red test tubes) contrast to the blank control (blue test tubes).**

### Bacterial PCR of MSR-1

To further demonstrate that the magnetotactic bacteria was truly grown in the liquid medium, bacterial PCR was carried out using specific primers designed from the 16srDNA of
*M. gryphiswaldense* and used *E.coli* and *Agrobacterium* as the negative control. As the results of bacterial PCR showed, there was a bright band in lane2,
while no bands in lane 3 and lane 4, demonstrating that MSR-1 was successfully grown up (Figure 12).

![](https://static.igem.org/mediawiki/2020/1/19/T--ZJU-China--bacterial_PCR_of_MSR-1.jpeg)

###### **Figure 12. Results of the bacterial PCR.**

## Future Work

Due to the spread of the COVID-19 virus, the majority of our lab work has been delayed. Though we have tried our best to carry out relative experiments to demonstrate our
design, there are still many lab work have not been completed. We do believe that the functionalized magnetosomes can make a great impact in the area of MRI contrast agent, and
we have listed the future plans for our project to obtain the genetic modification magnetosomes ultimately.

Our future plans are divided into two major parts: construction of functionalized magnetosomes and specificity validation.

### Construction of Functionalized Magnetosomes

#### Acquisition of Genetic Modification Magnetotactic Bacteria

To obtain the modified magnetosomes, we need to obtain the MSR-1 strain which is able to encoding mamC-ZZ protein, and this will be realized by the bi-parental conjugation of
the donor (*E.coli*) harboring recombinant plasmid and the recipient, MSR-1. To increase the amount of ZZ protein on the recombinant magnetosomes, we will use a *mamC*
mutant strain as our recipient, which is constructed by allelic gene replacement by a gentamicin resistance gene[[2]](#references). Both the recipient
strain and the recombinant strain will be selected by antibiotic resistant culture, respectively.

#### Bacterial Strains Cultures and the Purification of Magnetosomes

MSR-1 will be cultured in the Na-lactate medium at 30°C for 38–40 h. The contains and concentrates of the medium refers to some recent researchs[[3]](#references). Bacteria cells will be collected by centrifugation with PBS (pH 7.4) and then disrupted by two passes through a French Press. Magnetosomes from the disrupted cells will be collected at the bottom of a tube by neodymium-iron-boron magnets that produced an inhomogeneous magnetic field. After removing the supernatant, the collected magnetosomes should be washed eight to ten times with PBS while agitating via low level ultrasonication. Transmission electronic microscopy (TEM) will be used for the examination of purified magnetosomes. Besides that, the TEM image that shows the morphology of magnetosomes can be a preliminary basis for testing the functionalized magnetosomes and the normal. At last, the purified magnetosomes will be lyophilized using a freeze drier for 20 h and will be stored at -20°C as the previous purification and storage protocol guides[[4]](#references).

#### Self-assembly of ZZ Modified Magnetosomes and scFv

Based on our experiment results that the mamC-ZZ fusion protein and scFv-Fc fusion protein can bind together strongly and specifically *in vitro*, we will mix magnetosomes and the purified scFv-Fc proteins with different molar ratios under the same conditions as the previous experiments and use TEM and Zeta Potential Analyzer to detect the morphology of the magnetosome-scFv complexes and screen out the optimal ratio at which the magnetosomes can bind the most scFv on their surfaces.

#### Increase the Production of Magnetosomes

It is necessary that the yield of magnetosomes should be high enough for commercial applications in the future. At the genetic level, fine tuning such as enhance the expression of an ATPase gene around the putative elongation factor-G gene of the bacterial chromosome of MSR-1 can increase the production of magnetosomes[[5]](#references). In terms of mass cultivation, we have protocols for different scales of fermenters. The major restricting factor of the production of magnetosomes is the concentration of dissolved oxygen in the medium. Through changing the string speed by detecting the concentration of magnetotactic bacteria to regulated the concentration of dissolved oxygen timely and using chemostat culture technique, the highest yield could reach 88.59 mg/day, theoretically[[6]](#references).

#### Cytotoxicity and Immune Responses Assay

Although magnetosomes are biocompatible, there are several reports say that magnetosomes are toxic to mosquitos and plasmodium[[7]](#references), which means that they may also be toxic to human bodies. So, for the safety and thorough consideration, cytotoxicity and immune responses assay will be carried out by MTT assay in mouse cell lines such as BHK-21 cells. Cells will be seeded in 96-well plates and incubated for 24h after the addition of magnetosomes, and then accessed by the MTT colorimetric reaction. Absorbance of color should be measured at 485nm by a plate reader and the percent of cell viability should be calculated according to the following equation: Cell viability (%) = [OD490(Sample)/OD(490control)]\*100. Besides, as a bacterial product, the entry of magnetosomes into human bodies may causes immune responses, so we will carry out MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies to determine the immune responses of *in vivo* experiments. MTT assay for T cell proliferation is similarly operating as it is used for cytotoxicity testing, while ELISA can be simply operated using commercially available kits[[4]](#references).

### Specificity validation

#### *In vitro* and *in vivo* Fluorescence Imaging Assays of Magnetosome-scFv Complexes

For the validation of targeting ability and specificity of our ZZ-modified magnetosomes *in vitro* and *in vivo*, we will use confocal-laser scanning microscope or flow cytometry to analyze the fluorescence. We will use MDA-MB-468 cells and 293 A cells to carry out contrast experiments. All sample cells and control cells will be seeded at a same density into confocal dishes and cultured for a same period for the following imaging experiments. Though we have not obtained the magnetosomes, yet we are carrying out this part using mamC-ZZ and scFv-Fc complexes as a replacement of magnetosomes to validate its specificity. Sadly, we may not finish this experiment by the wiki freeze, this validation has to be written here.

For *in vivo* fluorescence imaging, HER2 tumor-baring nude mice will be obtained by injecting PBS containing about 1×107 tumor cells per 100 c into the right flank of the 6-7-week-old female BALB/c nude mice. *In vivo* fluorescence imaging will be performed 2 weeks after tumor implantation. 1 mg magnetosome-scFv complexes in 200 μL PBS are planned to be intravenously administered into the tumor-bearing nude mice via tail vein. And the fluorescence images of the mice and their main organs will be taken half an hour after the injection.

#### *In vivo* MR Imaging Study

To test the *in vivo* targeting ability of our magnetosome-scFv complexes, MR imaging will be performed on a 3.0T MR imaging system at the first affiliated hospital of Zhejiang University School of Medicine. The MR imaging study will be carried out on the same tumor-bearing nude mice for the *in vivo* fluorescence assay, and will be conducted by T2-weighted MR imaging with the strength field of 3.0T at different time points after the complexes administered via tail vein.

## Summary

All the experiment above are designed with detailed consideration and with the reference to large relative research. And we have all the instruments and proven protocols to carry out our future experiments, especially the *in vivo* experiments. Thankfully, Prof. Feng Chen from the Department of Radiology, the First Affiliated Hospital of Zhejiang University School of Medicine, provide us with the opportunity to use the MR imagine system and the nude mice kindly, so that we can carry out the in vivo experiments safely and scientifically. We do believe that our functionalized magnetosomes can make a great progress on the MRI detection of breast cancer and provide a new technology on specific imaging.

## References

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[6]. Liu Y, Li GR, Guo FF, Jiang W, Li Y, Li LJ. Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density. *Microb Cell Fact.* 2010 Dec 12;9:99.

[7]. Murugan K, Wei J, Alsalhi MS, Nicoletti M, Paulpandi M, Samidoss CM, Dinesh D, Chandramohan B, Paneerselvam C, Subramaniam J, Vadivalagan C, Wei H, Amuthavalli P, Jaganathan A, Devanesan S, Higuchi A, Kumar S, Aziz AT, Nataraj D, Vaseeharan B, Canale A, Benelli G. Magnetic nanoparticles are highly toxic to chloroquine-resistant Plasmodium falciparum, dengue virus (DEN-2), and their mosquito vectors. *Parasitol Res.* 2017 Feb;116(2):495-502.

[8]. Xiang Z, Yang X, Xu J, Lai W, Wang Z, Hu Z, Tian J, Geng L, Fang Q. Tumor detection using magnetosome nanoparticles functionalized with a newly screened EGFR/HER2 targeting peptide. *Biomaterials.* 2017 Jan;115:53-64.

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