iGEM 2023 — Team westlake-china

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{% block title %}Notebook{% endblock %}
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Notebook

Click!Read More

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* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)
* [October](#October)

---

# June

---

**6/23 BL21 Activation and Material Preparation**

Participant(s): Gehua WENG

BL21 activation was carried out by incubating in the shaker at 37 ℃ and 220 rpm for 1 day.
LB medium was packed into two 100 ml flasks, one 60 ml flask and one 150 ml flask which were prepared for
BL21 and BL21 was transferred with pGLO cultured with 10 mM and 1 mM FeCl3.
And we prepared 0.5 M 10 ml and saved at 4℃.
As pGLO carried with GFP which can help us easily observe if the bacteria is transferred and could express
the vector successfully. Additionally, in our preliminary stage, we planned to use pGLO to carry our
targeted protein.
We planned to add 0.1% arabinose to our LB medium for GFP carried by pGLO need arabinose to express GFP.
As the following figure:

![pGLO plasmid design](https://static.igem.wiki/teams/4702/wiki/description/notebook1.png)

Sited from BIO-RAD:
[pGLO Plasmid Map and Resources | Bio-Rad](https://www.bio-rad.com/zh-cn/applications-technologies/pglo-plasmid-map-resources?ID=NISQOC15)

So 50 ml 20% arabinose was prepared and saved at 4℃.

Recorded by Gehua WENG

**6/24 Ferric Ion Induced Nanoparticle Synthesis**

Participant(s): Gehua WENG, Yuqi SHEN

Our first experimental BL21 are mentioned in the table below:

|  | Bacteria | BL21 | | BL21+pGLO | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Bottle | 60ml LB | 150ml LB | 100ml LB | 100ml LB |
|  | FeCl3 | 1.2ml | 300μl | 2ml | 200μl |
|  | Arabinose | 300μl | 750μl | 500μl | 500μl |
|  | Ampicillin | - | - | + | + |

They were put into the shaker at 37℃ and were centrifuged at 200 rpm for 3 days.

Recorded by Gehua WENG

**6/27 Nanoparticle Expression Induced Strain Collection**

Participant(s): Gehua WENG

The bacteria from 6/24 were taken out and centrifuged at 3000 rpm 4℃ for 15 min. Besides, the supernatant
and the precipitate were stored at 4℃ and -80℃ respectively.

Recorded by Gehua WENG

# July

---

**7/3 Medium Preparation**

Participant(s): Shuoyi HU, Gehua WENG:

We set the gradient table and stepped in to prepare medium required.

**Medium**
**LB**

|  | Components | Content |
| --- | --- | --- |
|  | Tryptone | 10.0 g |
|  | Yeast Extract | 5.0 g |
|  | NaCl | 10.0 g |
|  | Deionized water | 1 L |

**LB low-salt**

|  | Components | Content |
| --- | --- | --- |
|  | Tryptone | 10.0 g |
|  | Yeast Extract | 5.0 g |
|  | NaCl | 5.0 g |
|  | Deionized water | 1 L |

**MH**
        Refer the [commercial protocols](https://meilunhelper.com/uploads/guide/MMT031.pdf) .
**TSB (standard one)**

|  | Components | Content |
| --- | --- | --- |
|  | Distilled water | 1.0 L |
|  | Tryptone (Pancreatic Digest of Casein) | 17.0 g |
|  | Soytone (Peptic Digest of Soybean) | 3.0 g |
|  | Glucose (= Dextrose) | 2.50 g |
|  | Sodium Chloride | 5.0 g |
|  | Dipotassium Phosphate | 2.5 g |

The final pH is about 7.3 ± 0.2 (you can measure the pH for amendment)

\*Adjusted and/or supplemented as required to meet performance criteria.

**9K Medium**

Solution 1:

|  | Components | Content |
| --- | --- | --- |
|  | Distilled water | 500 mL |
|  | Tryptone (Pancreatic Digest of Casein) | 17.0 g |
|  | (NH4)2SO4 | 3.0 g |
|  | KCl | 0.1 g |
|  | K2HPO4 | 0.5 g |
|  | MgSO4·7H2O | 0.5 g |
|  | Ca(NO3)2 | 0.01 g |

Solution 2:

|  | Components | Content |
| --- | --- | --- |
|  | Distilled water | 250 mL |
|  | FeSO4·7H2O | 44.22 g |
|  | H2SO4 | 1 mL |

Recorded by Gehua WENG

**7/12 MH medium Preparation**

Participant(s): Yixiao XIAO:

1 L MH medium were prepared for the second batch of bacteria.

Recorded by Gehua WENG

**7/15 Dialysis Bag Activation

        Ferric Ion Induced Culture Homogenization**

**Dialysis Bag Activation**

Participant(s): Shuoyi HU, Gehua WENG:

A reel of dialysis bag
[(Cat. No. FDM403-5m, Beyotime)](https://www.beyotime.com/product/FDM403-5m.htm) was activated by strictly
following the instruction manual provided by Beyotime and storing in 75% ethanol in 4ºC.

[Instruction manual was provided by Beyotime](https://www.beyotime.com/Manual/FDM403%20%E5%86%8D%E7%94%9F%E7%BA%A4%E7%BB%B4%E7%B4%A0%E9%80%8F%E6%9E%90%E8%A2%8B%2844mm%2C%203.5kDa%2C%20%E5%B9%B2%E5%9E%8B%29.pdf)

Recorded by Shuoyi HU

**Ferric Ion Induced Culture Homogenization**

Participant(s): Shuoyi HU, Gehua WENG:

Culture from 6/24 stored in -80ºC was taken out and homogenized by high
pressure homogenizer and stored at 4ºC for further TEM sample preparation in 7/16.

Recorded by Shuoyi HU

**7/16 TEM Sample Preparation**

Participant(s): Shuoyi HU

Culture from 6/24 whose bacteria pellet was homogenized in 7/15 was taken out for TEM sample
preparation.

In total, six samples were prepared as shown below:

1. Supernatant of 10 mM Fe3+ induced culture from 6/24.

2. Supernatant of 1 mM Fe3+ induced culture from 6/24.

3. Supernatant of 10 mM Fe3+ induced culture from 6/24, the **aqueous** phase from
CHCl3 extraction.

4. Supernatant of 10 mM Fe3+ induced culture from 6/24, the **organic** phase from
CHCl3 extraction.

5. From homogenized bacteria pellet of 10 mM Fe3+ induced culture
from 6/24, further treated by centrifugation at 11000 rcf for 30 minutes,
the **supernatant** was taken as sample 5.

6. From homogenized bacteria pellet of 10 mM Fe3+ induced culture
from 6/24, further treated by centrifugation at 11000 rcf for 30 minutes,
the **resuspended broken cell pellet** was taken as sample 6.

Recorded by Shuoyi HU

**7/17 Compentent Cell Preparation and pGLO Transformation**

Participant(s): Shuoyi HU, Shijie HE, Yuqi SHEN

Ten tubes of [SHuffle® Strain *E. coli*](https://www.neb.com/products/c3026-shuffle-t7-competent-e-coli#Product%20Information) (Strain the same as the one in the Link, while we got the strain
without
competence from Liang TAO's Lab at Westlake University) were induced to compentent state and
pGLO was transformed into SHuffle® strain *E. coli*. (Named as SHuffle-pGLO below)

![Clones observed after pGLO transformation](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-7-17-1.png)

Recorded by Shuoyi HU

**7/18 Monoclonal SHuffle-pGLO Preparation**

Participant: Shuoyi HU

Five single colonies of SHuffle-pGLO were picked up and incubated in 5 mL LB broth
(0.1 g/L ampicillin) respectively for 8 hours and sent for sequencing. (Sequence
properly aligned in the report later)

Recorded by Shuoyi HU

**7/19 Ferric Ion Induced Nanoparticles Biosynthesis

        TEM Analysis**

**Ferric Ion Induced Nanoparticles Biosynthesis**

Participant: Shuoyi HU

Three different ferric ion concentrations were
used to induce iron oxide nanoparticles synthesis.

|  | Concentration of FeCl3 | | |
| --- | --- | --- | --- |
|  | 5 mM | 10 mM | 15 mM |
| OD600 value when  adding FeCl3 | 0.133 | 0.168 | 0.097 |

Culture medium: [Mueller-Hinton Broth](https://meilunhelper.com/uploads/guide/MMT031.pdf),
0.2% Arabinose for protein expression induction,
0.1 g/L Ampicillin.
 Time of adding FeCl3: 00:00.

Condition of shaker: 37ºC, 220 rpm, change to 18ºC at 18:46.

Culture Volume: 100 mL per group of treatment.

Additionally, 0.25 M Fe2(SO4)3 was prepared and filtrated through
0.22 μm filtration membrane and distributed into 5 mL
sterilized tubes and stored at 4ºC.

Recorded by Shuoyi HU

**TEM analysis**

Participant(s): Gehua WENG

Six samples from 7/16 were sent for TEM analysis, but four samples got bacteria
contamination, the only one without contamination showed attachment with
biological structures.

Characteristic figures were shown below:

![TEM results of the sample without bacteria contamination](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/1-1.jpg)

**Figure 1.** Supernatant of 10 mM Fe3+ induced culture from 6/24

![TEM results of the sample without bacteria contamination](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/5-1.jpg)

**Figure 2.**Supernatant gained after broken cell centrifugation of 10 mM Fe3+
induced
culture from 6/24.

![TEM results of the sample with bacteria contamination](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/4-1.jpg)

**Figure 3.**Example figure of bacteria contamination.

Results here informed us that we should not keep the sample prepared for days before TEM analysis.

And we also noticed that few amount of deeper dots could be observed in isolated manner but most
were
attached to biological structures.
This informed us that we should focus on the cell pellets more
instead of supernatant in later nanoparticles separation work.

Recorded by Shuoyi HU

**7/20 Nanoparticle Separation

        Ferric Ion Induced Nanoparticle Synthesis**

**Nanoparticle Separation**

Participant(s): Shuoyi HU, Mingchun XU, Yuchen JIANG

The samples from 7/19 were collected at 18:00. Centrifugation was done
at 4000 rcf for 20 minutes, under UV light, differences between protein
expression level (Green Fluorescence Protein on pGLO) were observed as
shown below. (0 mM FeCl3 negative controls were collected other days before
and stored at 4ºC, just as a reference)

![GFP expression level under different concentration of ferric ion treatment](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-7-20-1.png)

From the result here, we decided to use 5 mM FeCl3 as the induction level for later
nanoparticle synthesis, as the external protein expression under 10 mM
and 15 mM FeCl3 were clearly repressed, this possibly indicated the
bacteria in stressed condition. And as the optimal vision of us for nanoparticles
synthesis is to have nanoparticles and antibody synthesized in the same system
and have them self-assemble, so we didn't want the iron ion concentration we
used to suppress protein expression. Out of this concern, the following nanoparticles
synthesis were done using 5 mM Fe3+ induction.

Additionally, 50 mL of the supernatant of 5 mM FeCl3 induced culture after centrifugation
by 4000 rcf was evenly distributed into two tubes and centrifuged by 12000 rcf for 20 minutes.
Then the supernatant was discarded and resuspended with 5 mL protein purification buffer and 5 mL 7.5 M
guanidine hydrochloride. After that it was kept on ice for 15 minutes. Samples are ultrafiltrated by using
100 kDa
ultrafiltration tubes and collected the flow through for TEM analysis.

In all, six samples were prepared for TEM analysis, detailed treatment methods are shown below:

1. 5 mM FeCl3 induced culture (SHuffle-pGLO) from 7/19, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **buffer
A** and centrifuged again by 18200g and **supernatant** was taken as sample 1.

2. 5 mM FeCl3 induced culture (SHuffle-pGLO) from 7/19, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **7.5 M
guanidine hydrochloride** and centrifuged again by 18200g and **supernatant** was taken as sample
2.

3. 5 mM FeCl3 induced culture (SHuffle-pGLO) from 7/19, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **Buffer
A** and centrifuged again by 18200g and **broken pellet** was resuspended by **guanidine
hydrochloride** as sample 3.

4. 5 mM FeCl3 induced culture (SHuffle-pGLO) from 7/19, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **Buffer
A** and centrifuged again by 18200g and **broken pellet** was resuspended by **buffer A** as
sample 4.

5. 10 mM FeCl3 induced culture (BL21) from 6/24, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **Buffer
A** and centrifuged again by 18200g and **broken pellet** was resuspended by **buffer A** as
sample 5.

6. 10 mM FeCl3 induced culture (BL21) from 6/24, homogenized by high pressure
homogenizer, centrifuged at 11000 rcf for 30 minutes, the **pellet** was resuspended by **Buffer
A** and centrifuged again by 18200g and **broken pellet** was resuspended by **guanidine
hydrochloride** as sample 6.
Samples prepared for TEM analysis were stored at 4ºC and sent for TEM analysis on 7/21.

Recorded by Shuoyi HU

**Ferric Ion Induced Nanoparticle Synthesis**

Participant(s): Shuoyi HU, Mingchun XU

To further confirm the proper ferric ion concentration for nanoparticle synthesis,
we use Fe2(SO4)3 with different concentrations to develop culture
medium.

|  | Concentration of Fe3+ (based on Fe2(SO4)3) | | |
| --- | --- | --- | --- |
|  | 0.1 mM | 1 mM | 5 mM | 10 mM |
| OD600 value when  adding Fe2(SO4)3 | 0.095 | 0.099 | 0.0099 | 0.107 |

Culture medium: [Mueller-Hinton Broth](https://meilunhelper.com/uploads/guide/MMT031.pdf),
0.2% Arabinose for protein expression induction,
0.1 g/L Ampicillin.
 Time of adding Fe2(SO4)3: 22:00.

Condition of shaker: 37ºC, 220 rpm, change to 18ºC at 18:46.

Culture Volume: 100 mL per group of treatment.

Recorded by Shuoyi HU

**7/21 TEM Analysis

        TEM Sample Preparation**

**TEM Analysis**

Participant(s): Yuqi SHEN, Yuchen JIANG

Six samples prepared on 7/20 were sent for TEM analysis.

Recorded by Shuoyi HU

**TEM Sample Preparation**

Participant(s): Shuoyi HU

Samples are resuspended by buffer A with 5 mM, 10mM, 15mM FeCl3
induced cell lysate were prepared as samples for TEM analysis in 7/26.

**7/22 Ferric Ion Induced Nanoparticle Separation**

Participant(s): Shuoyi HU

The culture from 7/20 was collected at 12:35 and bacteria pellets were stored at -80°C.

Recorded by Shuoyi HU

**7/26 TEM analysis**

Participant(s): Shuoyi HU

TEM samples from 7/21 were sent for TEM analysis.
A large number of crystal structures were observed in the view field,
which we guessed is caused by the salt in buffer A.

This way, we decided to use deionized water instead of buffer A for TEM sample preparation.

Example figures of salt interference were shown as below:

![TEM results of salt interference](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0001-20230726-1612-13500-x-ceta.jpg)

Recorded by Shuoyi HU

**7/27 Ferric Ion Induced Nanoparticle Synthesis

        TEM Sample Preparation**

Participant(s): Shuoyi HU, Songnan REN

5 mM Ferric Ion was used to induce IONPs synthesis.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Flask I | Flask II | Flask III |
| OD600 value when  adding FeCl3 | 0.093 | 0.105 | 0.110 |
| Time of adding FeCl3 | 13:25 | 14:10 | 15:28 |

Culture medium: LB Broth

Condition of the shaker: 28.5 ºC, 220 rpm.

Culture Volume: 100 mL per group of treatment.

Time of collecting samples: 7/30 13:43.

Recorded by Shuoyi HU

**7/28 TEM Analysis**

**Cell recovery of SK-BR-3 cells**

Participant: Shuoyi HU, Songnan REN

The samples from 7/27 induced by 5 mM FeCl3 were sent for TEM analysis.

Results were shown as below:

![TEM results of nanoparticle synthesis induced by 5 mM FeCl3](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-0728-1.jpg)

![TEM results of nanoparticle synthesis induced by 5 mM FeCl3](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-0728-2.jpg)

From the Results here we have the following observations:

1. The nanoparticles are not strictly spherical.

2. The nanoparticles are not strictly uniform in size

3. The nanoparticles are still attached to some biological structure.

And from this observation, we decided to use unltrasonic treatment to release
the nanoparticles from the biological structure.

Recorded by Shuoyi HU

Participant(s): Yixiao XIAO, Shijie HE, Shuoyi HU

Cells are recovered in a 15 ml tube with 8ml prewarmed [McCoy's 5a Medium Modified](https://www.thermofisher.cn/order/catalog/product/16600082) with [Fetal Bovine Serum](https://www.thermofisher.cn/order/catalog/product/10099141C) added to
a
final concentration
of 10% and 1% of Penicillin and streptomycin and centrifuged at 1200 rpm for 3 mins.
Medium were removed and cell pellet was resuspended with 4 ml medium and then cultured
in 2 T-75 plates with 22.5 ml medium each.
[SK-BR-3 [SKBR3] - HTB-30
|
ATCC](https://www.atcc.org/products/htb-30#detailed-product-information)

Cell conditions are as below:

![sk-br-3-day1-7-29](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day1-7-29.jpg)

Recorded by Yixiao XIAO

**7/29 Ferric Ion Induced Nanoparticle Synthesis**

**Cell Culturing Medium Exchange**

Participant: Shuoyi HU, Songyuan XUE, Songnan REN

Three flasks of 100 mL culture of ferric ion induced pGLO-SHuffle were prepared for
nanoparticle synthesis, the process was induced by adding 5 mM FeCl3.

Culture medium: [Mueller-Hinton Broth](https://meilunhelper.com/uploads/guide/MMT031.pdf),
0.2% Arabinose for protein expression induction,
0.1 g/L Ampicillin.

Condition of shaker: 220 rpm, 37°C

Participant(s): Yixiao XIAO

The medium of the cell culture were changed with 22.5 ml of medium in each plate. The cells
grew slowly and seemed to be infected by bacteria. A well of culture medium was
cultured in a 24-well plate and the same environment with previous culture but
no cells inside to be found as the cause of infection.

Recorded by Yixiao XIAO

**7/30 Culture Collection

Cell Condition Record**

**Culture Collection**

Participant(s): Shuoyi HU, Songyuan XUE, Gehua WENG

Culture induced from 7/27 was collected at 12:00 and stored at -20ºC.

**Cell Condition Record**

Participant(s): Yixiao XIAO

Cells got infected and was thrown out but the culture medium blank culture was still transparent
which means it didn't get infected.

Cell condition is as shown below:

![sk-br-3-day2-7-30](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day2-10x-7-30.jpg)

Recorded by Yixiao XIAO

**7/31 Culture Collection and Ultrasonication**

Participant(s): Shuoyi HU, Gehua WENG

Cultures from 7/29 were collected and lysed along with the cultures
collected from 7/30 by ultrasonication. The cell lysate were centrifuged
by 14000 rcf and the pellets were stored in -20°C before further treatment.

Recorded by Shuoyi HU

# August

---

**8/7 DLS analysis**

Participant(s): Mingchun XU, Yuchen JIANG

The biosynthesized nanoparticles from 7/27 were sent for DLS analysis to get the particle size
distribution analysis.

(Detailed treatment of the sample: 5 mM FeCl3
incubation, synthesized by pGLO-SHuffle, cell ultrasonication for effective
time 10 minutes, centrifugation by 10000 rcf for 30 minutes and the pellets
were resuspended by deionized water and dissociated in the ultrasonic cleaner
for 10 minutes, further ultrafiltration by 100 kDa filtration membrane by
5000 rcf for 20 minutes, flow through dissociated in the ultrasonic cleaner
for 10 minutes, and ultrafiltration by 3 kDa filtration membrane by 5000 rcf
for 20 minutes, concentrated solution was collected for DLS analysis)

Recorded by Shuoyi HU

**8/9 scFv-pGLO Transformation**

Participant(s): Shuoyi HU

Vector scFv-pGLO was transformed into SHuffle strain *E. coli*.

(Named as SHuffle-antibody below)

Recorded by Shuoyi HU

**8/10 Monoclonal SHuffle-antibody Preparation

        Nanoparticle Sample Treatment**

Participant(s): Shuoyi HU, Mingchun XU, Yuchen JIANG

Three single colonies of SHuffle-antibody were picked up and incubated
in 5 mL LB broth (0.1 g/L ampicillin) for 8 hours and sent for sequencing.

(Sequence properly aligned in later report)

The biosynthesized nanoparticle synthesized from 7/27 was further
ultrafiltrate by 100 kDa filtration membrane again as the result of DLS
measurement in 8/7 show the interference of large particles and we guessed
it may be dusts.

Recorded by Shuoyi HU

**8/11 SHuffle-antibody Culture Amplification

        scFV-cys-his and scFV-his-cys Transformation**

Participant(s): Shuoyi HU

In total 150 mL culture of SHuffle-antibody were developed.
Substrate was LB broth with 0.1 g/L ampicillin and SHuffle-antibody
*E. coli* was inoculated by 1:100 dilution. (Bacteria culture from the
single colony developed in 8/10, sequence aligned properly)
After inoculation at 12:00, the OD600 of the culture reached 0.7 at 16:30
(which was not ideal but we continued the steps following), 20% Arabinose stock
solution was added by 1:100 dilution to the culture.

Additionally, two vectors contain scFV-cys-his and scFV-his-cys respectively
were transformed into SHuffle strain *E. coli* separately.

(Named as SHuffle-antibody-cys-his and SHuffle-antibody-his-cys below)

![Clones observed after pGLO-antibody-cys-his transformation](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-1.png)

![Clones observed after pGLO-antibody-his-cys transformation](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-2.png)

Recorded by Shuoyi HU

**8/12 SHuffle-Antibody Culture Collection**

Participant(s): Shuoyi HU

Culture of SHuffle-Antibody developed in 8/11 was collected through centrifugation
by 4000 rcf for 20 minutes and stored in -80ºC for protein extraction.

Recorded by Shuoyi HU

**8/13 Monoclonal SHuffle-antibody-his-cys and
SHuffle-antibody-cys-his
Preparation**

Participant(s): Shuoyi HU

Five single colonies of SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
respectively were picked up and incubated in 5 mL LB broth (0.1 g/L ampicillin)
for 8 hours and sent for sequencing.
(Sequence properly aligned in later report)

Then the culture was stored at 4ºC for later use.

Recorded by Shuoyi HU

**8/14 Bacteria Strain Storage**

Participant(s): Shuoyi HU

In total seven tubes of bacteria were stored in -80ºC in glycerol.

(pGLO-SHuffle for one tube, SHuffle-Antibody, SHuffle-antibody-his-cys
and SHuffle-antibody-cys-his for two tubes respectively)

Recorded by Shuoyi HU

**8/15 scFV-his Protein Purification**

Participant(s): Shuoyi HU

The scFV domain of the antibody was purified through the AKTA
system with culture collected from 8/12. Detailed steps could be
found in the protocol of [His-tagged protein
purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4).
BCA test was done but the 1 M iminazole in the elution buffer B was found to
be not compatible with the BCA protein concentration measurement kit.
(Solution would turn blue when the protein solution was added, while the
existence of protein could be verified as the absorbance at 560 nm for extracted
protein was greater than the buffer B negative control for multiple times)

Then, protein was stored in 20% glycerol and 80% buffer B in -20ºC.

Recorded by Shuoyi HU

**8/16 Nanoparticle Treatment for DLS Analysis**

Participant(s): Shuoyi HU

The biosynthesized nanoparticle synthesized from 7/27 and further ultrafiltrate
by 100 kDa filtration membrane again in 8/10 was sent for DLS particle size
distribution analysis.

Recorded by Shuoyi HU

**8/17 SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
Culture
Amplification**

Participant(s): Shuoyi HU

Two strains of *E. coli* including SHuffle-antibody-his-cys and
SHuffle-antibody-cys-his were expanded into a total volume of 300 mL
culture respectively for protein extraction [His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4)

Recorded by Shuoyi HU

**8/18 SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
Culture
Collection**

Participant(s): Shuoyi HU

The expanded culture of SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
were collected through centrifugation by 4000 rcf for 20 minutes and stored in
-80 ºC for protein extraction.

Recorded by Shuoyi HU

**8/19 SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
Protein
Purification**

Participant(s): Shuoyi HU, Songnan REN

Bacteria pellet of SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
collected from 8/18 were crushed in high pressure homogenizer. Antibodies
with cystine-poly-his tag and poly-his-cystine tag were got extracted following
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4). Protein
electrophoresis
were also conducted and the result showed existence of our targeted protein.
Sampling sequence of the figure below: No.1-11 SHuffle-antibody-cys-his protein collection
tube, protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from
top to bottom.

![Antibody-cys-his protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-1.png)

Sampling sequence of figure n+1: Tube No. 1-5 SHuffle-antibody-his-cys protein,
Tube No.5-8 SHuffle-antibody (Extracted from 8/15, only used as a supplementary
information), protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD,
17kD from top to bottom.

![Antibody-his-cys and antibody protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-2.png)

Recorded by Shuoyi HU

**8/20 Protein Concentration Determination**

Participant(s): Shuoyi HU, Songnan REN

BCA tests were done for the protein extracted from 8/19, but unfortunately all
the samples show no significant difference in Ab570 comparing to the negative control
(Buffer B), even the incubation time was elongated to several hours, still no clear
change in Ab570 could be observed. This indicated no protein exist in the preservation
tube. We suspected protein get degraded or the fact that we didn't have the protein
snap freeze or snap thaw get protein out of solution system. So, buffer for preservation
was changed to buffer A with 0.1 mM PMSF in later experiments.

Recorded by Shuoyi HU

**8/21 SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
Amplification

        SHuffle-antibody-str-his Transformation**

**2nd round of cell culture of SK-BR-3**

Participant(s): Shuoyi HU, Songnan REN

SHuffle-antibody-his-cys strain was expanded into a total volume of 400 mL culture
and SHuffle-antibody-cys-his was expanded into a total volume of 450 mL culture for
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4) again.

Additionally, the vector containing scFV-streptavidin-his was transformed into SHuffle
strain *E. coli* separately. (Named as SHuffle-antibody-str-his below)

![Clones observed after SHuffle-antibody-str-his transformation](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-21-1.png)

Recorded by Shuoyi HU

Participant: Yixiao XIAO

[SK-BR-3](https://www.atcc.org/products/htb-30#detailed-product-information) cells were
recovered in two 100 mm cell culture plates using
the same medium used in 7/28
(10 ml medium for each culture plate).

Recorded by Yixiao XIAO

**8/22 Monoclonal SHuffle-antibody-str-his Preparation

        Antibody-cys-his and Antibody Protein Purification**

Participant(s): Shuoyi HU, Mingchun XU, Songnan REN, Yuchen JIANG

Three single colonies of SHuffle-antibody-str-his were picked up and incubated
in 5 mL LB broth (0.1 g/L ampicillin) for 8 hours and sent for sequencing.
(Sequence properly aligned in later report)
SHuffle-antibody-str-his was expanded into a total volume of 300 mL culture
respectively for [His-tagged protein
purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4) in the morning.
Then the culture was stored at 4ºC for later use.
Besides, the expanded culture of SHuffle-antibody-his-cys and SHuffle-antibody-cys-his
were collected through centrifugation by 4000 rcf for 20 minutes.
Bacteria pellets of SHuffle-antibody-his-cys and SHuffle-antibody-cys-his collected
then were crushed in high pressure homogenizer. Antibodies with cystine-poly-his tag
and poly-his-cystine tag were got extracted following [His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4)
again. As the threshold of protein collection was set to
be UV280 greater than 0 in elution step, while the concentration of SHuffle-antibody-his-cys
protein was really low and the UV280 was always lower than 0 in this purification step,
so no SHuffle-antibody-his-cys protein was collected this time. And then protein
electrophoresis was also conducted and the result showed existence of our targeted protein
besides SHuffle-antibody-his-cys protein.

Sampling sequence of figure below: No.1-11 SHuffle-antibody-cys-his protein collection tube,
protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from top to bottom.

![Antibody-cys-his protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-1.png)

Sampling sequence of figure below: Tube No. 2-12 SHuffle-antibody protein,
protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD
from top to bottom.

![Antibody protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-2.png)

Additionally, as we found that under 5 mM FeCl3 induction for biological
nanoparticle synthesis, there would have precipitated underlying in the culture
after centrifugation. To examine the nanoparticle we observed under TEM microscopy,
two cultures were developed. One was developed following the Nanoparticle biosynthesis
protocol(hyperlink). For the other one as negative control, when OD reached 0.1 and FeCl3
was added, the culture was distributed into 50 mL centrifugation tube and bacteria pellet
was collected through centrifugation by 4000 rcf and stored in -80 ºC.

Recorded by Shuoyi HU

**8/23 Protein Concentration Determination and Storage**

Participant(s): Shuoyi HU, Mingchun XU, Songnan REN, Yuchen JIANG

BCA protein concentration test of SHuffle-antibody-cys-his protein
No. 2,3,4 and SHuffle-antibody protein No. 6,7,8 was conducted. Results were shown
in the table below:

|  | Sample Measured | Protein concentration (mg/mL) |
| --- | --- | --- |
|  | SHuffle-antibody-cys-his-2 | 0.512 |
|  | SHuffle-antibody-cys-his-3 | 0.546 |
|  | SHuffle-antibody-cys-his-4 | 0.378 |
|  | SHuffle-antibody-6 | 0.758 |
|  | SHuffle-antibody-7 | 1.362 |
|  | SHuffle-antibody-8 | 0.504 |

Besides, the culture for biological nanoparticle synthesis developed in 8/22 was
collected. Then the 5 mM FeCl3 induced culture along with the negative control bacteria
pellets stored in -80 ºC were used for [Nanoparticle
Separation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#9).
The expanded culture of SHuffle-antibody-str-his from 8/22 was collected through centrifugation
by 4000 rcf for 20 minutes and resuspended by Buffer A with 0.1 mM PMSF and crushed in high
pressure homogenizer. Antibodies with streptavidin and poly-his tag were got extracted following
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4). While the
concentration of
SHuffle-antibody-str-his protein was really low and the UV280 was always lower than 0 in
this purification step, so no SHuffle-antibody-str-his protein was collected this time.
We guessed that the protein may be included in the inclusion Body . So, we modified the
method to extract antibody-streptavidin-his protein next time by using guanidine hydrochloride.

Recorded by Shuoyi HU

**8/24 TEM Sample Preparation**

Participant(s): Gehua WENG, Songnan REN

TEM sample of the nanoparticle extracted from 5 mM FeCl3 induced
culture and the negative control were prepared on Formvar Carbon
Support Film on Square Grids (Catalog number: FCF300-CU-50)

Recorded by Shuoyi HU

**8/25 SHuffle-antibody-str-his Culture
Amplification
        TEM analysis

        Biologically Synthesized Nanoparticle-antibody
Conjugation by NHS-PEG-Maleimide Linking**

**SHuffle-antibody-str-his Culture Amplification**

Participant(s): Shuoyi HU, Yixiao XIAO

SHuffle-antibody-str-his was expanded into a total volume of 300 mL culture
respectively for [His-tagged protein
purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4) in the morning.

TEM analysis was conducted by the samples prepared from 8/24.
No existence of nanoparticle for negative control sample was confirmed.
(There do have some spots in the view field while the spots would go
when a higher intensity of electron beam exposure applied).
And clear existence
of nanoparticle could be observed in the sample extracted from 5 mM
FeCl3 induced culture.

![TEM result for negative control](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-1.png)

![TEM result for for sample extracted from 5 mM FeCl<sub>3</sub> induced culture](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-2.png)

The expanded culture of SHuffle-antibody-str-his was collected through centrifugation
by 4000 rcf for 20 minutes and stored in -80 ºC for protein extraction in the evening.

Recorded by Shuoyi HU

**Biologically Synthesized Nanoparticle-antibody
Conjugation by NHS-PEG-Maleimide Linking**

Participant: Songnan REN

Two groups were set up, one adopted the antibodies with the addition of cysteine
on the end of the antibodies, before the his-tag, while the other group adopted
antibodies without this cysteine. For each group, 25 µL nanoparticle was taken
and added in 50 µL 100 µM NHS-PEG-Maleimide DMSO solution, reacting for 1h under
room temperature. After it was finished, proper amount of DMSO solution was added
and 5000g ultrafiltration in 3kDa tube was applied in 4 degree Celsius for
20 minutes to wash out unreacted NHS-PEG-Maleimide molecules. After the
modification, 25µL maleimide-modified nanoparticle was taken for each group,
and mixed with 50 µg 30 µg/mL antibodies, reacting for 1.5h under room temperature.
After it finished, proper amount of Buffer A solution was added and 14000g
ultrafiltration in 10kDa tube was applied in 4 degree Celsius for 20 minutes
to wash out unlinked antibodies. After it accomplished, the ultrafiltration
tube was converted to collect the left liquid by centrifuging at 1000g for
1 minute.

|  |  | Nanoparticle | NHS-PEG-Mal (µM) | Antibody |
| --- | --- | --- | --- | --- |
|  | Group 1 | Biologically | 100 | Cys-his end |
|  | Group 2 | Biologically | 100 | His end |

Recorded by Songnan REN

**8/26 SHuffle-antibody-str-his Protein Extraction

        Chemically Synthesized Nanoparticle-antibody
Conjugation by NHS-PEG-Maleimide Linking**

**SHuffle-antibody-str-his Protein Extraction**

Participant(s): Shuoyi HU, Mingchun XU, Yuchen JIANG, Yixiao XIAO, Songnan REN

Bacteria pellets of SHuffle-antibody-str-his collected from 8/25 were resuspended
by Buffer A with 0.1 mM PMSF and crushed in high pressure homogenizer. This time,
after centrifugation by 11,000 rcf for 30 minutes, the precipitate was rinsed
with 10 ml of 2 mM EDTA, 30 mM Tris-HCI (pH 8.0), 0.1% Triton X-100 and then
centrifuged as previously described. Next, the precipitate was dissolved in 10 ml
of 6 M guanidine hydrochloride (pH 1.5) with gentle stirring and subsequently
dialyzed against 6 M guanidine hydrochloride (pH 1.5). The resulting dialysate
was further dialyzed overnight against 0.2 M NaHCO3 (pH 8.7) to eliminate guanidine
hydrochloride.
(Reference: Sano T, Cantor CR. Expression of a cloned streptavidin
gene in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):142-6.
doi: 10.1073/pnas.87.1.142. PMID: 2404273; PMCID: PMC53216.)

SHuffle-antibody-str-his was expanded into a total volume of 150 mL culture
respectively for [His-tagged
protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4) again.

Recorded by Shuoyi HU

**Chemically Synthesized Nanoparticle-antibody
Conjugation by NHS-PEG-Maleimide Linking**

Participant: Songnan REN

Two groups were set up, one adopted the antibodies with the addition of cysteine on the
end of the antibodies, before the his-tag, while the other group adopted antibodies without
this cysteine. For each group, 5µL 50mg/mL nanoparticle was taken and diluted to 10mg/mL
by DMSO solution. 50µL 100µM NHS-PEG-Maleimide DMSO solution was then added, reacting for
1h under room temperature. After it finished, proper amount of DMSO solution was added
and 5000g ultrafiltration in 3kDa tube was applied in 4 degree Celsius for 20 minutes to
wash out unreacted NHS-PEG-Maleimide molecules. After the modification, 25µL maleimide-modified
nanoparticle was taken for each group, and mixed with 50µg 30 µg/mL antibodies,
reacting for 1.5h under room temperature. After it finished, proper amount of Buffer
A solution was added and 14000g ultrafiltration in 10kDa tube was applied in 4 degree
Celsius for 20 minutes to wash out unlinked antibodies. After it accomplished, the
ultrafiltration tube was converted to collect the left liquid by centrifuging at 1000g for 1 minute.

|  |  | Nanoparticle (mg/mL) | NHS-PEG-Mal (µM) | Antibody (mg/mL) |
| --- | --- | --- | --- | --- |
|  | Group 1 | Chemically, 10 mg/mL | 100 | Cys-his end, 0.03 mg/mL |
|  | Group 2 | Chemically, 10 mg/mL | 100 | His end, 0.03 mg/mL |

Recorded by Songnan REN

**8/27 SHuffle-antibody-str-his Protein Purification**

Participant(s): Mingchun XU, Shuoyi HU, Yuchen JIANG, Chenxu LIU

The dialysate from 8/26 was centrifuged at 18,000 rcf for 30 minutes,
and the resulting supernatant was utilized for his-tag protein purification.
While this time we still had the concentration of SHuffle-antibody-str-his
protein was really low and the UV280 was always lower than 0 in this purification
step, so no SHuffle-antibody-str-his protein was collected this time.
We guessed that the protein may be in the renatured precipitate,
so we preserve the protein for further electrophoresis examination.

The expanded culture of SHuffle-antibody-str-his was collected through centrifugation
by 4000 rcf for 20 minutes and treated by 6 M guanidine hydrochloride for 1 hour
and then dialyzed against two solutions: the first one is 500 mL water including
20 mM PBS + 50 mM Tris-HCl for 6 hours and the second one is 500 mL water based
solution with 50 mM Tris-HCl ,150 mM NaCl ,10 mM EDTA ,0.5% Triton X-100 ,0.2 M
ammonium sulfate ,0.3 M glycine and 0.2 M glucose for 24 hours (process ended in 8/28)

Recorded by Shuoyi HU

**8/28 SHuffle-antibody-str-his Protein Purification**

**Cell recovery of BT-474**

Participant(s): Mingchun XU, Chenxu LIU

The dialysate from 8.27 was centrifuged at 18,000 rcf for 30 minutes,
and the resulting supernatant was utilized for his-tag protein purification.
However, the UV280 was always lower than 0 in this purification,
which indicated that still no SHuffle-antibody-str-his protein was collected this
time. It was possible that the target protein remained in the precipitate, so
the precipitate was resuspended by buffer A. And then we added 5X SDS-PAGE loading buffer
into the solution and 95℃ heated it for 5 min. The solution was preserved for
electrophoresis examination in 8/29.

Recorded by Mingchun XU

Participant: Yixiao XIAO

Recover cells in a 15 ml tube with 6ml prewarmed Roswell Park Memorial Institute (RPMI) 1640 medium
added
with [Fetal Bovine Serum](https://www.thermofisher.cn/order/catalog/product/10099141C) to
a final concentration of 10%, 2μg/ml of insulin and 1% of Penicillin and streptomycin. Cells
were then centrifugate the cells at 1000rpm for 3 mins. Remove medium and resuspend cell pellet with
6 ml
medium and then culture them in a 100mm cell culture plates with 10 ml medium.

Recorded by Yixiao XIAO

**8/29 Protein Electrophoresis**

**Cell Culturing Medium Exchange**

Participant(s): Yanbo MAO

Protein electrophoresis was also conducted and the result showed existence
of our targeted protein besides SHuffle-antibody-his-cys protein.
Sampling sequence of the figure below: 1. maker, 2. Uninduced, 3. Undialysised,
4. Super, 5. pellet after dialysis, 6. sample4, 7. pellet, 8. Supernatant
after dialysis tube No. 3, 9. Supernatant after dialysis tube No. 4

![Protein electrophoresis result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-29-1.png)

Recorded by Yanbo MAO

Participant: Yixiao XIAO

BT-474 grew slowly but no bacteria infected. Culture medium was changed with
another 10 ml medium with 20% of [Fetal Bovine Serum](https://www.thermofisher.cn/order/catalog/product/10099141C) to
support the normal growth rate of BT-474 cells.

Cell condition under 10x microscope as below:

![bt474-10x-day1-8-29](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day1-10x-8-29.jpg)

Recorded by Yixiao XIAO

**8/30 Antibody-conjugated Nanoparticle Solution Protein Concentration
Determination**

**Cell Culturing Medium Exchange**

Participant: Songnan REN, Yanbo MAO, Songyuan XUE

BCA test was conducted following the protocol of [BCA test](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#6).
Standard Curve of BCA test was shown below:

![Standard Curve of BCA test](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-30-bsa-standard-curve.png)

Using the standard curve, the concentration of the sample was calculated as below:

| Nanoparticle Category | Antibody Category | Protein Concentration |
| --- | --- | --- |
| Chemically No.G01,H01 | Cys-his end | 0.018 mg/mL |
| Chemically No.G02,H02 | His end | 0.013 mg/mL |
| Biologically No.G03,H03 | Cys-his end | 0.038 mg/mL |
| Biologically No.G04,H04 | His end | 0.025 mg/mL |

Though high deviations between repeated measurements lead to insignificant differences
between cys-his end and his end antibodies, these data still gave us a plausible inspiration
that the addition of cysteine might have the effect of facilitating the conjugation of
antibodies on nanoparticle.

Recorded by Songnan REN

Participant(s): Yixiao XIAO

Culture medium was changed with another 10 ml medium
with 20% of [Fetal Bovine
Serum](https://www.thermofisher.cn/order/catalog/product/10099141C)
to support the normal growth rate of BT-474 cells.

Cell condition under 10x microscope as below:

![bt474-10x-day2-8-30](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt474-10x-day2-8-30.jpg)

Cell Experiment Recorded by Yixiao XIAO

**8/31 Cell Culturing Medium Exchange**

Participant(s): Yixiao XIAO

Culture medium was changed with another 10 ml medium
with 20% of [Fetal Bovine Serum](https://www.thermofisher.cn/order/catalog/product/10099141C)
to support the normal growth rate of BT-474 cells.

Cell condition under 10x microscope as below:

![bt-474-10x-day3-8-31](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-10x-day3-8-31.jpg)

Cell Experiment Recorded by Yixiao XIAO

# September

---

**9/1 Sub culture of BT-474**

Participant(s): Yixiao XIAO

The cell culture did not reach 70-75% confluency but the sub culture was held on
account of the limitation of time.
Cells were washed with 10 ml of Dulbecco's Phosphate Buffered Saline (PBS) and
digested with 1 ml of trypsin/EDTA for about 10 min at 37°C. 6 ml of media was
used to neutralize the trypsin and cells were spinned down at 1200rpm for 3 mins.
6 ml of media was added to the 15ml tube with cell pellet. The subculture ratio
was 1:2.

Recorded by Yixiao XIAO

**9/2 EDC/NHS Conjugation**

**Cell Condition Record**

**EDC/NHS Conjugation>**

Participant(s): Shijie HE

1) 15 mL 0.2M EDC·HCl and 20 mL 0.2M NHS were made in 50 mL tubes.

2) 250 µL of our bio-synthetic nanoparticles(DLS with dust) and 8 µL 0.4mg chemo-synthetic
nanoparticles coated with citric acid were transferred to two 5 ml tubes(EDC/NHS US & EDC/NHS PC).

3) 250 µL 0.1M PBS was added to the second tube to make up for the difference.

4) Then 4ml 0.1M PBS was added to each of the tubes to mix it thoroughly.

5) Then, 60 µL of EDC·HCl 0.2 M and 120 µL of NHS 0.2 M were added to each tube.

6) After 10 min, 0.041mg antibody(AB-7) was added to the EDC/NHS-activated NPs solutions.

7) Then the tubes were stored in 4℃ refrigerator.

**Cell Condition Record**

Participant(s): Yixiao XIAO

Cell condition under 10x microscope as below:

![bt-474-10x-g2-day1-9-2](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-10x-g2-day1-9-2.jpg)

Recorded by Yixiao XIAO

**9/3 EDC/NHS conjugation continued from 9/2**

**Culture Medium Change**

Participant(s): Shijie HE

After 24 h, the reaction system from 9/2 was centrifuged for 25 min at 14000 RPM to remove the
unbound antibodies.

Then the left solutions was sucked down to 200 µL by the pipette.

Recorded by Shijie HE

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria was infected. Culture medium was changed with another 10ml medium
with 20% of fbs to support the normal growth rate of BT-474 cells.
Cell recovery of MDA-MB-231 cells
Recover cells in a 15 ml tube with 6ml prewarmed medium ( a 1:1 mixture of dulbecco's modified eagle
medium(DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium added with fbs to a final
concentration of 10%, 2μg/ml of insulin and 1% of Penicillin and streptomycin). Cells were then
centrifugate the cells at 1000rpm for 3 mins. Remove medium and resuspend cell pellet with 6 ml
medium and then culture them in a 100mm cell culture plates with a 10 ml medium.

Recorded by Yixiao XIAO

**9/4 Culture medium change**

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria was infected. Culture medium was changed with another 10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
10ml of medium change was conducted for MDA-MB-231 cells.

Cell condition under 10x microscope as below:

![bt-474-10x-g2-day3-9-4](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-10x-g2-day3-9-4.jpg)

Recorded by Yixiao XIAO

**9/5 Culture medium change**

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria was infected. Culture medium was changed with another 10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
10ml of medium change was conducted for MDA-MB-231 cells.

Cell condition under 10x microscope as below:

![bt-474-10x-g2-day4-9-5](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-g2-day3-10x-9-5.jpg)

Figure 1. Cell Condition of BT-474

![mda-mb-231-20x-day1-0905](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-20x-day1-0905.jpg)

Figure 2. Cell Condition of MDA-MB-231

Recorded by Yixiao XIAO

**9/6 Culture medium change**

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria was infected. Culture medium was changed with another 10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
10ml of medium change was conducted for MDA-MB-231 cells.

Recorded by Yixiao XIAO

**9/7 EDC/NHS conjugation

        Subculture of MDA-MB-231 in 6 mm dish and of BT-474 in
10 mm dish

        Cell seeding in 96-wells plate

        Preparation of nps/antibodies reagents for
cytotoxicity
test and flow cytometry test**

**EDC/NHS Conjugation**

Participant(s): Shijie HE

200 µL of our bio-synthetic nanoparticles(DLS with dust) and 0.4mg 8 µL chemo-synthetic
nanoparticles coated with citric acid were transferred to two eppendorf tubes(EN US&EN PC).

200 µL 0.1M PBS was added to the second tube to make up for the difference.

Then 200 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

Then, 10 µL of EDC·HCl 0.2 M and 20 µL of NHS 0.2 M were added to each tube.
After 10 min, 0.041mg antibody(AB-7) was added to the EDC/NHS-activated NPs solutions.

Then the tubes were stored in 4℃ refrigerator.

Recorded by Shijie HE

**Subculture of MDA-MB-231 in 6 mm dish and of BT-474 in 10 mm dish
& Cell seeding in 96-wells plate & Preparation of nps/antibodies reagents for
cytotoxicity test and flow cytometry test**

Participant(s): Yixiao XIAO, Chenxu LIU, Songyuan XUE

Remove the spent medium and wash both plates of MDA-MB-231 and BT-474 with 3 ml PBS. Then, remove the
PBS solution
and digest with 1 ml of trypsin/EDTA for about 10 min for BT-474 and 4 min for MDA-MB-231cells at
37°C. Then,
stop digestion with 3 ml of the corresponding culture medium for each kind of cells. And centrifugate
cells at
1200 rpm for 3 min for BT-474 cells and 1000 rpm for MDA-MB-231 cells. Remove the liquid supernatant and
resuspend the cell pellet with 6ml corresponding medium. Then, 10 ul of the cells suspension was taken
out and
mixed with 10ul of trypan blue and counted the cell density and living rate, which were 4.58\*10^5
cells/ml
and 89% for BT-474 and 2.24\*105 cells/ml and 94% for MDA-MB-231. For subculture, the seeding
rate were 1:4
for BT-474 and 1:10 for MDA-MB-231.And for 96-well plate, there should be 100 ul cell suspension with
10000
cells in it for each well. Therefore, 2800 ul of medium was added to 786 ul of BT-474 suspension and 3.6
ml
of medium was added to 3.6 ml of MDA-MB-231 cell suspension. And two 96-well plates wer added with 36
wells
of 100ul of BT-474 in each plate and the same to MDA-MB-231.

Reagents preparation was handled as the following table.

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | UCNPs\_1 | UCNPs\_2 | UCNPs\_3 | Bio\_AB | Bio\_CH | Chem-AB | Chem-CH | CH | AB |
| BT-474 Culture Medium (μL) | 540 | 582 | 598.2 | 580 | 580 | 580 | 580 | 580 | 580 |
| Sample volumn added (μL) | 60 | 18 | 1.8 | 20 | 20 | 20 | 20 | 20 | 20 |
| MDA-MB-231 Culture Medium (μL) | 540 | 582 | 598.2 | 580 | 580 | 580 | 580 | 580 | 580 |
| Sample volumn added (μL) | 60 | 18 | 1.8 | 20 | 20 | 20 | 20 | 20 | 20 |

Note that UCNPs represents Unlinked Chemically Synthesized Nanoparticles.

Bio-AB represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2
antibody with poly-his tag attached at the end.

Chem-AB represents Chemically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody
with poly-his tag attached at the end.

Bio-CH represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2
antibody with cystine-poly-his attached at the end.

CH represents scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.

AB represents scFv domain of anti HER2 antibody with poly-his tag attached at the end.

Recorded by Yixiao XIAO

**9/8 SHuffle-pGLO Amplification

EDC/NHS Conjugation continued**

**Drug Treatment, Antibody Incubation and Confocal Analysis of Cells**

**SHuffle-pGLO Amplification**

Participant(s): Gehua WENG

SHuffle-pGLO stored in 4°C was activated in 5 mL LB Broth with Ampicillin
(0.1 g/L). The culture was incubated in 37°C shaker for one day and used in 9/9.

Recorded by Shuoyi HU

**EDC/NHS Conjugation continued from 9/7**

Participant(s): Shijie HE

After 24 h, the solution was transferred to 10 KD ultrafiltration tubes and centrifuged for 10
min at 14000 g to remove the unbound antibody.

But mistakenly the samples were discarded.

Then, the EDC/NHS conjugation was conducted again.

200 µL of our bio-synthetic nanoparticles(DLS with dust) and 0.4mg 8 µL chemo-synthetic
nanoparticles coated with citric acid were transferred to two eppendorf tubes(EN US&EN PC).

400 µL 0.1M PBS was added to the second tube to make up for the difference.

Then 200 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

Then, 10 µL of EDC·HCl 0.2 M and 20 µL of NHS 0.2 M were added to each tube.

After 10 min, 0.015mg antibody(AB-8) was added to the EDC/NHS-activated NPs solutions.

Then the tubes were stored in 4℃ refrigerator.

Recorded by Shijie HE

Participant: Yixiao XIAO, Shuoyi HU, Gehua WENG

**Drug Treatment of Cells**

After about 6 hours, all cells have adhered to the plate/dish wall from the 96 wells plate
prepared in 9/7,
remove the spent medium in each well and add 100ul of prepared medium mixed with different reagents.

Cell condition before drug treatment in 96 wells plate was shown as below:

![bt-474-10x-g2-day4-9-8](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-20x-in-96-well-plate-0908.jpg)

Figure 1. Cell condition of MDA-MB-231

![bt-474-10x-g2-day4-9-8](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-in-96-well-plate-10x-0908.jpg)

Figure 2. Cell condition of BT-474

Recorded by Yixiao XIAO

**Antibody Incubation and Confocal Analysis of Cells**

1. Cells treated with drug were centrifuged at 350 rcf for 3 mins and then the supernatant was
removed.

2. A total volume of 100 μL Dulbecco's PBS was added in each well for washing,
then it was centrifuged at 350 rcf for 3 mins again.

3. The supernatant was removed and the cells were fixed with -20°C methanol (10 minutes)
and then with -20°C acetone in 4°C (1 minute).

4. A total volume of 100 μL Dulbecco's PBS containing 1% BSA was added in each
well for washing and blocking, then it was centrifuged at 350 rcf for 3 mins twice.

5. 50 μL Monoclonal Anti-polyHistidine in PBS, Catalog Number SAB4200620, was
added into each well and incubated for 60 minutes.

6. A total volume of 100 μL Dulbecco's PBS was added in each well and kept for
5 minutes for washing, then it was centrifuged at 350 rcf for 3 mins twice.

7. 50 μL Anti-Mouse-IgG in PBS , Catalog Number ab150115, was added into two replica
of each treatm and leave one well of each treatment for negative control and
incubated for 60 minutes away from light.

8. Then the cells were ready for confocal analysis.

Recorded by Shuoyi HU

**9/9 Ferric Ion Induced Nanoparticle Synthesis

        Antibody and Antibody-cys-his Protein
Purification**

**BT-474 and MDA-MB-231 spent medium change

Cytotoxicity Examination of Biosynthesized Nanoparticle and Chemically Synthesized
Nanoparticle**

Participant(s): Shuoyi HU

**Ferric Ion Induced Nanoparticle Synthesis**

5 mM ferric ion concentration was used to induce iron oxide nanoparticle synthesis.

|  |  |  |
| --- | --- | --- |
|  | Flask I | Flask II |
| OD600 value when  adding FeCl3 | 0.095 | 0.093 |

Culture medium: LB broth
0.2% Arabinose for protein expression induction
0.1 g/L Ampicillin.
 Time of adding FeCl3: 17:00.

Condition of the shaker: 37ºC, 220 rpm.

Culture Volume: 150 mL per flask and in total 300 mL.

**Antibody and Antibody-cys-his Protein Purification**

Bacteria pellets of SHuffle-antibody and SHuffle-antibody-cys-his
collected from 8/18 were crushed in high pressure homogenizer. Antibodies
with cystine-poly-his tag and poly-his tag were got extracted following
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4). Protein
electrophoresis
was also conducted and the result shows the existence of our target protein.
Sampling sequence of the figure below: No.A03-A09 SHuffle-antibody protein collection
tube, supernatant after centrifugation of broken cell culture, uninduced SHuffle-Antibody, protein
ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from
top to bottom.

![Antibody protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-9-9-antibody-final-used-on-website-tif.jpg)

Sampling sequence of the figure below: No.A03-A09 SHuffle-antibody-cys-his protein collection
tube, supernatant after centrifugation of broken cell culture, uninduced SHuffle-Antibody-cys-his,
protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from
top to bottom.

![Antibody-cys-his protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-9-9-2-c-h-final-used-on-website.jpg)

Recorded by Shuoyi HU

Participant: Yixiao XIAO, Songyuan XUE

**BT-474 and MDA-MB-231 spent medium change**

10 ml of medium change of BT-474 and 6 ml of medium change of MDA-MB-231 were conducted.

Cell conditions were shown as below:

![mda-mb-231-g2-day2-10x-0909](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-g2-day2-10x-0909.jpg)

Figure 1. Cell condition of MDA-MB-231, Second Generation.

![bt-474-day2-10x-0909](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day2-10x-0909.jpg)

Figure 2. Cell condition of BT-474, Second Generation.

**Cytotoxicity Examination of Biosynthesized Nanoparticle and Chemically
Synthesized Nanoparticle**

After 24 hours of co-culture of the cells and our substances were added, 10 ul of cck-8 medium
was added to each well in the plate. And after 3 hours of culture, optical density of wells
in the plate was measured at 460 nm.

Recorded by Yixiao XIAO

**9/10 EDC/NHS Linkage

        NHS-PEG-Maleimide Linking

        BCA Test

        Nanoparticle Separation

        Culture medium change**

**EDC/NHS Linkage**

Participant(s): Shuoyi HU

Two reaction systems which contained *Biologically synthesized nanoparticles, EDC, NHS, antibody*
and *Chemically synthesized nanoparticles, EDC, NHS, antibody* were ultrafiltrated
through 10kD filtration membrane to remove unreacted samples for 20 minutes and washed
with PBS solution for two times and stored at 4°C.

Recorded by Shuoyi HU

**NHS-PEG-Maleimide Linking**

Participant(s): Songnan REN, Chenxu LIU, Songyuan XUE

Three groups were set up, one adopted the antibodies with the addition of cysteine
on the end of the antibodies, before the his-tag, while the other group adopted antibodies
without this cysteine. Besides, there was one group of nanoparticles without the addition
of antibody solution. For each group 5µL 50 mg/mL nanoparticles was taken and diluted to
10 mg/mL by DMSO solution. 50 µL 100 µM NHS-PEG-Maleimide DMSO solution was then added for
each group, reacting for 1h under room temperature. After it was finished, proper amount of DMSO
solution was added and 5000g ultrafiltration in 3kDa tube was applied to all the groups in 4
℃ for 20 minutes to wash out unreacted NHS-PEG-Maleimide molecules. After
ultrafiltration, ultrafiltration tubes were heated in 37ºC incubator for 10 minutes to melt
the frozen DMSO solution. During the ultrafiltration, nanoparticle solution had been diluted
to about 4 mg/mL. 10 µL nanoparticle solution was taken for each group, and mixed with 40µL
1.245 mg/mL antibodies except for Blank NP group, reacting for 1.5h under room temperature.
After it was finished, all the three tubes were stored in 4ºC fridge.

|  |  | Chemically Synthesized Nanoparticle (mg/mL) | NHS-PEG-Mal (µM) | Antibody (mg/mL) |
| --- | --- | --- | --- | --- |
|  | Group 1 (His-Cys end NP) | 10 mg/mL before Ultrafiltration 4 mg/mL after Ultrafiltration | 100 | 1.245 mg/mL |
|  | Group 2 (Poly-His end NP) | 10 mg/mL before Ultrafiltration 4 mg/mL after Ultrafiltration | 100 | 1.245 mg/mL |
|  | Group 3 (Blank NP) | 10 mg/mL before Ultrafiltration 4 mg/mL after Ultrafiltration | 100 | 0 mg/mL |

Recorded by Songnan REN

**Ferric Ion Induced Nanoparticle Separation**

Participant(s): Shuoyi HU, Chenxu LIU, Songyuan XUE

The ferric ion induced pGLO-SHuffle were used for nanoparticle separation following
the protocol of [Biologically
Synthesized Nanoparticle Separation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#10) (Except this time we didn't plan to
use the sample for TEM analysis, so we used Buffer A instead of deionized water
in steps in the protocol)

Recorded by Shuoyi HU

**BCA Test**

Participant(s): Shuoyi HU, Chenxu LIU

BCA test was conducted following the protocol of [BCA
test](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#6).
Standard Curve of BCA test was shown below:

![BCA standard curve in 9/10](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/result-for-notebook/10092023.png)

Using the standard curve, the concentration of the sample was calculated as below:

|  | Protein category | Protein Concentration |
| --- | --- | --- |
|  | Antibody with poly his end No. A04 | 2.725 mg/mL |
|  | Antibody with poly his end No. A05 | 4.69 mg/mL |
|  | Antibody with poly his end No. A06 | 1.795 mg/mL |
|  | Antibody with cystine-poly-his end No. A04 | 0.645 mg/mL |
|  | Antibody with cystine-poly-his end No. A05 | 1.245 mg/mL |
|  | Antibody with cystine-poly-his end No. A06 | 0.425 mg/mL |

Recorded by Shuoyi HU

**Culture medium change**

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria infection had happened. Culture medium was changed with another 10ml
medium with
20% of fbs to support the normal growth rate of BT-474 cells.
6ml of medium change was conducted for MDA-MB-231 cells.

Recorded by Yixiao XIAO

**9/11 NHS-PEG-Maleimide Linking Negative Control Supplement

        EDC/NHS Conjugation

        Culture Medium Change**

**NHS-PEG-Maleimide Linking Negative Control Supplement**

Participant(s): Songnan REN

In order to provide negative control group in subsequent cytotoxicity examination,
two additional groups were set up, one was antibodies with the addition of cysteine
on the end of the antibodies, before the his-tag, while the other group was antibodies
without this cysteine. This part of operation skipped the modification and ultrafiltration
of nanoparticles. 10µL DMSO solution was taken for each group, and mixed with 40 µL 1.245 mg/mL
antibodies, while for Blank NP group, DMSO solution was mixed with 40µL Buffer C,
staying under room temperature for 1.5h. After it was finished, both two tubes were
stored in 4ºC .

Recorded by Songnan REN

**EDC/NHS Conjugation**

Participant(s): Shijie HE

 0.4mg 8 µL of our chemo-synthetic nanoparticles and nothing were transferred to two eppendorf
tubes(EN PC 8&EN a 8).

200 µL 0.1M PBS was added to the second tube to make up for the difference.

Then 400 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

Then, 10 µL of EDC·HCl 0.2 M and 20 µL of NHS 0.2 M were added to each tube.

After 10 min, 0.015mg antibody(Ab-8) was added to the second tube.

Bio-synthetic nanoparticles(NP 9、10) was transferred into a 5kD ultrafiltration tube and centrifuged
for 85min at 5000g to remove the unwanted substance.

The obtained approximate 500 µL bio-synthetic NPs is collected in original tube.

Double 0.036mg 8 µL of our chemo-synthetic nanoparticles and nothing were transferred to three
eppendorf tubes(EN PC-a 5&EN PC 5&EN a 5) .

Then 400 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

Then, 10 µL of EDC·HCl 0.2 M and 20 µL of NHS 0.2 M were added to each tube.

After 10 min, 0.039mg antibody(Ab-5) was added to the first and third tubes.

Then the tubes were stored in 4℃.

Recorded by Shijie HE

**Culture Medium Change**

Participant(s): Yixiao XIAO

BT-474 grew slowly but no bacteria infected. Culture medium was changed with another 10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
6ml of medium change was conducted for MDA-MB-231 cells.

Cell condition was shown as below:

![mda-mb-231-g2-day3-10x-0911](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-g2-day4-10x-0911.jpg)

Figure 1. Cell condition of MDA-MB-231, Second Generation.

![bt-474-day3-10x-0911](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day4-10x-0911.jpg)

Figure 2. Cell condition of BT-474, Second Generation.

Recorded by Yixiao XIAO

**9/12 EDC/NHS Conjugation conjugation continued from 9/11

        Preparation of NPs/antibodies reagents for
cytotoxicity test and flow cytometry test

        Culture Medium Change**

**EDC/NHS Conjugation continued from 9/11**

Participant(s): Shijie HE

After 24 h, the solution(EN PC 8&EN a 8&EN PC-a 5&EN PC 5&EN a 5) was transferred to 10kD
ultrafiltration tubes and centrifuged for 10 min at 14000 g to remove the unbounded antibody.

Then 200 µL 0.1M PBS was added to the tubes to run 14000 g ultrafiltration for 10 min and the
sample was washed in this way twice.

Finally, the obtained approximate 150 µL samples are collected in EP tubes.

Recorded by Shijie HE

**Preparation of nps/antibodies reagents for cytotoxicity test and flow cytometry test**

Participant(s): Yixiao XIAO

The spent medium was removed and both plates were washed of MDA-MB-231 and BT-474 with 3 ml PBS. Then,the
PBS solution was removed
and digested with 1 ml of trypsin/EDTA for about 10 min for BT-474 and 4 min for MDA-MB-231cells at
37°C. Then,
the digestion was stopped with 3 ml of the corresponding culture medium for each kind of cells. And cells
were centrifugated
at 1200
rpm for 3 min for BT-474 cells and 1000 rpm for MDA-MB-231 cells. The liquid supernatant was removed and
the
cell pellet was resuspended with 6ml corresponding medium. Then, 10 ul of the cells suspension was taken
out and mixed
with 10ul
of trypan blue and counted the cell density and living rate, which were 7.17\*10^5 cells/ml and 90.7% for
BT-474
and 1.94\*10^5 cells/ml and 97.1% for MDA-MB-231. There should be 100 ul cell suspension with 10000 cells
in it
for each well. Therefore, 4 ml of medium was added to 2 of BT-474 suspension and 5 ml of medium was
added to 3 ml of
MDA-MB-231 cell suspension. And two 96-well plates were added with 36 wells of 100ul of BT-474 in each
plate and
the same to MDA-MB-231.

Reagents preparation was handled as the following table.

For treatment 1 (T1):

|  | Samples(μL) | Medium (μL) |
| --- | --- | --- |
| Bio-unlinked (1:10) | Bio+AB-8 (1:10) | 100ug/ml np1 | 40ug/ml np1 / np1+AB / np2+AB / np2 | 20ug/ml np1 / np1+AB / np2+AB / np2 | 5ug/ml np1 / np1+AB / np2+AB / np2 |
| 30 | 30 | 30 | 12 | 6 | 1.5 |
| 270 | 270 | 270 | 288 | 294 | 298.5 |

For treatment 2 (T2):

|  | Samples(μL) | Medium (μL) |
| --- | --- | --- |
| 40ug/ml np1+AB / np1+CH / np1 | 20ug/ml np1+AB / np1+CH / np1 | 5ug/ml np1+AB / np1+CH / np1 | CH(1:10) |
| 90 | 45 | 11.2 | 30 |
| 210 | 255 | 288.8 | 270 |

**Culture Medium Change**

Participant(s): Yixiao XIAO, Songyuan XUE

BT-474 grew slowly but no bacteria was infection had happened. Culture medium was changed with another
10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
6ml of medium change was conducted for MDA-MB-231 cells.

Cell conditions were shown as below:

![mda-mb-231-g2-day4-10x-0912](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-g2-day5-10x-0912.jpg)

Figure 1. Cell condition of MDA-MB-231, Second Generation.

![bt-474-day4-10x-0912](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day5-10x-0912.jpg)

Figure 2. Cell condition of BT-474, Second Generation.

**9/13 Anti HER2 scFv domain application as immunostaining toxic
addition**

**Anti HER2 scFv domain application as immunostaining pre-treatment**

Participant: Yixiao XIAO

After about 6 hours, all cells have adhered to the plate/dish wall,
the spent medium was removed in each well and 100ul of prepared medium was added mixed with different
reagents.

Before drug treatment, cell conditions were recorded as below:

![mda-mb-231-g2-day5-10x-0913](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-10x-in-96-well-plate-0913.jpg)

Figure 1. Cell condition of MDA-MB-231, Second Generation before Drug Treatment.

![bt-474-day5-10x-0913](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-in-96-well-plate-10x-0913.jpg)

Figure 2. Cell condition of BT-474, Second Generation before Drug Treatment.

Recorded by Yixiao XIAO

**9/14 Immunostaining, confocal and flow cytomotry examination**

**Immunostaining, confocal and flow cytomotry examination**

Participant: Shuoyi HU, Yixiao XIAO

Our experiment design was shown as below:

| Cell Strain | Treatment of the cells | | |
| --- | --- | --- | --- |
|  | Anti HER2 scFv domain antibody | Anti His tag antibody (mouse derived) | Goat Anti-Mouse-IgG |
| BT-474 treatment 1  (three replica) | + | + | + |
| BT-474 treatment 2 | + | + | - |
| BT-474 treatment 3 | - | + | + |
| MDA-MB-231 treatment 1  (two replica) | + | + | + |
| MDA-MB-231 treatment 2  (two replica) | + | + | - |
| MDA-MB-231 treatment 3 | - | + | + |

After treating the cells with scFv domain of anti HER2 antibodies, the cells in 96 well plate were
washed with
100 uL PBS with 1% BSA for 2 times, and the PBS with 1% BSA was removed after centrifugation through 400
rcf for 5 minutes.
After that cells were fixed with 100 uL -20°C methanol for 10 minutes and removed after centrifugation
through 400 rcf for 5 minutes.

100 uL PBS with 1% BSA washing was conducted for 3 times after incubation.

50 uL anti-polyHistidine antibody was added to each well and incubated for 60 minutes.
100 uL PBS
Washing was conducted for 3 times after incubation.

Then, 50 uL Goat derived Anti-Mouse-IgG was added to each well and incubated for 60 minutes.
100
uL PBS Washing was conducted for 3 times after incubation.

Here cells were prepared for confocal analysis and flow cytometry analysis.

Recorded by Shuoyi HU

**cck8-test**

Participant: Yixiao XIAO

After 24 hours of co-culture of the cells and our added substances, 10 ul of cck-8 medium was added to
each well in the plate. And after 3 hours of culture, optical density of wells in the plate was measured
at 460 nm.

Recorded by Yixiao XIAO

**9/15 Ferric Ion Induced Iron Oxide Nanoparticle Synthesis

         SHuffle-Antibody-cys-his Amplification**

**Ferric Ion Induced Iron Oxide Nanoparticle Synthesis**

Participant: Shuoyi HU

5 mM ferric ion concentrations was used to induce iron oxide nanoparticle synthesis.

|  |  |  |
| --- | --- | --- |
|  | Flask I | Flask II |
| OD600 value when  adding FeCl3 | 0.125 | 0.105 |

Culture medium: LB broth
0.2% Arabinose for protein expression induction
0.1 g/L Ampicillin.
 Time of adding FeCl3: 17:00.

Condition of shaker: 37ºC, 220 rpm.

Culture Volume: 150 mL per flask and in total 300 mL.

Recorded by Shuoyi HU

**SHuffle-Antibody-cys-his Amplification**

Participant: Shuoyi HU

SHuffle-antibody-cys-his of *E. coli* was expanded into a total volume of 300 mL
culture for protein extraction
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4)

Recorded by Shuoyi HU

**9/16 Ferric ion induced culture collection and
SHuffle-Antibody-cys-his culture collection**

Participant: Shuoyi HU

In total 300 mL culture of ferric ion induced pGLO-SHuffle, 150 ml SHuffle-Antibody-cys-his
culture developed under 18°C for 16 hours and 150 mL SHuffle-Antibody-cys-his culture
developed under 37°C were collected and centrifuged at 4000 rcf for 20 minutes, cell
pellets were stored at -80°C for future use.

Recorded by Shuoyi HU

**9/17 Nanoparticle Separation

        DLS and Zeta Potential Analysis of Biologically
Synthesized Nanoparticles and Chemically Synthesized Nanoparticles

        Antibody-cys-his Protein Purification**

**Nanoparticle Separation and DLS analysis of Biologically Synthesized Nanoparticles
and Chemically Synthesized Nanoparticles**

Participant: Shuoyi HU, Mingchun XU

The ferric ion induced pGLO-SHuffle were used for nanoparticle separation following
the protocol of [Biologically
Synthesized Nanoparticle Separation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#10)

Recorded by Shuoyi HU

**DLS analysis of Biologically Synthesized Nanoparticles and Chemically Synthesized Nanoparticles**

Participant: Shuoyi HU

From DLS analysis, we can see that the equivalant hydrodynamic size of our biologically
synthesized nanoparticle solution system is 6.37 nm with an average baseline index 8.8, and the detailed
reported result could be found in the pdf file below:

DLS analysis of Biologically Synthesized Nanoparticles

While if we look into the report, we have the largest intensity of the peak lay around 7.5 nm,
as the peak below one was more likely to be the attachment dropped when ultrasonication was conducted.

For the Chemically synthesized nanoparticle solution system, the equivalant hydrodynamic size
is 348.92 nm with an average baseline index 1.27, and the detailed reported result could
be found in the pdf file below:

DLS analysis of Chemically Synthesized Nanoparticle

This result for Chemically synthesized nanoparticle was less trustable as in detailed
peak of intensity graph there had clumps could be observed, indicating too long storage time before
doing DLS analysis.

Recorded by Shuoyi HU

**Zeta Potential analysis of Biologically Synthesized Nanoparticles and Chemically Synthesized
Nanoparticles**

Participant: Shuoyi HU

From Zeta Potential analysis, we can see that the zeta potential of our biologically
synthesized nanoparticle solution system is -19.98 mV, and the detailed reported result could be found
in the pdf file below:

Zeta Potential analysis of Biologically Synthesized Nanoparticles

For the reliability of the data, the following parameter could be analysed:

Zeta Potential Range: Looking at the range of zeta potential values (-31.19 mV to -14.51 mV),
we can see that there is a reasonable variation in the data, suggesting that the measurements
cover a diverse range of samples.

Sample Count Rate and Ref. Count Rate: The sample count rate and reference count rate values
seem to be consistent across the measurements, indicating that the measurements were taken under similar
conditions and with stable instrument performance.

Mean and Standard Deviation: The mean zeta potential value is -19.98 mV, and the standard
deviation is 5.44 mV. The relatively low standard deviation suggests that the measurements are
relatively consistent and reproducible.

RMS Residual: The RMS residual values provide an indication of the goodness of fit for the
zeta potential measurements. Lower values indicate a better fit. The provided RMS residual
values range from 1.4009E-02 to 3.5592E-02, which suggests a reasonable fit for the data.

Standard Error: The standard error values provide an estimation of the variability of the mean.
Lower values indicate a more precise estimation. The provided standard error values range from 1.81
to 2.3527E-03, which suggests a relatively small variability in the mean zeta potential.

For Chemically
synthesized nanoparticles, the zeta potential is -18.46 mV, and the detailed reported result could be
found in the pdf file below:

Zeta Potential analysis of Chemically Synthesized Nanoparticle

For the reliability of the data, the following parameter could be analysed:

Zeta Potential Range: The range of zeta potential values in the data set is -25.60 mV to -9.41 mV,
indicating a reasonable variability in the measurements.

Sample Count Rate and Ref. Count Rate: The sample count rate and reference count rate values appear
consistent across the measurements, suggesting stable instrument performance and similar measurement
conditions.

Mean and Standard Deviation: The mean zeta potential value is -18.46 mV, and the standard deviation
is 4.75 mV. The relatively low standard deviation suggests that the measurements have relatively low
variability around the mean.

RMS Residual: The RMS residual values provided in the data set range from 1.1134E-02 to 6.3507E-02.
Lower values indicate a better fit for the zeta potential measurements. The provided values suggest a
reasonable fit for the data.

Standard Error: The standard error values provide an estimation of the variability of the mean. Lower
values indicate a more precise estimation. The provided standard error values range from 1.58 to
5.9294E-03, suggesting a relatively small variability in the mean zeta potential.

From the above analysis, we can see that the zeta potential of our biologically
synthesized nanoparticle solution system is -19.98 mV, and the zeta potential of our
chemically synthesized nanoparticle solution system is -18.46 mV, indicating that both
of our nanoparticle solution system are relatively stable in aqueous soluiton, and
the biologically synthesized nanoparticle have extra stability comparing to the chemically
synthesized one.

Recorded by Shuoyi HU

**Antibody-cys-his Protein Purification**

Participant(s): Shuoyi HU, Mingchun XU

Bacteria pellets of SHuffle-antibody-cys-his
collected from 9/15 were crushed in high pressure homogenizer. Antibodies
with cystine-poly-his tag and poly-his tag were got extracted following
[His-tagged protein purification](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#4). Protein
electrophoresis
were also conducted and the result showed existence of our targeted protein.
Sampling sequence of the figure below: Protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD,
34kD, 26kD, 17kD, Uninduced bacteria, supernatant after centrifugation of broken cell culture
of 37°C, collection tube No. A05, A06, A07, B05 of 37°C Culture, protein ladder: 180kD, 130kD,
95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD.
supernatant after centrifugation of broken cell culture of 18°C, collection tube No. A04, A05,
A06 of 18°C Culture.

![Antibody-cys-his protein purification result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/marker-uninduced-18-c-sup-a04-a05-a06-b05-marker-37-c-uninduced-a05-a06-a07.jpg)

Recorded by Shuoyi HU

**9/18 Nanoparticle Extraction from Supernatant of Ferric Ion
Induced
Culture

         BCA Protein Concentration Test

         Chemically Synthesized Nanoparticle NHS-PEG-Maleimide
Modification**

**Nanoparticle Extraction from Supernatant of Ferric Ion Induced Culture**

Participant: Shuoyi HU

The supernatant cell homogenization after centrifugation of 5 mM
Fe2(SO4)3 induced culture were filtrated through the 0.22 μm filtration
membrane
and then filtrated through 100 kD ultrafiltration membrane for future treatment and analysis.

Recorded by Shuoyi HU

**BCA Protein Concentration Test**

Participant(s): Shuoyi HU

BCA Protein Concentration Test were conducted following the protocol of
[BCA Protein Concentration Test](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#5).
Standard Curve of BCA test was shown below:

![Antibody-cys-his protein concentration result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/0918-bca.png)

Using the standard curve, the concentrations of the samples were calculated as below: (the dillution
factor
before BCA test was considered already)

|  |  |
| --- | --- |
| Sample Name | Protein concentration (mg/ml) |
| 18°C induced A05 | 4.236 |
| 18°C induced A06 | 1.517 |
| 18°C induced A07 | 0.707 |
| 37°C induced A06 | 1.654 |
| 37°C induced A07 | 2.269 |

Recorded by Shuoyi HU

**Chemically Synthesized Nanoparticles NHS-PEG-Maleimide Modification**

Participant(s): Songnan REN, Haotian SHEN

Three groups were set up, one adopted the antibodies with
the addition of cysteine on the end of the antibodies, before
the his-tag, while another group adopted antibodies without this
cysteine, and the last group set up for negative control, without
the addition of antibodies. For each group, 5µL 50mg/mL nanoparticles
was taken and 15µL 100µM NHS-PEG-Maleimide DMSO solution was then added,
reacting for 1h under room temperature. After it was finished, proper amount
of buffer C was added and 5000g ultrafiltration in 3kDa tube was
applied in 4 ℃ for at least 20 minutes to wash out
unreacted NHS-PEG-Maleimide molecules. After ultrafiltration, the
solution was diluted to 100 µL, indicating the concentration of
nanoparticles had declined to 2.5mg/mL

Recorded by Songnan REN

**9/19 Cell Recovery of SK-BR-3 and MDA-MB-231**

Participant(s): Yixiao XIAO

**Cell recovery of MDA-MB-231 cells**

Cells were recovered in a 15 ml tube with 4 ml prewarmed medium
(a 1:1 mixture of dulbecco's modified eagle medium (DMEM) and
Roswell Park Memorial Institute (RPMI) 1640 medium added with FBS
to a final concentration of 10% and 1% of Penicillin and streptomycin).
Cells were then centrifugate the cells at 1200rpm for 3 mins.
Remove medium and resuspend cell pellet with 3 ml medium and then
culture them in a 100mm cell culture plates with a 10 ml medium.

**Cell recovery of SK-BR-3 cells**

Recover cells in two 15 ml tubes with 4 ml of each of prewarmed culture
medium (McCoy's 5a Medium Modified added with fetal bovine serum (fbs) to
a final concentration of 10% and 1% of Penicillin and streptomycin and
Roswell Park Memorial Institute (RPMI) 1640 medium added with fbs to a final concentration of 10%, 10μg/ml
of insulin and 1% of Penicillin and streptomycin). Cells were then centrifugate the cells at 1200rpm for 3
mins. Remove medium and resuspend cell pellet with 3 ml medium and then culture them in two 100mm cell
culture plates with 10 ml medium.

Recorded by Yixiao XIAO

**9/20 DLS Analysis for Chemically Synthesized
Nanoparticles and Biologically Synthesized Nanoparticles Extracted from Supernatant of
Ferric Ion Induced Culture

        TEM Sample Preparation

        SHuffle-pGLO Activation

        Cell Medium Change**

**DLS Analysis for Chemically Synthesized
Nanoparticles and Biologically Synthesized Nanoparticles Extracted from Supernatant of
Ferric Ion Induced Culture**

Participant(s): Shuoyi HU, Gehua WENG

The supernatant of 5 mM Fe2(SO4)3 induced culture cell lysis were
filtrated through the 0.22 μm filtration membrane
and then filtrated through 100 kD ultrafiltration membrane in 9/18, further
it was filtrated through 3kDa ultrafiltration membrane and collected the unfiltrated part.

Later, after ultrasonication treatment, we conducted DLS analysis for the unfiltrated part.
The hydrodynamic size of the nanoparticle was 6.76 nm with an average baseline index 8.3, and the detailed
reported result could be found in the pdf file below:
Results were shown in the pdf file below:

DLS analysis of Biologically Synthesized Nanoparticles Extracted
from Supernatant of
Ferric Ion Induced Culture<< /div>

Also, as the data from 9/17 for chemically synthesized nanoparticles was less trustable as the baseline
index of the data was not high,
we conducted DLS analysis for the chemically synthesized nanoparticles right after 2 minutes
ultrasonication
treatment again.
The hydrodynamic size of the nanoparticle was 345.38 nm with an average baseline index 9.4, and the
detailed
reported results could be found in the pdf file below:
Results were shown in the pdf file below:

DLS analysis of Chemically Synthesized Nanoparticles

Besides, zeta potential analysis was also conducted for the chemically synthesized nanoparticles right
after
DLS measurements, which was tested to be -23.28 mV on average.
Detailed results could be found in the pdf file below:

Zeta Potential analysis of Chemically Synthesized Nanoparticles

Here the zeta potential of the biologically synthesized nanoparticle was not tested as the sample was too
dilute to be tested.
We decided to expand the culture again and concentrate the sample for future analysis.

Recorded by Shuoyi HU

**TEM Sample Preparation**

Participant(s): Shuoyi HU, Gehua WENG

The chemically synthesized samples and the biologically synthesized nanoparticles extracted from
supernatant of
ferric ion induced culture were used for TEM sample preparation. (These two samples were also used for
the
DLS analysis above)

Recorded by Shuoyi HU

**SHuffle-pGLO Activation**

Participant(s): Shuoyi HU, Chenxu LIU

The SHuffle-pGLO culture stored in 4°C were activated by adding 50 μL stored culture into 5 mL
fresh LB with 0.1 g/L Ampicillin.
Culture here were put into the 37°C shaker with 220 rpm for usage tomorrow.

Recorded by Shuoyi HU

**Cell Medium Change**

Participant(s): Yixiao XIAO

10ml cell culture medium change was conducted for all the three plates (MDA-MB-231 cells and the other
two
plates of Sk-Br-3 cells)
However, some special contaminants were found in the plate of Sk-Br-3 cells cultured by McCoy's 5a
Medium.

The figures showing conditions of the cells were shown below:

![MDA-MB-231 cells cultured by McCoy's 5a Medium](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-10x-0920.jpg)

**Figure 1.** MDA-MB-231 cells cultured by McCoy's 5a Medium under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with insulin added](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day1-10x-insulin-0920.jpg)

**Figure 2.** Sk-Br-3 cells cultured by McCoy's 5a Medium with insulin added under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Contamination Observed](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day1-10x-0920.jpg)

**Figure 3.** Sk-Br-3 cells cultured by McCoy's 5a Medium with Contamination Observed under 10x
Object

Recorded by Yixiao XIAO

**9/21 Cell Contaminants treatment**

Participant(s): Yixiao XIAO

the plate of Sk-Br-3 cells cultured by McCoy's 5a Medium was terribly contaminated by nanobacteria.
They
were then treated by 70% ethanol and paraformaldehyde (PFA).
Cell medium change
10ml cell culture medium change was conducted for both plates (MDA-MB-231 cells and Sk-Br-3 cells).

The figures showing condition of the cells were shown below:

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Contamination Observed](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/polluted-40x-9-21.jpg)

**Figure 1.** Sk-Br-3 cells cultured by McCoy's 5a Medium with Contamination under 20x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day2-20x-9-21.jpg)

**Figure 2.** Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added under 40x Object

![MDA-MB-231 cells cultured by McCoy's 5a Medium with Insulin Added](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-day2-20x-9-21.jpg)

**Figure 3.** MDA-MB-231 cells cultured under 20x Object

Recorded by Yixiao XIAO

**9/22 Ferric Ion Induced Nanoparticle Synthesis with Time
Gradient

        Subculture of MDA-MB-231 in 10 mm dish**

**Ferric Ion Induced Nanoparticle Synthesis with Time Gradient**

Participant(s): Shuoyi HU

Three flasks of 150 mL culture of ferric ion induced pGLO-SHuffle were prepared for
nanoparticle synthesis, the process was induced by adding 5 mM FeCl3at 11:00
when the OD600 of three cultures were all in the range of 0.095-0.100.

Time gradient of the synthetic process was set to be 24 hours, 36 hours and 48 hours.

This means that the first flask would be collected at 11:00 9/23, the second flask would be
collected
at 23:00 9/23,
and the third flask would be collected at 11:00 9/24.

The collected supernatant and pellet of the cultures would be used for TEM and DLS analysis later.

Recorded by Shuoyi HU

**Subculture of MDA-MB-231 in 10 mm dish**

Participant(s): Yixiao XIAO

The confluency of the MDA-MB-231 reached 90% and the subculture was conducted. The spent medium was
removed
and
both plates of MDA-MB-231 were washed with 3 ml PBS. Then, the PBS solutionwas removed and digested with 1
ml of
trypsin/EDTA for about 3 min at 37°C. Then, the digestion was stopped with 2 ml of the corresponding
culture
medium.
And cells were centrifugated at 1200 rpm for 3 min. The liquid supernatant was removed and the cell
pellets were resuspended
with 4 ml corresponding medium. For subculture, the seeding rate was 1:10 for MDA-MB-231 and 500 μl of
cell
suspension was added into two 100mm cell culture plate with 10 ml culture medium in each.

**9/23 TEM Analysis of Chemically Synthesized Nanoparticles and
Biologically Synthesized Nanoparticles Extracted from Supernatant of
Ferric Ion Induced Culture

        Ferric Ion Induced Nanoparticle Synthesis with Time
Gradient Culture Collection

        EDC/NHS Conjugation

        Cell Medium Change**

**TEM Analysis of Chemically Synthesized Nanoparticle and Biologically Synthesized Nanoparticle
Extracted
from Supernatant of
Ferric Ion Induced Culture**

Participant(s): Shuoyi HU

TEM analysis of two samples above was conducted.

The chemically synthesized one was observed to be in average around 22 nm and showed square shape.
And
the biologically synthesized sample was observed to be around 3-6 nm and showed round shape.

One thing to note is that under the TEM, the Chemically synthesized nanoparticles showed a lot of
aggregation, which could explained why the DLS analysis before gave much larger hydrodynamic diameter
than the diameter observed in TEM analysis.

 Below are two representative figures of the two samples:

![Chemically synthesized nanoparticle TEM result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0003-20230923-0936-57000-x-ceta-1.jpg)

Figure 1. Chemically synthesized nanoparticles' TEM result

![Biologically synthesized nanoparticle TEM result](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0023-20230923-1051-390-kx-ceta.jpg)

Figure 2. Biologically synthesized nanoparticles' TEM result

Recorded by Shuoyi HU

**Ferric Ion Induced Nanoparticle Synthesis with Time Gradient Culture Collection**

Participant(s): Mingchun XU, Shuoyi HU

Two flasks of 150 mL culture of ferric ion induced pGLO-SHuffle prepared in 9/22 were collected
respectively at 11:00 9/23 and 23:00 9/23.

Then the supernatant was stored at 4°C, the pellets were stored at -80°C for future use.

The supernatant and pellets of the cultures would be used for TEM and DLS analysis later.

Recorded by Shuoyi HU

**EDC/NHS Conjugation**

Participant(s): Shijie HE

EDC/NHS Conjugation was conducted following the protocol of
[EDC/NHS Conjugation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#6).

 1)Double 0.036mg 8 µL of our chemical-synthesized nanoparticles and double 235µL newly
bio-synthesized
nanoparticles(in Fe2(SO4)3) were transferred to four eppendorf tubes(EN PCn-ab5 1&EN PCn-ab5 2&EN
BNP-ab5
1&EN BNP-ab5 2) .

 2)Then 100 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

 3)Then, 20 µL of 0.2M EDC·HCl and 20 µL of 0.2M NHS were added to each tube.

 4)Then 135 µL 0.1M PBS was added to first two tubes to make compensate for the solution volume.

 5)After 10 min, 0.038mg antibody(AB-5) was added to each tube.

 6)Then the tubes were stored in 4℃ .

Recorded by Shijie HE

**Cell Medium Change**

Participant(s): Yixiao XIAO

10ml cell culture medium change was conducted for all three plates (2 MDA-MB-231 plates and the Sk-Br-3
plate).

The figures showing conditions of the cells were shown below:

![MDA-MB-231 cells culture](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day4-10x-0923-1.jpg)

**Figure 1.** MDA-MB-231 cells cultured under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day3-10x-0923g2-2.jpg)

**Figure 2.** Sk-Br-3 cells cultured under 10x Object

Recorded by Yixiao XIAO

**9/24 Nanoparticle Separation from Both Supernatant and Cell
Lysis
of
Ferric Ion Induced Culture

        DLS Analysis of Nanoparticle Separation from Both
Supernatant and Cell Lysis of Ferric Ion Induced culture

        EDC/NHS Linking

        Cell Medium Change**

**Nanoparticle Separation from Both Supernatant and Cell Lysis of Ferric Ion Induced Culture**

Participant(s): Chenxu LIU, Songyuan XUE

In total we had three samples with culturing time 24 hours, 36 hours and 48 hours respectively, each
containing both supernatant and cell lysis.

For the supernatant, each sample was filtrated through 0.45 μm filtration membrane and 0.22 μm
filtration membrane to avoid jamming in ultrafiltration.
Then 15 mL sample filtrated through 100 kD ultrafiltration membrane.

Further, the sample was filtrated through 3 kD ultrafiltration membrane and the unfiltrated part was
collected for DLS and TEM analysis.

For the cell pellet, all the cell pellet was resuspended with no more than 40 mL water. Then it
was
first homogenized in high pressure homogenizer and centrifugation by 14000 rcf for 30 minutes followed.

After that, both the supernatant and the resuspended pellet of each time gradient induced culture was
filtrated through 0.45 μm filtration membrane and 0.22 μm filtration membrane to avoid jamming in
ultrafiltration.

 Then 15 mL sample filtrated through 100 kD ultrafiltration membrane. Now samples were prepared for
DLS
and TEM analysis.

In total we had 9 samples, including the supernatant of the culture with
24 hours, 36 hours and 48 hours culturing time, the supernantant of cell lysis of the culture
with 24 hours, 36 hours and 48 hours culturing time, and the resuspended and ultrasonic
treated cell lysate part of the culture with 24 hours, 36 hours and 48 hours culturing time.

From these 9 samples, we hope to find out the optimized synthetic time and the
relative amount of the nanoparticles released from the cell, the amount of unattached
nanoparticles kept in the cell and the amount of nanoparticles attached to the biomass in the
cell.

Recorded by Shuoyi HU

**DLS and Zeta Potential Analysis of Nanoparticle Separation from Both Supernatant and Cell Lysis of
Ferric Ion Induced Culture**

Participant(s): Shuoyi HU

As said above, 9 samples were prepared for DLS and Zeta Potential analysis.
Raw data of each samples could be found in the following pdf files.

DLS analysis of 5 mM FeCl3
24 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Culture

DLS analysis of 5 mM FeCl3
36 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Culture

DLS analysis of 5 mM FeCl3
48 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Culture

DLS analysis of 5 mM FeCl3
24 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Cell Lysate

DLS analysis of 5 mM FeCl3
36 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Cell Lysate

DLS analysis of 5 mM FeCl3
48 Hours Induction Biologically Synthesized Nanoparticle from Supernatant of Cell Lysate

For the resuspended cell pellet groups, in DLS measurement we found that the concentration
of the samples were too low since the ultrafiltration today leaves total volume of each sample
multiple times than the ultrafiltration conducted before. Future concentrating treatment
is required to reconduct the DLS analysis. While today's DLS informed us that the concentration
of iron oxide nanoparticles are far more concentrated directly in the supernatant of the
cell lysate.

Recorded by Shuoyi HU

**EDC/NHS Linking**

Participant(s): Shijie HE

EDC/NHS Linking was conducted following the protocol of
[EDC/NHS Linking](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#6).

1)After 24 h, the solutions (EN PCn-ab5 1&EN PCn-ab5 2&EN BNP-ab5 1&EN BNP-ab5
was transferred to 10kD ultrafiltration tubes and centrifuged for
10 min at 14000 g to remove the unbounded antibodies.

2)Then 200 µL 0.1M PBS was added to the tubes to run 14000 g ultrafiltration
for 10 min and the samples were washed in this way twice.

3)Finally, the obtained approximate four 150 µL samples were collected in
ultrafiltration tubes by reversing the inner tubes of ultrafiltration in
new ultrafiltration tubes and running them at 1000g for 2 mins.

4)Then the samples were stored in 4℃.

Recorded by Shijie HE

**Cell Medium Change**

Participant(s): Yixiao XIAO

10ml cell culture medium change was conducted for all three plates (2 MDA-MB-231 plates and the Sk-Br-3
plate).

The figures showing conditions of the cells were shown below:

![MDA-MB-231 cells culture](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-10x-0924-2.jpg)

**Figure 1.** MDA-MB-231 cells cultured under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-10x-0924.jpg)

**Figure 2.** Sk-Br-3 cells cultured under 10x Object

Recorded by Yixiao XIAO

**9/25 TEM Analysis of Ferric Ion Induced Nanoparticle Synthesis
with
Time Gradient

        Subculture of MDA-MB-231 and Sk-Br-3 in 10 mm
plates**

**TEM Analysis of Ferric Ion Induced Nanoparticle Synthesis with Time Gradient**

Participant(s): Shuoyi HU

9 samples used for DLS analysis were also used for TEM analysis.
There was too much salt in the resuspended cell pellet groups and
cell culture groups, which interfered the TEM analysis.

Only the resuspended cell pellet groups showed clear nanoparticle groups,
but we only measured the 48 hours sample as other samples were not dry
enough to be observed under TEM.
from the average of the diameters we observed in multiple fields, the average diameter
was found to be 14.63 nm.

Representative figure of 48 hours resuspended cell pellet group was shown below:

![TEM result of 48 hours resuspended cell pellet group](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/result-for-notebook/0005-20230925-2150-190-kx-ceta.jpg)

Figure 1. TEM result of 48 hours resuspended cell pellet group

Recorded by Shuoyi HU

**Subculture of MDA-MB-231 and Sk-Br-3 in 10 mm plates**

Participant(s): Yixiao XIAO

The confluency of the MDA-MB-231 reached 90% and Sk-Br-3 reached 75%-80% so that the subculture was
conducted.

The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS.
Then,
the
PBS
solution was removed and digested with 1 ml of trypsin/EDTA for about 3 min for MDA-MB-231 and 9 min for
Sk-Br-3 at
37°C. Then, the digestion was stopped with 2 ml of the corresponding culture mediums. And cells were
centrifuged at
1200
rpm for 3 min. The liquid supernatant was removed and the cell pellet was resuspended with 4 ml
corresponding
medium.
For subculture, the seeding rates were 1:10 for MDA-MB-231 and 1:4 for Sk-Br-3. Therefore, 500 μl of
MDA-MB-231 suspension and 1 ml of Sk-Br-3 suspension were added into two 100mm cell culture plates for
each
cells with 10 ml corresponding culture medium in each.

Recorded by Yixiao XIAO

**EDC/NHS Linking**

Participant(s): Shijie HE

1) 0.036mg 8 µL of our chemo-synthetic nanoparticles was transferred to an eppendorf tube(EN PCn) .

2) Then 100 µL 0.1M PBS was added to each of the tube to mix it thoroughly.

3) Then, 20 µL of 0.2M EDC·HCl and 20 µL of 0.2M NHS were added to each tube.

4) Then 135 µL 0.1M PBS was added to first two tubes to make compensate for the solution volume.

5) Then the tubes were stored in 4℃ .

Recorded by Shijie HE

**9/26 Cell medium change

        EDC-NHS Linking**

Participant(s): Yixiao XIAO

10ml cell culture medium change was conducted for all three plates (2 MDA-MB-231 plates and the Sk-Br-3
plate).

Recorded by Yixiao XIAO

**EDC/NHS Linking**

Participant(s): Shijie HE

1) After 24 h, the solution (EN PCn) was transferred to 10kD ultrafiltration tubes and centrifuged for
10 min at 14000 g to remove the unbounded antibody.

2) Then 200 µL 0.1M PBS was added to the tubes to run 14000 g ultrafiltration for 10 min and wash the
sample in this way twice.

3) Finally, the obtained approximate four 70 µL samples were collected in ultrafiltration tubes by
reversing the inner tubes of ultrafiltration in new ultrafiltration tubes and running them at 1000g for 2
mins.

Recorded by Shijie HE

**9/27 Immunostaining, Confocal and Flow Cytomotry Examination

        Antibody Conjugation for Previously
NHS-PEG-Maleimide
Modified Chemically Synthesized Nanoparticles

        Cell Medium Change**

**Immunostaining, Confocal and Flow Cytomotry Examination**

Participant(s): Shuoyi HU, Shijie HE

| Cell Strain | Treatment of the cells | | |
| --- | --- | --- | --- |
|  | Anti HER2 scFv domain antibody | Anti His tag antibody (mouse derived) | Goat Anti-Mouse-IgG |
| SK-BR-3 treatment 1  (three replica) | - | + | + |
| SK-BR-3 treatment 2 (three replica) | + | + | + |
| SK-BR-3 treatment 3 (three replica) | + | + | - |
| SK-BR-3 treatment 4 (three replica) | + | - | + |
| MDA-MB-231 treatment 1  (four replica) | - | + | + |
| MDA-MB-231 treatment 2  (three replica) | + | + | + |
| MDA-MB-231 treatment 3 (two replica) | + | + | - |
| MDA-MB-231 treatment 4 (two replica) | + | - | + |

After treating the cells with scFv domain of anti HER2 antibodies, the cells in 96
well plate were washed with 100 uL PBS with 1% BSA for 2 times, and the
PBS with 1% BSA was removed after centrifugation through 400 rcf for 5 minutes.
After that cells were fixed with 100 uL -20°C methanol for 10 minutes and removed after centrifugation
through 400 rcf for 5 minutes.
100 uL PBS with 1% BSA washing was conducted for 3 times after incubation.
50 uL anti-polyHistidine antibody was added to each well and incubated for 60 minutes.
100 uL PBS Washing was conducted for 3 times after incubation.
Then, 50 uL Goat derived Anti-Mouse-IgG was added to each well and incubated for 45 minutes.
100 uL PBS Washing was conducted for 3 times after incubation.
Here cells were prepared for confocal analysis and flow cytometry analysis.

Recorded by Shuoyi HU

**Antibody Conjugation for Previously NHS-PEG-Maleimide Modified Chemically
Synthesized Nanoparticles**

Participant(s): Songnan REN

Each group was mixed with its corresponding antibodies with mole
number ratio of nanoparticle and antibody to be 1:2. For the
blank group, the same volume of buffer C was added. After reacting
for 1.5h under room temperature, proper amount of Buffer C was
added and 14000g ultrafiltration in 10kDa tube was applied in
4℃ for at least 20 minutes to wash out unlinked antibodies.
After it was accomplished, the ultrafiltration tube was converted to
collect the left liquid by centrifuging at 1000g for 1 minute.

|  | Nanoparticles | Antibody |
| --- | --- | --- |
| Group 1 | Chemically, 2.5 mg/mL, 100µL | Cys-His end, 4.69 mg/mL, 71µL |
| Group 2 | Chemically, 2.5 mg/mL, 100µL | His end, 4.24 mg/mL, 78µL |
| Group 3 | Chemically, 2.5 mg/mL, 100µL | Blank |

Recorded by Songnan REN

**Cell Medium Change**

Participant(s): Yixiao XIAO

10ml cell culture medium change was conducted for all three plates (2 MDA-MB-231 plates and the Sk-Br-3
plate).

The figures showing condition of the cells were shown below:

![MDA-MB-231 cells culture](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-10x-g3-day3-0927.jpg)

**Figure 1.** MDA-MB-231 cells cultured under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added under 10x Object](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-10x-g2-day3-0927.jpg)

**Figure 2.** Sk-Br-3 cells cultured under 10x Object

Recorded by Yixiao XIAO

**9/28 TEM Sample Repreparation

        Preparation of Types of Treated Nanoparticle for
Cell
Treatment**

**TEM Sample Repreparation**

Participant(s): Shuoyi HU

Since the result from 9/25 were not pleasant, we decided
to reconduct the TEM analysis for all the samples with larger
sample loading by increasing the loading volumn of the
sample to the copper membrane.

Recorded by Shuoyi HU

**Preparation of Drug for Cell Treatment**

Participant(s): Gehua WENG

The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS. Then,
the
PBS
solution was removed and digested with 1 ml of trypsin/EDTA for about 10 min for Sk-Br-3 and 4 min for
MDA-MB-231cells
at 37°C. Then, the digestion was stopped with 3 ml of the corresponding culture medium for each kind of
cells. And
cells were centrifuged at 1200 rpm for 3 min for Sk-Br-3 and MDA-MB-231 cells. The liquid supernatant was
removed
and
the cell pellets were resuspended with 3 ml corresponding medium. Then, 10 ul of the cells suspension was
taken
out
and mixed with 10ul of trypan blue and counted the cell density and living rate, which were
2.87\*106
cells/ml and 96.7% for Sk-Br-3 and 6.77\*106 cells/ml and 97.1% for MDA-MB-231. There should
be
100 ul cell
suspension with 10000 cells in it for each well. Therefore, 11.58 ml of medium was added to 418 μl of
Sk-Br-3 suspension and 11.82 ml of medium was added to 180 ul of MDA-MB-231 cell suspension. And two
96-well
plates were added with 72 wells of 100ul of BT-474 in each plate and the same to MDA-MB-231.
Reagents preparation was handled as the following table.

And cell condition before plating was shown as the figures below:

![MDA-MB-231 cells culture](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/mda-mb-231-10x-g3-day4-0928.jpg)

**Figure 1.** MDA-MB-231 cells cultured under 10x Object

![Sk-Br-3 cells cultured by McCoy's 5a Medium with Insulin Added under 10x Object](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-10x-g4-day3-0928.jpg)

**Figure 2.** Sk-Br-3 cells cultured under 10x Object

**9/29 Ferric Ion Induced Nanoparticle Synthesis with Ferric Ion
Concentration Gradient**

**Ferric Ion Induced Nanoparticle Synthesis with Ferric Ion Concentration Gradient**

Participant(s): Shuoyi HU

Four flasks of 150 mL culture of ferric ion induced pGLO-SHuffle
were prepared for nanoparticle synthesis, the process was induced respectively by
adding 0, 2.5, 5, 7.5 mM FeCl3 at 11:35 when the OD600 of four cultures
were all in the range of 0.095-0.100. Then all four flasks were incubated
in the 37°C shaker by 220 rpm for 24 hours.

Recorded by Shuoyi HU

**Drug Treatment of the SK-BR3 and MDA-MB-231 Cells**

Participant(s): Gehua WENG

The drug prepared yesterday were used to treat cells. Two plates were prepared,
one is for CCK-8 cytotoxicity examination and the other one is for confocal analysis
and flow cytometry analysis. We have three technical replica for each treatment.

Drug was added at 16:00 and kept for 24 hours before further CCK-8 or staining treatment.

Recorded by Shuoyi HU

**9/30 Separation of Ferric Ion Induced Nanoparticle with Ferric
Ion
Concentration Gradient

        Immunostaining, Confocal and Flow Cytomotry
Examination

        CCK-8 Cytotoxicity Examination

        scFv Incubation Treatment of SKBR-3 and
MDA-MB-231**

**Separation of Ferric Ion Induced Nanoparticle with Ferric Ion Concentration Gradient**

Participant(s): Shuoyi HU, Shijie HE

At 11:30, the four flasks of 150 mL culture of ferric ion induced
pGLO-SHuffle were collected respectively. Same steps as 9/24 were conducted towards the resuspended
cell lysate groups and supernatant after cell lysate centrifugation groups. Finally in total 8 samples
were used for DLS and TEM analysis later.

Recorded by Shuoyi HU

**Immunostaining, Confocal and Flow Cytomotry Examination**

Participant(s): Shuoyi HU, Shijie HE

Here all samples treated with drugs with different linking methods and
concentration gradient were stained with both secondary antibody and tertiary antibodies.

General steps were the same as 9/27, including the PBS washing,
cold methanol fixation, PBS washing and immunostaining steps.

Recorded by Shuoyi HU

**CCK-8 Cytotoxicity Examination**

Participant(s): Gehua WENG

After 24 hours of co-culture of the cells and our added substances,
10 ul of cck-8 medium was added to each well in the plate. And after
1 hour of culturing, optical density of wells in the plate was measured at 450 nm.

1 hour later, the optical density of wells in the plate was measured at 450 nm again.

1 hour later, the optical density of wells in the plate was measured at 450 nm again.

1 hour later, the optical density of wells in the plate was measured at 450 nm again.

Recorded by Shuoyi HU

**scFv Incubation Treatment of SKBR-3 and MDA-MB-231**

Participant(s): Gehua WENG

100 μL of scFv solution with concentration 4.236 μg/μL (scFv-cys-polyhis
extracted from 9/17, 18°C induced, tube No. A05) was added to one plate of SKBR-3 and
MDA-MB-231 cells respectively.

Recorded by Shuoyi HU

# October

---

**10/1 Western Blot Analysis**

Participant(s): Shuoyi HU, Gehua WENG

Cells incubated in scFv solution for 24 hours were collected by cell scraper
and centrifuged by 400 rcf for 5 minutes. Then the supernatant was replaced by 2 mL
PBS with 0.5 mM PMSF. After that, the cells were sonicated for 10 minutes by
ultrasonication (effective sonification time 3.3 minutes) and concentrated by
ultrafiltration through 10kD membrane by 14000 rcf for 30 minutes in total.

Then the concentrated sample was mixed with 5X loading buffer and boiled for 10 minutes.

After that, the sample was loaded into the SDS-PAGE gel and run for 1 hour.

Then the gel was transferred to PVDF membrane and blocked with 1% BSA for 1 hour.

After washing the membrane for 30 minutes with TBS-T, the membrane was
incubated with Anti-His antibody in 4°C overnight.

Since we ran two identity gel at the same time, one of the gel was used for
Coomassie Brilliant Blue staining and the result is shown below, where the sampling
sequence is marker, MDA-MB-231 cell lysate, SK-BR-3 cell lysate, negative control
and scFv-cys-polyhis protein, the same as the sampling sequence of the other gel used for Western Blot.:

![Coomassie Brilliant Blue staining result](https://static.igem.wiki/teams/4702/wiki/results/results/marker-mda-mb231-cell-lysate-sk-br-3-cell-lysate-neg-c-h-protein-tif.jpg)

Figure 1. Coomassie Brilliant Blue staining result

The result showed that the relative concentration of scFv-cys-polyhis protein used for
binding induction is high.

Recorded by Shuoyi HU

**10/2 TEM Sample Preparation

        DLS Analysis of Ferric Ion Induced Nanoparticle with
Ferric Ion Concentration Gradient

        Western Blot Analysis Continued**

**TEM Sample Preparation**

Participant(s): Shuoyi HU

TEM sample preparation of the 8 samples prepared in 9/30 were conducted for further TEM analysis in
10/5.

Recorded by Shuoyi HU

**DLS Analysis of Ferric Ion Induced Nanoparticle with Ferric Ion Concentration Gradient**

Participant(s): Shuoyi HU

DLS analysis of the 8 samples prepared in 9/30 were conducted. We found that the
samples from the supernatant of cell lysate have low concentration of nanoparticles, and not
suitable for DLS analysis. While the samples from the resuspended cell lysate show high concentration.

In summary, we found that the optimized concentration of ferric ion for nanoparticle synthesis is
2.5
mM,
as no significant difference in average hydrodynamic diameter and polydispersity index was found between
2.5
mM
and 5 mM induced group but the absolute value of zeta potential of 2.5 mM induced group is
higher than 5 mM induced group, indicating a higher stability. Summarized data were shown in the
following
figures:

![hydrodynamic summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/hydrodynamic-diameter-1.jpg)

Figure 1. Hydrodynamic diameter summary figure

![polydispersity index summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/polydispersity-1.jpg)

Figure 2. Polydispersity index summary figure

![zeta potential summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/zeta-potential-1.jpg)

Figure 3. Zeta potential summary figure

Raw datas of each samples could be found in the following pdf files.

DLS Result of 0 mM FeCl3 induced Sample

DLS Result of 2.5 mM FeCl3 induced Sample

DLS Result of 5 mM FeCl3 induced Sample

DLS Result of 7.5 mM FeCl3 induced Sample

Zeta Potential Result of 0 mM FeCl3 induced Negative
Control Sample

Zeta Potential Result of 2.5 mM FeCl3 induced Sample

Zeta Potential Result of 5 mM FeCl3 induced Sample

Zeta Potential Result of 7.5 mM FeCl3 induced Sample

Recorded by Shuoyi HU

**Western Blot Analysis Continued**

Participant(s): Shuoyi HU

After incubation with Anti-His antibody, the membrane was washed with TBS-T for 30 minutes.

Then the membrane was incubated with Goat Anti-Mouse-IgG antibody under room temperature for 1 hour.

After washing the membrane for 30 minutes with TBS-T, finally the membrane
could be observed by adding exposing agent under the exposure machine.

The protein bands were observed as shown below:

![Western Blot result](https://static.igem.wiki/teams/4702/wiki/results/results/1002-scfv-c-h-0-3s-2023-10-02-11-54-53-ch-marker.jpg)

Figure 1. Western Blot result for scFv domain staining

Here only the standard protein control band could be observed to have been stained,
while the other bands were not stained. This may be due to the low concentration of protein
scontent in cell protein samples or poor binding affinity of the
scFv domain synthesized by us. (Sampling sequence: marker,
MDA-MB-231 cell lysate, SK-BR-3 cell lysate, negative control and scFv-cys-polyhis protein)

Recorded by Shuoyi HU

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

Engineering Success

Click!Read More

# Overview

---

Since the target of our project is to synthesize the Iron Oxide Nanoparticles using biological method and
achieve specific targeting towards the breast cancer cell, we designed four different plasmid expressing
the carefully selected scFv domain of anti-HER2 antibody and the specific functional domain to enable the
purification and conjugation of the antibody, specific
bacteria strain was selected to ensure the proper folding of the antibody.

For the future work, when the coating composition of the iron oxide nanoparticles is better
determined and the synthetic mechanism of IONPs is better understood, designs of the plasmid can be
further optimized to enabling the expression termial of antibody to be the same site as IONPs, along with
addition of functional groups in the plasmid design, self assembly of the antibody and IONPs may become
possible, which will greatly improve the efficiency for large scale prodection.

![](https://static.igem.wiki/teams/4702/wiki/engineering/layout-2.jpg)

# Design and Build

---

![](https://static.igem.wiki/teams/4702/wiki/engineering/e-coli-2.png)

# *E. coli* Strain Adoption

Antibodies were required to achieve our goal to target cancer cells. We managed to synthesize antibodies
in
*E. coli* by constructing, inducing external plasmids to express desired antibodies and took
advantage of its low cost,
simplicity, and high yield.
However, it was not easy to validly express an eukaryotic gene in a prokaryotic cell due to its simpler
inner cell structure, lacking of endoplasmic reticulum and Golgi apparatus, which were crucial
to processing protein. These deficiencies might lead to incorrect structural features of antibodies.
Besides, in wild type strain of *E. coli*, the reducing cytoplasm would cause the formation of
inclusion
bodies,
a kind of abnormal structure.
Considering that oxidizing environment was important to form disulfide bond, which was indispensable
for the biological activity of antibodies. To ensure antibody validity, we therefore adopted SHuffle
strain
of *E. coli*, whose cytoplasm was semi-oxidizing, facilitating successful expression of biologically
active
antibodies in *E. coli*.

![](https://static.igem.wiki/teams/4702/wiki/engineering/antibody-type-adoption.png)

# Antibody Type Adoption

In consideration of relatively small size of our iron-oxide nanoparticles, to ensure the binding
efficiency
between antibodies and nanoparticles, full-length recombinant antibodies might not be a decent option
because of their large size, which had the potential to lead to steric hinderance, and furthermore to
restrict the binding efficiency. Single-chain variable fragments (scFv) was composed of variable regions
of
heavy (VH) and light (VL) chains of immunoglobulins, linked via a flexible short peptide linker. Its lack
of
Fc domain enabled itself to possess a smaller size, which reduced the possibility of steric hinderance
issue
to happen. Besides, due to scFv's structural simplicity compared to full-length recombinant antibodies,
the
probability of incorrect folding dramatically decreased since slight changes would be applied on the scFv,
since we were aiming to link it on our iron-oxide nanoparticles.

![](https://static.igem.wiki/teams/4702/wiki/engineering/plasmid-contruction.jpg)

# General Idea for Plasmid Construction

To fulfill successful expression of antibodies, plasmid construction was a crucial part. Focusing on
breast
cancer, we selected HER2 as our targeting site. The pGLO plasmid we used had been verified to be able to
express protein since the expressed green-fluorescent protein (GFP) was visible to naked human eyes.

It was plausible that our antibodies could also be expressed since we only altered the protein-coding
region. Therefore, we replaced the original protein coding gene with anti-HER2 scFv coding gene. The
coding gene was derived by protein-DNA sequence conversion optimization, in frame with 6x His-tag to
detect and also purify the expressed protein.

---

![](https://static.igem.wiki/teams/4702/wiki/engineering/layout-1-three-vector-design-by-mal-linking.jpg)

## Specific Design in Plasmid Construction for NHS-PEG-Maleimide Conjugation

Beyond the elaborate design above, several other designs for better fit of NHS-PEG-Maleimide conjugation
using the thiol group were also prepared.

In the first one, three codons coding cysteine were added between the end of anti-HER2 scFv gene and the
head
of His-tag coding gene. The intention of the addition of the cysteine at this position was to provide a
free thiol group for maleimide-modified iron-oxide nanoparticles to bind, while without the interference
of biological activity of anti-HER2 scFv and capacity of being purified of His-tag. The cloning of this
modified anti-HER2 scFv gene was achieved through seamless cloning technology.

The second one was to move the added cysteine to the end of the His-tag, which might have the
effect to
enhance the binding activity of thiol group on the cysteine.

---

![](https://static.igem.wiki/teams/4702/wiki/engineering/20231010204848.jpg)

## Specific Design in Plasmid Construction for Streptavidin-Biotin Conjugation

As the Streptavidin-Biotin linkage is a very strong interaction in nature, we also had a vector design
trying to utilize this binding effect.
By combining the scFv, streptavidin, and His tag into a single
plasmid, we can leverage the advantages of each component to enhance our research and development efforts.
The scFv provides us with a highly specific and versatile antibody fragment, capable of recognizing
various targets of interest. Streptavidin, on the other hand, offers a strong and stable interaction with
biotinylated nanoparticles, allowing for efficient targeting and delivery. Lastly, the His tag serves as a
well-established purification tag, enabling rapid and reliable protein purification using affinity
chromatography.

# Test

---

![](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/engineering/engineering-test.jpg)

# Test Results of Our Plasmids

We introduced our plasmids into *E. coli* respectively and induced them to express the protein. The
results showed that except for the scFV-streptavidin-his protein was found to be in the inclusion body,
all other types of the protein was successfully expressed and purified and gave a satisfying concentration
around 1-5 mg/ml after solution displacement to storage buffer.

Then, We conducted the experiment of linking the antibody to the chemically synthesized Iron Oxide
Nanoparticles, comparing to the chemically synthesized Iron Oxide Nanoparticles without antibody addition,
the cytotoxicity of the group with antibody linkage treatment was higher than the group without the
linkage process, indirectly indicating partial bioactivity of the scFv protein.

And the detailed result of our experiment could be found in our [result page](https://2023.igem.wiki/westlake-china/results), especially the cytotoxicity test result and protein purification result.

# Future

![](https://static.igem.wiki/teams/4702/wiki/result-for-notebook/e310b35721f23d15526cc50af17868d.png)

# Future from Our Tests and Other Prospects

For the results of our tests, one thing we could do for the alternative of the scFv-streptavidin-his
design is to remove the streptavidin in the design but using chemical method to link the biotin to the
antibody, and then link the biotinylated antibody to the avidin linked iron oxide nanoparticles.

One promising direction for future work is to optimize the design of plasmids to enable the expression
terminal of antibodies to be located at the same site as the IONPs. This strategic modification would
facilitate the self-assembly of antibodies and IONPs if the process is made possible in the future,
potentially leading to more efficient and targeted
delivery systems. By incorporating functional groups into the plasmid design, we could enhance
the binding affinity between the antibodies and IONPs, thereby improving the overall performance of the
nanocomplex.

The successful self-assembly of antibodies and IONPs holds great potential for large-scale production.
With this advancement, we could streamline the manufacturing process and significantly increase
the efficiency of nanoparticle production. Additionally, the ability to self-assemble the components may
offer new opportunities for tailoring the properties of the nanocomplex, such as controlling the release
kinetics of drugs or enabling specific interactions with biological targets.

The present development in biologically synthesized IONPs offers promising avenues for future research. By
further refining the coating composition, understanding the synthetic mechanism, and optimizing plasmid
design, scientists are paving the way for improved efficiency, stability, and functionality of these
nanoparticles. Ultimately, these advancements hold great potential for revolutionizing various fields,
including targeted drug delivery, biomedical imaging, and therapeutics.

{% endblock %}

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

Experiments

Click!Read More

---

* [Competent Cell Preparation](#1)
* [Competent Cell Transformation](#2)
* [Culture Development for Protein Extraction](#3)
* [His-tagged Protein Purification](#4)
* [Protein Electrophoresis](#5)
* [BCA Test](#6)
* [Nanoparticle Biosynthesis](#7)
* [NHS-PEG-Maleimide Linking](#8)
* [EDC/NHS Conjugation](#9)
* [Biologically Synthesized Nanoparticle Separation for TEM Analysis](#10)
* [Cell Culture Development](#11)
* [CCK-8 Cytotoxicity Testing](#12)
* [Western Blot](#13)
* [Author & Reference](#14)

---

# Competent Cell Preparation [[1]](#a1)

---

Material

1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.

2. Stock Ampicillin

3. 0.1M MgCl2

4. 0.1M CaCl2

5. Glycerol Stock Bacterial Cells (SHuffle strain in our case)

Day 1

1. Take out one tube of frozen glycerol stock bacterial cells from -80ºC storage. (SHuffle strain in our
case)
2. Thaw the cell and spread onto an antibiotic free LB plate.
3. Grow the plate overnight at 37°C.

Day 2

1. Pick up a single colony of SHuffle strain from fresh LB plate

2. Inoculate a 5 mL starter culture of LB without antibiotics.

3. Grow culture at 37°C in shaker at 220 rpm overnight.

Day 3

1. Inoculate 150 mL of LB media with 1.5 mL starter culture (1:100) and grow in 37°C shaker.

2. Measure the OD600 till it reaches 0.4-0.6(no exceeding 0.6), collect the culture in 50 ml
centrifugation tubes. Then immediately put the cells on ice. Chill the culture on ice for 20-30 minutes,
swirling
occasionally to ensure that the cultrue is evenly cooled.

3. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C.

4. Decant the supernatant and gently resuspend the pellet in 50 mL of pre-chilled 0.1M MgCl2.
Then keep it on
ice for 20 minutes.

5. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C. Decant the supernatant and
resuspend the pellet in 50 mL of pre-chilled 0.1M CaCl2. Keep this suspension on ice for 20
minutes.

6. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C. Decant the supernatant and
resuspend the pellet in 5 mL of pre-chilled ice cold.0.1M CaCl2,15% glycerol solution.

7. Aliquot 100 μL compentent cell into sterile 1.5 mL microfuge tubes and snap freeze with liquid
nitrogen. Store frozen
cells in the -80°C freezer.

# Competent Cell Transformation [[2]](#a1)

---

Material

1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.

2. Stock Ampicillin

3. Glycerol Stock Bacterial Cells (SHuffle strain in our case)

4. 10 ng vectors for transformation (pGLO and genetically modified pGLO in our case)

5. LB plate with anphicilin (0.1 g/L in our case)

Day 1

1. Take out one tube of frozen glycerol stock competent bacterial cells from -80ºC storage. (SHuffle
strain in our case)

2. Thaw the cell on ice untill little crystalloid could be observed in the tube.

3. Add 10 ng vectors for transformation into the tube.

4. Heat shock at 42ºC water bath for 90 seconds.

5. Keep on ice for two minutes.

6. Add 500 μL antibiotic free LB broth into the 1.5 mL EP tube. Incubate the tubes for 30 minutes at
37ºC shaker at 220 rpm to
allow antibiotic resistance gene expressed.

7. Concentrate the cell by conducting centrifugation at 4000 rcf for one minute.

8. Discard the supernatant and leave around 100 μL cell culture, spread the cell onto LB plate with
corresponding antibiotics. (Ampicillin in our case)

9. Incubate the plate at 37ºC.

Day 2

1. Pick up a single colony of SHuffle strain from LB plate into 5mL antibiotics containing liquid LB medium.
Incubate in 37ºC shaker at 220 rpm for 8 hours.

2. Send the culture for sequencing.

# Culture Development for Protein Extraction [[3]](#a1)

---

Material

1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.

2. Stock Ampicillin

3. 20% Arabinose solution (mass fraction)

4. Stock bacteria culture (SHuffle strain in our case)

Day 1

1. Taking 50 μL culture from 5 mL antibiotics containing liquid LB medium into new 5 mL antibiotics
containing liquid LB medium. Incubate at 37ºC in the shaker at 220rpm overnight. (If the original culture
has been stored in the 4ºC for some days, otherwise if directly following the competent cell transformation
step, start from Day 2)

Day 2

1. Prepare the autoclaved LB medium, for 500 mL Erlenmeyer flask, filled with 150 mL LB medium, for 1 L
Erlenmeyer flask, filled with 250 mL LB medium.

2. Add ampicillin to the medium to make the concentration to be 0.1g/L.

3. Add bacteria culture using 1:100 dilution by volume. (For 150 mL LB medium add 1.5 mL bacteria strain
prepared in Day 1)

4. Incubate at 37ºC in the shaker at 220 rpm untill the OD600 of the culture reach 0.4-0.6.

5. Immediately add 20% Arabinose solution (mass fraction) using 1:100 dilution by volume. (For 150 mL LB
medium add 1.5 mL 20% Arabinose solution prepared as stock solution, filtrate through 0.22 μL filter
membrane)

6. Incubate at 37ºC in the shaker at 220 rpm for 8 hours or at 18 ºC in the shaker at 220 rpm for 16-18
hours.

Day 3

1. Distribute the culture into 50 mL centrifugation tube and collect the cell by centrifuge at 4000 rcf for
20 minutes.

2. If not broken immediately after this step, discard the supernatant and store the cell pellet at -80
ºC refrigerator.

# His-tagged Protein Purification [[4]](#a1)

---

Materials

1. **Buffer A**:
   150 mM NaCl,
   20 mM Tris-HCl, pH =8.0
2. High-pressure homogenizer (Union-Biotech(shanghai)Co., Ltd.)
3. Filtration membrane (0.45 μm, Millipore, Catalog number: SLHPR33RB)
4. AKTA purification system (Union-Biotech(shanghai)Co., Ltd.)
5. HisTrapTM HP His tag protein purification column (Cytiva, Catalog number:17524701)
6. **Buffer B**:
   150 mM NaCl,
   20 mM Tris-HCl,
   1 M imidazole, pH adjusted to 8.0 by HCl
7. PMSF (Phenylmethylsulfonyl fluoride)

Procedure

1. Prepare the buffer system using Buffer A as substrate, and add extra PMSF untill a final concentration of
0.1 mM. (Buffer A components: 150 mM NaCl, 20 mM Tris-HCl pH= 8.0)

2. Resuspend the cell pellet with around 10 mL buffer system prepared above for high pressure
homogenization. (For 150 mL culture system, homogenizing at 750 bar for 3 minutes, using homogenizer from
Union-Biotech(shanghai)Co., Ltd.)

3. Centrifugation at 11000 rcf for 30 minutes.

4. Collect the supernatant and filtrate through 0.45 μm filtration membrane for the preparation of
running AKTA purification system.

5. HisTrapTM HP His tag protein purification column (Catalog number:17524701) are applied onto the AKTA
system
(Union-Biotech(shanghai)Co., Ltd.). The program is set to pre-charge the flow path with 10 column volume
with Buffer A, then 12 column volume of the supernatant containing sample protein is applied onto the
column. After that apply 10 column volume buffer A to wash out the unbonded protein. Finally, apply the
linear gradient of the mixture of buffer A and buffer B (starting ratio is Buffer A: Buffer B = 70%:30%,
final ratio is Buffer A: Buffer B = 0%:100%) to wash out the his-tagged protein and collected.
(Buffer B components: 150 mM NaCl, 20 mM Tris-HCl, 1 M iminazole, pH adjusted to 8.0 by HCl) Then collect
eluted samples
by fixed volume collection with 1 mL per EP tube.

6. After that, conduct electrophoresis and Coomassie blue staining to see the purity and relative
concentration of the protein collected. Further, collect ones with high purity and concentration
through ultrafiltration to substitute the buffer B to buffer A with 0.1 mM PMSF added. And after the solvent
replacement, perform BCA test to tell the protein concentration. (For detailed information of this
step, see the protocol for electrophoresis, ultrafiltration and BCA test)

# Protein Electrophoresis [[5]](#a1)

---

Materials

1. 5x SDS-PAGE Sample Buffer
2. Protein samples
3. Running Buffer (SDS-PAGE 10× SDS Running Buffer: Tris base 30.3 g, Glycine 144.4 g, Sodium Dodecyl
   Sulfate 10 g
   Dissolve in 1 L of ddHO, composition could be different under different cases, dilluted
   before use)
4. Protein ladder
5. Electrophoresis system
6. Power supply
7. Coomassie blue dye

Note: Amount of protein sample used for electrophoresis could be adjusted according to the loading capacity
of the gel used under specific circumstance.

Procedure

1. Prepare 16ul protein samples and add 4ul 5x SDS-PAGE Sample Buffer to each protein sample.

2. Heat up the protein sample to 95ºC and keep for 10 minutes.

3. Prepare the electrophoresis chamber and running buffer. Take out required number of 12-well precast
gels and set
up the system. Fill the chamber with running buffer.

4. Load all the protein samples onto the gel. And add a protein ladder to one well to serve as a
molecular weight marker for reference.

8. Transfer all the rest of the running buffer into the chamber and making sure that the gel is
completely
submerged.

9. Connect the power supply to the electrophoresis system and run the system by 80 V for 20 minutes to
check if the circuit is well developed. Then switch the voltage to 140 V and run for another 1 hour. The
specific running time will depend on the size of the gel and the separation requirements of specific
experiment. Generally, stop running when the dye front reaches the bottom of the gel or reaches a desired
distance.

10. Take down the chamber and wash the gel with deionized water by snap-boiling the system in micro-wave
oven twice.(Turn down the micro-wave immediately when the deionized water boil)

11. Stain the gel using Coomassie blue dye for 30 minutes.

12. Decolor the gel with deionized water by snap-boiling the system in micro-wave oven multiple times
when background coloring is insignificant.

13. Observe the bands to see the purity of the purified protein.

# BCA Test [[6]](#a1)

---

Our BCA test are conducted by using [Enhanced BCA
Protein Assay Kit from Beyotime (Catalog number: P0009)](https://www.beyotime.com/product/P0010.htm).

Standard Instruction protocol could be found in the [link](https://www.beyotime.com/Manual/P0009_Enhanced%20BCA%20Protein%20Assay%20Kit.pdf)

And we strictly follow the instruction given by Beyotime.

# Nanoparticle Biosynthesis [[7]](#a1)

---

Materials

1. SHuffle-pGLO bacteria single colony
2. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.
3. Stock Ampicillin
4. 0.5 M FeCl3 solution
5. 0.22 μL filter membrane

Day1

Pick up a single colony of SHuffle-pGLO bacteria from LB plate (with 0.1 g/L ampicillin) into 5 mL LB
Broth (0.1 g/L ampicillin). Incubate at 37ºC in the shaker at 220rpm overnight.

Day2

1. Prepare the autoclaved LB medium. For 500 mL Erlenmeyer flask, fill with 150 mL LB medium. For 1 L
Erlenmeyer flask, fill with 250 mL LB medium.

2. Add ampicillin to the medium to make the concentration to be 0.1g/L.

3. Add bacteria culture using 1:100 dilution by volume. (For 150 mL LB medium, add 1.5 mL bacteria
strain
prepared in Day 1)

4. Incubate them at 37ºC in the shaker at 220 rpm untill the OD600 of the culture reaches
0.1.

5. Immediately add FeCl3 solution to a final concentration of 5 mM Fe3+. (In our
case, 0.5 M FeCl3
solution are prepared as stock solution which was filtrated through
0.22 μL filter membrane, and was added with a 1:100 dilution)

6. Incubate at 37ºC in the shaker at 220 rpm for one day in 37ºC.

Day3

1. Distribute the culture into 50 mL centrifugation tubes and collect the cell by centrifuge at 4000 rcf for
20 minutes.

2. If they don't break immediately after this step, discard the supernatant and store the cell pellet in
the -80
ºC refrigerator.

# NHS-PEG-Maleimide Linking Protocol [[8]](#a2)

---

Material

1. NHS-PEG-Maleimide (MW 3400)

2. DMSO

3. 3kDa Ultrafiltration tube (Catalog number: UFC9003, Millipore)

4. 10kDa Ultrafiltration tube (Catalog number: UFC5010BK, Millipore)

5. Stock Nanoparticle solution

Preperation

1. Dissolve 0.05/0.10g NHS-PEG-Maleimide in DMSO with a concentration of 100µM and store

Primary Part

1. Mix maleimide solution and nanoparticle solution with a volume ratio 2:1, reacting
for 1 h at room temperature

2. Dilute the solution to a proper volume for ultrafiltration. Ultrafiltrate (3kDa), centrifuging at
5000g for at least 20 mins. Finish until the liquid volume above the filtrate is less than 1.5mL.

3. Collect the liquid above the filtrate and combine the solution with the liquid before, add 30 µg/mL
antibody solution the same volume as maleimide solution consumed before, reacting for 1.5 h at room
temperature

4. Ultrafiltrate (10 kDa), centrifuging at 14000g for at least 20 mins. Finish until the liquid above
the filtrate less than 100 µL.

5. Collect the liquid above the filtrate by converting the ultrafiltration tube, centrifuging at 1000g
for 1 min.

# EDC/NHS Conjugation [[9]](#a4)

---

Material

1. 0.1M PBS
2. 0.2M EDC·HCl
3. 0.2M NHS
4. 10kDa Ultrafiltration tube (Catalog number: UFC5010BK)
5. Stock Nanoparticle solution
6. Antibody solution

Primary Part

1) Transfer 200 μL of our bio-synthetic nanoparticles and 0.4mg (8μL in volume) chemo-synthetic
nanoparticles
to two eppendorf tubes.

 2) Add 200 μL 0.1M PBS to the second tube to make up for the difference

 3)Then add 200 μL 0.1M PBS to each of the tubes to mix it thoroughly.

 4)Then, add 60 µL of 0.2 M EDC·HCl and 120 µL 0.2 M NHS to each tube.

 5)After 10 min, add 30 μg antibody to the EDC/NHS-activated NPs solutions.

 6)After 24 h, transfer the solution to 10 KD ultrafiltration tubes and centrifuge for 20 min
at 14000 rpm to remove the unbounded antibody.

 7)Finally, collect the obtained antibody-conjugated NPs (IONPs-antibody) in EP tubes.

# Biologically Synthesized Nanoparticle Separation for TEM Analysis [[10]](#a1)

---

Materials

1. Deionized water
2. High-pressure homogenizer (Union-Biotech(shanghai)Co., Ltd.)
3. 100 kDa Ultracentrifugation tube (Catalog number: UFC9100, Millipore)
4. 3 kDa Ultracentrifugation tube (Catalog number: UFC9003, Millipore)
5. Buffer A:
   150 mM NaCl,
   20 mM Tris-HCl
6. Ultrasonic Cleaner

Procedure

1. Resuspend the cell pellet for each 50 mL centrifuge tube with around 10 mL deionized
water for high pressure homogenization. (For 150 mL culture system, homogenize at 750 bar for 3 minutes,
the broken cell system could be concentrated in one 50 mL centrifugation tube after homogenization, using
homogenizer from Union-Biotech(shanghai)Co., Ltd.)

2. Centrifugation at 11000 rcf under 4ºC for 30 minutes.

3. Discard the supernatant, add 10 ml deionized water, vortex the system for 1 minutes. (Notice that now
there should still have a lot iron salt precipitate at the bottom of the tube, while the cell debris should
already be resuspended, this could prevent jamming in the ultrafiltration step followed).

4. Carefully put the tube into an ice bath and then put the whole system into ultrasonic cleaners and
run the cleaners for 15 minutes.

5. Without disrupting the precipitate filtrate, the supernatant through 0.45 μm and 0.22 μm filtration
membranes
as a prevention for ultrafiltration membrane blockage, then move the filtrated supernatant into activated
100 kDa Ultracentrifugation
tube.

6. Centrifuge at 5000 rcf under 4ºC for 25 minutes. Note that if there still have more than 2 mL
liquids above the filtration membrane, centrifuge at 5000 rcf under 4ºC for another 20 minutes.

7. Discard the filtration membrane and carefully put the tube into an ice bath and then put the whole
system into ultrasonic cleaners and run the cleaners for 15 minutes.

8. Transfer the flow through into an activated 3 kDa Ultracentrifugation tube.

9. Centrifuge at 5000 rcf under 4ºC for 25 minutes. Note that if there still have more than 5 mL
liquids above the filtration membrane, centrifuge at 5000 rcf under 4ºC for another 20 minutes untill
there only have around 2 mL liquid above the filtration membrane.

10. Collect the liquid above the filtration membrane. Carefully put the tube into an ice bath and then
put the whole system into ultrasonic cleaners and run the cleaners for 15 minutes. This sample could be used
for TEM characterization.
Note: In this experiment design, we use deionized water for the solution system to avoid the interference of
mineral salt in TEM analysis. But if the target is to get nanoparticle with all membrane protein on the
surface biologically active, please use Buffer A to take place of deionized water.

# Cell Culture Development [[11]](#a3)

---

Material

1. DMEM High Glucose H-21 Media (Catalog number: 11965084)

2. RPMI 1640 basal Media (Catalog number: 12633012)

3. Fetal Bovine Serum (Catalog number:10099141C)

4. Penicillin, Streptomycin

5. Trypsin/EDTA

6. Dulbecco's Phosphate Buffered Saline

7. Cell strains

8. Tissue culture dish

9. 15 ml Falcon tube

10. 37ºC water bath

11. 37ºC 5% CO2 incubator

12. Insulin (Catalog number: I860440-25mg)

MDA-MB-231 (HER2 negative cells) Culture

Culture medium: 450 ml DMEM High Glucose H-21 Media, 50 ml (to 10%) [Fetal Bovine Serum (Catalog
number:10099141C)](https://www.thermofisher.cn/order/catalog/product/10099141C) , 5 ml Pen Strep

Thawing Cells:

 1. Take one vial immediately in 37ºC water bath. Transfer the content to a 15 ml Falcon tube already
containing 10 ml of fresh medium.

 2. Spin down at 1200 rpm for 3 min at 4ºC. Aspirate supernatant and resuspend cells in fresh medium
and transfer to 10cm tissue culture dish and cultured at 37°C in 5% CO2. Change medium about
every 3 days.

 Subculture

 After cell culture reaches 80-85% confluence, subculture is conducted.

 1. Remove the medium from the dish.

 2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline (without Ca2+ or
Mg2+)

 3. Add 1 ml of 0.05% Trypsin and trypsinize for 3-5 min at 37°C.

 4. Add 8 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down
4-5 times to detach the cells off.

 5. Spin down at 1200rpm for 3 min. Aspirate supernatant.

 6. Add 8 ml of medium to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture
ratio is about 1:10.

 7. Put the cells into 37°C with 5% CO2.

[Reference
Linkage](https://genome.ucsc.edu/ENCODE/protocols/cell/human/MDA-MB231-S_Farnham_protocol.pdf#:~:text=Open%20the%20vial%20and%20transfer%20the%20content%20to,and%20medium%20is%20changed%20about%20every%203%20days.)

BT474 (HER2 positive cells) Culture

Culture medium: 500 ml [RPMI
1640 basal Media](https://www.thermofisher.cn/order/catalog/product/12633012#:~:text=Advanced%20RPMI%20%28Roswell%20Park%20Memorial%20Institute%29%201640%20is,with%20no%20change%20in%20growth%20rate%20or%20morphology.), 100 ml (to about 20%) [Fetal Bovine Serum](https://www.thermofisher.cn/order/catalog/product/10099141C), 5 ml
Penicillin, Streptomycin, 2μg/ml insulin

Thawing Cells:
1. Take one vial immediately in 37℃ water bath. Transfer the content to a 15 ml Falcon tube already
containing 8 ml of fresh medium.

 2. Spin down at 1000 rpm for 3 min. Aspirate supernatant and resuspend cells in fresh medium and
transfer
to 10cm tissue culture dish and culture at 37°C in 5% CO2. Change medium about every 2 days.

Subculture

After cell culture reaches 70-75% confluence, subculture is conducted.

 1. Remove media from dish.

2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline.

3. Add 1 ml of trypsin/EDTA for about 10 min at 37°C.

4. Add 6 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down
4-5
times to detach the cells off.

5. Spin down at 1200rpm for 3 min. Aspirate supernatant.

6. Add 6 ml of media to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture
ratio
is about 1:3/1:4.

7. Put the cells into 37°C with 5% CO2.

[Reference Linkage](https://www.atcc.org/products/htb-20)

# CCK-8 Cytotoxicity Assay [[12]](#a3)

---

Material

1. Cell Counting Kit-8 (CCK-8) (Catalog number: ab228554, Abcam)

2. 96-well plate

3. 37ºC, 5% CO2 incubator

4. 37ºC water bath

5. 96-well plate reader

6. WST-8 Solution

Primary Steps

1. Plate 10,000 cells in 100 µl per well for a 96 well plate

2. Add test compounds into cells and incubate for 24 hours in a 37ºC, 5% CO2
incubator.

 3. For blank wells (medium without cells), add the same amount of test compounds.

4. Add 10 µl/well (96 well plate) or 5 µl/well (384-well plate) of WST-8 Solution to each well.
Protect from the light and incubate for 1-4 hours at 37ºC.

5. Measure the absorbance increasement at 460 nm.

[Reference
1](https://www.abcam.com/ps/products/228/ab228554/documents/Cell-Counting-Kit-protocol-book-v2d-ab228554%20%28website%29.pdf)
[Reference
2](https://www.abcam.com/products/assay-kits/cell-counting-kit-8-wst-8--cck8-ab228554.html)

# Western Blot Analysis [[13]](#a1)

---

Material

1. SDS-PAGE gel

2. PVDF membrane

3. BSA blocking solution

4. TBS-T solution

5. Primary antibody

6. Secondary antibody

7. Exposing reagent

8. Electrophoresis system

9. Power supply

10. Protein ladder

Primary Steps

1. Prepare the SDS-PAGE gel and electrophoresis system.

2. Prepare the protein samples and add 5x SDS-PAGE Sample Buffer to each protein sample.

3. Heat up the protein sample to 95ºC and keep for 10 minutes.

4. Load all the protein sample onto the gel. And add a protein ladder to one well to serve as a
molecular weight marker for reference.

5. Transfer the protein onto PVDF membrane.

6. Block the membrane with 1% BSA in TBS-T solution for 1 hour.

7. Incubate the membrane with primary antibody at 4ºC overnight.

8. Wash the membrane with TBST for 3 times, 10 minutes each time.

9. Incubate the membrane with secondary antibody at room temperature for 1 hour.

10. Wash the membrane with TBST for 3 times, 10 minutes each time.

11. Develop the membrane with exposing reagent and observe the bands.

# Author & Reference

### Author

* Shuoyi HU:
  [[1]](#1)
  [[2]](#2)
  [[3]](#3)
  [[4]](#4)
  [[5]](#5)
  [[6]](#6)
  [[7]](#7)
  [[10]](#10)
  [[13]](#13)
* Songnan REN:[[8]](#8)
* Shijie HE:[[9]](#9)
* Yixiao XIAO:
  [[11]](#11)
  [[12]](#12)

{% endblock %}

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

Results

Click!Read More

---

* [Summary](#1)
* [TEM Characterization of IONPs](#2)
* [DLS/Zeta Potential Characterization of IONPs](#3)
* [Impact of Induction Concentration and Induction Time on IONPs](#4)
* [Conjugation Examination of scFv Domain](#5)
* [Cytotoxicity Examination of the IONPs](#6)
* [Protein Purification](#7)
* [Author Contribution](#13)

---

# Summary [[1]](#a1)

---

Explanation about Parameters Tested by Us

Since we are a team that focus on the biosynthesis and application of
the Iron Oxide Nanoparticles, specifically the specific targeting of the
antibody linked Iron Oxide Nanoparticles to the HER2 positive cell,
following important things were tested by us:

1. Whether the bacteria strain chosen by us could properly
synthesize the Iron Oxide Nanoparticles, and which magnetic state it would be in,
what would the average diameter and polydispersity of the biologically
synthesized nanoparticles be, whether the method we used to separate IONPs was good.
This should be tested by TEM and DLS analysis
after using the method we designed to separate the nanoparticles.

2. Whether the scFv domain of the anti-HER2 antibody could be properly
expressed in the bacteria strain chosen by us, and whether the protein could be
biologically active after purification (which means that binding with HER-2 positive
cells should occur). This should be tested by immunostaining, flow cytometry
analysis and western blot analysis by setting proper comparison
groups and conduct the same staining to HER-2 negative cells.

3. Whether the biologically synthesized nanoparticles could be conjugated with
the scFv domain of the anti-HER2 antibody effectively. This should be tested also by
immunostaining if the second task could be proven. Or we could conduct the conjugation
method on the chemically synthesized nanoparticles which shows significant cytotoxicity,
and compare the result with unlinked groups to indirectly show both the biological activity
of scFv synthesized by us and the success of the conjugation methods.

Major Achievements

1. We successfully synthesized the Iron Oxide Nanoparticles (IONPs) *E. coli*
and the method used to separate IONPs are designed and tested by ourselves.

2. We successfully characterized the IONPs using TEM and DLS and found that using
ok our method, the IONPs got by us were generally in superparamagnetic state and suitable for
application need magnetic induction and won't let particles aggregrate.

3. We successfully synthesized the scFv domain of anti HER2 antibody in *E. coli*
and purified the protein using His-tag purification column.

4. We showed that the biologically synthesized IONPs have no obvious cytotoxicity to both the HER2
positive cells and the HER2 negative cells, which might be caused by its low concentration. In comparison,
the bare chemically synthesized IONPs show obvious cytotoxicity to both the HER2 positive cells and the HER2
negative cells. Also, the linkage of antibodies with scFv domain to the chemically synthesized IONPs could
give them specificity against HER2 positive breast cancer cells. This indicates that the biologically
synthesized nanoparticles are better for application if higher concentration and good specificity could be
achieved in the future.

Approaches Without Success / Unexplained Confusion in Data We Got

1. We tried to use the immunostaining to examine the conjugation of the scFv domain
to the HER2 positive cell. But the result shows that the
[anti-polyhis antibody](https://www.sigmaaldrich.cn/CN/zh/product/sigma/sab4200620)
doesn't have much specificity to the primary antibody, indicating we couldn't
test the conjugation of the scFv domain to the HER2 positive cell using
this method or to say this antibody.

2. Now the binding affinity of the antibody designed by us could only be
partially given from the cytotoxicity examination, which is not quite convincing.

3. In one of our DLS characterization, the hydrodynamic diameter of the
nanoparticles were much greater than other repeats through exactly
the same treatment, which is unexpected. And the only difference observed by us is that,
only this time there did exist partially ropy stuff
(like crude DNA extract) in the supernantant after
centrifugation of the cell lysate. We are not sure whether this is directly
related to the unusual hydrodynamic diameter.
One assumption of us is that
since the viscosity of the sample we got that time is much larger,
that would affect the speed of the brownian motion, thus affect the final
hydrodynamic radius measured.

Future Work

1. As the existance of the biologically synthesized nanoparticles was proven,
we could further examine the coating composition by using Surface Plasmon Resonance or
Raman spectroscopy to see if the predicted composition or chemical groups
do exist on the membrane, this could further help us design new conjugation methods
towards both antibodies and drugs.

2. We could use the same conjugation method to further attach drugs like Doxorubicin to the
biologically synthesized nanoparticles to see if the cytotoxicity could be
increased, and further examine the specificity of the antibodies.

3. We could renew our antibody design by expanding the design to outside the scFv domain,
as full structure of the antibody may improve the biological activity of the antibody.
But since the supporter of protein expression chose by us was still bacteria,
the proper folding of the whole protein could be unreliable.
So we could express the protein in eukaryote cells like 293F.
(one type of human embryonal kidney (HEK) cells)

4. We could further examine the conjugation of the scFv domain to the HER2
positive cell by attaching a fluorescent group to the scFv domain and directly
stain the cell to see if the conjugation is successful.

# TEM Characterization of Biologically Synthesized Nanoparticles [[2]](#a1)

---

Method Description

In our examination,
[Talos L120C G2 Transmission Electron Microscope](https://www.thermofisher.com/us/en/home/electron-microscopy/products/transmission-electron-microscopes/talos-l120c-tem.html) was used for the characterization of the
nanoparticles.

Basically, this microscope utilizes a beam of electrons instead of light to visualize the sample,
enabling incredibly
detailed images with a resolution down to the sub-nanometer scale.

To begin the analysis, we prepare a sample of the nanoparticles by depositing a thin layer onto a
carbon-coated copper
grid. This grid is carefully loaded into the Talos L120C G2 TEM, ensuring proper positioning for imaging and
analysis.

Once the sample is inserted, we set the imaging parameters according to our specific requirements. The
TEM employs a
high-energy electron beam that is directed onto the sample. As the electrons interact with the
nanoparticles, they
scatter and interact with the internal structure of the particles.

We observe and capture the scattering patterns produced by the interaction of the electrons with the
nanoparticles.
These patterns are collected by a detector and transformed into high-resolution images, providing us with
detailed
information about the morphology, size, and distribution of the nanoparticles.

In addition to imaging, we also utilize Selected Area Electron Diffraction (SAED) analysis with the
Talos
L120C G2 TEM. By selecting a specific area of interest on the sample using an aperture, we focus the
electron beam
onto that region. The resulting diffraction pattern reveals the crystalline properties and orientation of
the nanoparticles,
enabling us to determine their structural characteristics.

They provide us with valuable information about the size, shape, and distribution of the nanoparticles,
as well as give us one way to compare the sample with uninduced control to show the success of the synthetic
process.

Results / Data of 5 mM FeCl3 Induced Culture

The representative TEM figures of the biologically synthesized nanoparticles and the negative control with
no ferric ion induction are shown below.

![TEM Result for Biologically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/results/tem-characterization-of-biologically-synthesized-nanoparticles/0009-20230825-1107-92000-x-ceta.jpg)

**Figure 1.** Biologically Synthesized Nanoparticle by 5 mM FeCl3 Induction Attached to
Biomass.

![TEM Result for Biologically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/results/tem-characterization-of-biologically-synthesized-nanoparticles/0017-20230823-1011-190-kx-ceta.jpg)

**Figure 2.** Biologically Synthesized Nanoparticle by 5 mM FeCl3 Induction Detached from
Biomass
through ultrasonication.

![TEM Result for Negative Control](https://static.igem.wiki/teams/4702/wiki/results/tem-characterization-of-biologically-synthesized-nanoparticles/0009-20230825-0939-310-kx-ceta.jpg)

**Figure 3.** Negative Control for Nanoparticle without Ferric Ion Induction.

To get the size distribution of the nanoparticles, we use ImageJ to analyze the TEM images.
Multiple figures were chosen for data processing. In total fifty to seventy particles were randomly selected
in each figure,
the size distribution statistical chart of the nanoparticles is shown below.

![Biologically Synthesized Nanoparticle Size Distribution](https://static.igem.wiki/teams/4702/wiki/results/tem-characterization-of-biologically-synthesized-nanoparticles/biologically-synthesized-nanoparticle-distribution-figure.jpg)

**Figure 4.** Size Distribution of the Biologically Synthesized Nanoparticles.

From the size distribution statistical data, the Polydispersity Index of the samples in each view field
was calculated and shown as below:

![Polydispersity Index of the Samples in Each View Field](https://static.igem.wiki/teams/4702/wiki/results/tem-characterization-of-biologically-synthesized-nanoparticles/pdi-samples.jpg)

**Figure 5.** Polydispersity Index of the Samples in Each View Field.

All the figures used for data processing are labelled and shown in the supplementary data section
below.

Analysis / Discussion

Firstly, the comparison between ferric induced culture and uninduced culture shows the success of the
biological
synthesis of the nanoparticles. There are no nanoparticles observed in the uninduced culture, (the faint
grain in
the TEM figures for uninduced culture were actually some inorganic salt as when electron-beams were fired
to the sample,
the salt grain would disappear), while the induced culture shows a large amount of nanoparticles
attached to the biomass or off the biomass.

Secondly, the size distribution of the nanoparticles shows that the nanoparticles are in the
range of 5-10 nm, this means that
almost all the nanoparticles synthesized by us are in superparamagnetic state, indicating the
applicability of biologically synthesized nanoparticle in tumor
penetration or tumor accumulation.

Thirdly, the comparison between the sample attached to the biomass and the sample detached from
the biomass shows
that in separation of the biological synthesized nanoparticles, ultrasonication chosen by us is a proper
method to detach
the nanoparticles from the biomass.

Fourthly, the Polydispersity Index of the samples in each view field shows that the
nanoparticles are relatively uniform in size.

Selective Electron Diffraction Results and Analysis

Beyond simply observing the morphology of the nanoparticles, we also utilize
Selected Electron Diffraction analysis to gain a deeper understanding of their
structural properties.
The raw data of the Selected Electron Diffraction analysis of the biologically
synthesized nanoparticle was shown below:

![Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/result/results/0035-20230823-1123-420-mm-ceta.jpg)

**Figure 6.** Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticles Taken
in 8/23

![Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/result/results/0016-20230825-1132-530-mm-ceta.jpg)

**Figure 7.** Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticles Taken
in 8/25

The result shows that the sample is a type of polycrystal with 6 crystal faces. The crystal faces from
inner to the outer are numbered 1 to 6. The distance between each layer is 3.6nm, 1.03nm, 0.42nm, 0.65nm,
0.36nm. It means that the samples don't have a uniform structure and the specific crystal type of our
sample can't be further analyzed. Maybe it is because there still remains some biological structures
attached on our samples which corresponds to the layer 3,4,5,6 on the picture. More accurate separation
process could be done to further separate the unwanted biological structures. In all it proved the
existance of crystal structure in our samples.

Supplementary Datas

The supplementary figures and statistical information of the TEM characterization of the biologically
synthesized nanoparticles could be found in the pdf file below.

TEM Characterization of Biologically Synthesized
Nanoparticles and Size Distribution Statistics

# DLS and Zeta Potential Characterization of Biologically Synthesized Nanoparticles and Zeta Potential Measurement [[3]](#a1)

---

Method Description

For the DLS characterization and Zeta Potential measurement of the biologically synthesized
nanoparticles, we use
[Brookhaven NanoBrook
Series](https://www.brookhaveninstruments.com/product/nanobrook-series/).

This advanced instrument utilizes Dynamic Light Scattering (DLS) technology, which allows us to
measure the
size distribution of nanoparticles in a liquid suspension. By analyzing the fluctuations in the
intensity of scattered
light caused by the random motion of nanoparticles, we can obtain valuable information about their size,
size distribution, and polydispersity.

When it comes to analyzing our biologically synthesized nanoparticles, the NanoBrook Series offers a
user-friendly and efficient workflow. Firstly, we prepare a sample of our nanoparticles in a suitable
liquid
medium, ensuring that the sample is well-dispersed. Then we introduce the sample into the NanoBrook
instrument,
where it is carefully illuminated with a laser beam.

The scattered light from the nanoparticles is collected and analyzed, allowing us to obtain
valuable data
on the size distribution of the particles. This information is crucial for evaluating the quality of our
synthesized
nanoparticles and understanding their behavior in biological systems.

Besides, the NanoBrook Series enables us to measure the Zeta Potential of our nanoparticles, \
which is a key parameter that provides insights into the stability
and surface charge of nanoparticles. Also, by assessing the electric potential at the particle's
surface, we can gain
a deeper understanding of their interactions with their environment.

Results / Data of 5 mM FeCl3 Induced Culture

**DLS Analysis**

The size distribution pattern of the biologically synthesized nanoparticles using log scale is shown
below:

![Biologically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/bio-dls.jpg)

**Figure 1.** Biologically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.

From the size distribution pattern, the average hydrodynamic diameter of nanoparticles is 7.23 nm.

The size distribution pattern of the chemically synthesized nanoparticle using log scale is
shown below:

![Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/chem-dls.jpg)

**Figure 2.** Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.

From the size distribution pattern, the average hydrodynamic diameter of nanoparticles is 252.41 nm.

Actually the real size of the chemically synthesized nanoparticles by TEM analysis is around 20 nm,
but
the
hydrodynamic diameter of the nanoparticles sythesized by us is much larger than their real size due to the
aggregration of the nanoparticles,
this also indicates that the chemically synthesized nanoparticles
are not that stable and are easier to get precipitated out.

For clearer representation, the linear scale size distribution pattern of the chemically synthesized
nanoparticles is shown below:

![Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/chem-linear-scale-dls.jpg)

**Figure 3.** Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.

**Zeta Potential Measurement**
For biologically synthesized nanoparticles, the Zeta Potential measured
by us shows an average of -21.52 mV, which is larger than the chemically
synthesized nanoparticles in its absolute value .
Raw data of the Zeta Potential measurement of the biologically synthesized nanoparticles is shown below:
![Zeta Potential Measurement of the Biologically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/result/results/zeta-biooo.jpg)

**Figure 4.** Zeta Potential Measurement of the Biologically Synthesized Nanoparticles.

And the supplementary all field data of the Zeta Potential
measurement of the biologically synthesized nanoparticles is
shown in the pdf file below:

Zeta Potential Measurement of the Biologically Synthesized
Nanoparticles

As a comparision, for the chemically synthesized nanoparticle, the Zeta Potential measured
by us shows an average of -20.26 mV, which is smaller than the biologically
synthesized nanoparticles in its absolute value.
Raw data of the Zeta Potential measurement of the chemically synthesized nanoparticles is shown below:
![Zeta Potential Measurement of the Chemically Synthesized Nanoparticle](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/chem-zeta.jpg)

**Figure 5.** Zeta Potential Measurement of the Chemically Synthesized Nanoparticles.

And the supplementary all field data of the Zeta Potential
measurement of the chemically synthesized nanoparticles is
shown in the pdf file below:

Zeta Potential Measurement of the Chemically Synthesized
Nanoparticles

Analysis / Discussion

Firstly, for the DLS result of biologically synthesized nanoparticles, the results give the
average hydrodynamic diameter to be 7.23 nm, which is in superparamagnetic state and not
easy to form aggregation. This is consistent with the result of TEM analysis.

While for the chemically synthesized nanoparticles, the results give the average hydrodynamic
diameter to be 252.41 nm, which is much larger than the real size of the
nanoparticles, indicating that the nanoparticles are easier to form aggregation and not easy to
get separated by untrasonic treatment. This is also consistent with the result of TEM analysis.

Though here we only have one biologically synthesized nanoparticle sample tested,
the consistent result of three samples from 10/2 showed good reproducibility of
the biological synthesis process, separation method and feasibility of the synthetic process.

Secondly, for the Zeta Potential measurement of the biologically synthesized nanoparticles,
the average Zeta Potential is -21.52 mV, in absolute value larger than
the zeta potential of chemically synthesized nanoparticles.

As the Zeta Potential is a parameter that indicates the stability of the nanoparticles,
the larger absolute value of the Zeta Potential of the biologically synthesized nanoparticles
indicates that the biologically synthesized nanoparticles are more stable than the chemically
synthesized nanoparticles. This result was also shown good reproductbility by comparing
with results in the other days.

# Characterization of Nanoparticles Synthesized by Different Times and in different Ferric Ion Induction Concentrations [[4]](#a1)

---

Description

To optimize the large scale biological nanoparticles synthesis, we tested the effect of
different ferric ion induction concentrations and different induction time on the nanoparticles synthesis.
Three time gradient including 24 hours, 36 hours and 48 hours were tested, and four ferric ion
induction concentration gradient including 0 mM, 2 mM, 5 mM and 7.5 mM were tested. These designs came from
our preliminary test that iron concentration over 10 mM would greatly inhibit the growth of the bacteria
and its protein expression level.

For each group, the general processes were controlled to be the same as the protocol
written in our experiment page. And characterizations were conducted by TEM and DLS analysis as introduced
above.

Iron Concentration Gradient

By DLS and Zeta Potential measurement, we found the optimized concentration of ferric ion for nanoparticle
synthesis is 2.5 mM,
as no significant difference in average hydrodynamic diameter and polydispersity index was found between 2.5
mM
and 5 mM induced group but the absolute value of zeta potential of 2.5 mM induced group is
higher than 5 mM induced group, indicating a higher stability. Summarized data were shown in the following
figures:

![hydrodynamic summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/hydrodynamic-diameter-1.jpg)

**Figure 1.** Hydrodynamic diameter summary figure

![polydispersity index summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/polydispersity-1.jpg)

**Figure 2.** Polydispersity index summary figure

![zeta potential summary figure](https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/zeta-potential-1.jpg)

**Figure 3.** Zeta potential summary figure

Time gradient

Here as the Concentrations of the samples were too low, we failed to get reliable zeta potential
data.
And the hydrodynamic diameter and polydispersity index data were shown below:

![hydrodynamic diameter summary figure](https://static.igem.wiki/teams/4702/wiki/results/results/bio-nps-diameter-cell-lysate-sup.jpg)

**Figure 4.** Hydrodynamic diameter summary figure

![polydispersity index summary figure](https://static.igem.wiki/teams/4702/wiki/results/results/bio-nps-polydispersity-cell-lysate-sup.jpg)

**Figure 5.** Polydispersity index summary figure

Here we could see that the diameter of the IONPs varies according to different induction time,
fulfilling
different requirements for different usage. And as for specific targeting with
the requirement of superparamagnetism, 24 hours induced group is the best choice.

Analysis

Unfortunately, the vacuum facilities of the TEM machine in our school was broken
during the time we were doing the final characterization for iron concentration gradient
and time gradient, so we could not get the TEM images for these two groups.

For the ferric ion gradient examination, the hydrodynamic diameter of all groups were
larger than the expected data and the former data we got. As said in the summary above, there
were one unusual phenomenon that after the centrifugation of the cell lysate, there were
some ropy stuff (like crude DNA extract) in the supernantant. Since the viscosity of
this is much larger than water, would affect the speed of the brownian motion.

But the result of the zeta potential measurement still show significant difference between samples
with negative control and samples with ferric ion induction, indicating success of the synthesis.

And as all four samples shown similar phenomenon when conducting nanoparticle separation,
and the negative control for 0 mM ferric ion induction group have zeta potential of nearly zero,
such that the trend could still give a preliminary result that 2.5 mM
is the optimized concentration for nanoparticle synthesis as the
zeta potential and polydispersity of 2.5 mM
induced group is the lowest among all the induced groups.

# Examination of the Conjugation with scFv Domain to the HER2 Positive Cell [[5]](#a1)

---

Method Description

Basically, we used the immunostainning to examine the conjugation of the scFv domain to the HER2
positive cell.

As we only synthesized the scFv domain of the anti-HER2 antibody with his-tag attached to
the end, we could not
directly find a secondary antibody with fluorescent tag to bind to the scFv domain. Therefore,
we use the mouse
derived anti-polyhis antibody to attach the scFv domain synthesized by us and then use the
tertiary goat derived
anti-mouse antibody to label the anti-polyhis antibody.

For convenience of explanation, here we called the scFv domain to be the primary antibody, the
[anti-polyhis antibody](https://www.sigmaaldrich.cn/CN/zh/product/sigma/sab4200620)
to be the secondary antibody and the [anti-mouse
antibody](https://www.abcam.com/products/secondary-antibodies/goat-mouse-igg-hl-alexa-fluor-647-ab150115.html) to be the tertiary antibody.

Generally, to test the conjugation, we added these three antibodies to the HER2 positive cell
and HER2 negative cell.
And to see if the secondary antibody has relatively good specificity to the primary
antibody, we also added one group
that only had the secondary antibody and the tertiary antibody to the HER2 positive cell and
HER2 negative cell.
Also, the background and unstained fluorescent signal were also tested by two groups, one is
only added the primary and secondary
antibody to the HER2 positive cell and HER2 negative cell, the other is only added the secondary
and
tertiary antibody to the HER2 positive cell and HER2 negative cell.

Final results were tested by the confocal microscopy and flow cytometry analysis to see the mean
fluorescence intensity of the cells.

We have the detailed design of the test as below:

| Cell Strain | Treatment of the cells | | |
| --- | --- | --- | --- |
|  | Anti HER2 scFv domain antibody | Anti His tag antibody (mouse derived) | Goat Anti-Mouse-IgG |
| SK-BR-3 treatment 1  (three replica) | - | + | + |
| SK-BR-3 treatment 2 (three replica) | + | + | + |
| SK-BR-3 treatment 3 (three replica) | + | + | - |
| SK-BR-3 treatment 4 (three replica) | + | - | + |
| MDA-MB-231 treatment 1  (four replica) | - | + | + |
| MDA-MB-231 treatment 2  (three replica) | + | + | + |
| MDA-MB-231 treatment 3 (two replica) | + | + | - |
| MDA-MB-231 treatment 4 (two replica) | + | - | + |

Result

Sadly, the results of the confocal microscopy and flow cytometry analysis show that the
secondary
antibody don't have much specificity to the primary antibody, indicating we couldn't test the
conjugation of
the scFv domain to the HER2 positive cell using this method. The group only added the
secondary and
tertiary antibody also shows strong fluorescent signal, which should not occur if the secondary
antibody is a
good one.

The mean fluorescent intensity of the SK-BR3 under different treatments are shown
below:

![Mean Fluorescent Intensity of the SK-BR3 under Different Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/skbr3-9-28.jpg)

**Figure 1.** Mean Fluorescent Intensity of the SK-BR3 under Different Treatments.

Note that as the background comparision groups were not included in the figures as the
effective
counted area were over 100 times smaller than positive groups, this way the mean fluorescent
intensity is not useful.

To further show the success of negative result, the figures of the confocal microscopy
result after setting the photo to 8 bit and auto the threshold are shown below:

![Confocal Microscopy Result of the SK-BR3 under Primary and Secondary Antibody Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/skbr3-b3-12-airyscan-processing-09-c2.jpg)

**Figure 2.** Confocal Microscopy Result of the SK-BR3 under Primary and Secondary
Antibody Treatment.

The figure shows that the background signal is weak, indicating the background
fluorescence is weak.

And the corresponding figure under bright field is shown below:

![Bright Field Channel Result of the SK-BR3 under Primary and Secondary Antibody Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/skbr3-b3-12-airyscan-processing-09-c3.jpg)

**Figure 3.** Bright Field Channel Result of the SK-BR3 under Primary and Secondary
Antibody Treatment.

![Confocal Microscopy Result of the SK-BR3 under Primary and Tertiary Antibody Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/skbr3-b4-13-airyscan-processing-12-c2.jpg)

**Figure 4.** Confocal Microscopy Result of the SK-BR3 under Primary and Tertiary
Antibody Treatment.

The figure shows that the background signal is weak, indicating the specificity of
tertiary antibody is high
and was washed thoroughly.

And the corresponding figure under bright field is shown below:

![Bright Field Channel Result of the SK-BR3 under Primary and Tertiary Antibody Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/skbr3-b4-13-airyscan-processing-12-c3.jpg)

**Figure 5.** Bright Field Channel Result of the SK-BR3 under Primary and Tertiary
Antibody Treatment.

The flow cytometry results also support the conclusion that the
secondary antibody didn't show specificity towards the scFv domain.

![Flow Cytometry Result of the SK-BR3 under Different Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/28-sep-2023-layout.jpg)

**Figure 6.** Flow Cytometry Result of the SK-BR3 under Different Treatment.

The figure shows no obvious difference between the group adding all three
antibodies and
the group only adding secondary and tertiary antibodies, while two negative
controls show
normal results.

Method Two by Western Blot Analysis

Besides, we conducted western blot test to see if the scFv domain could bind to the
HER2 protein.
This was done by firstly adding the scFv domain to the cell well for 12-24 hours,
then washing the
well with PBS several times to remove the unbound scFv domain, then extracting the
protein for electrophoresis,
membrane transformation and staining. Comparison groups were the HER2 positive cell,
HER2 negative cell and empty well. Possible drawback of this design is that since
western
blot analysis required proper amount of protein content, the amount of scFv domain
added to the
cell plate may not be enough to show the band in the western blot analysis. We added
180 μL scFv-cys-polyhis
with 4.236 μg/μL concentration to the cell plate, which is 762 μg scFv-cys-polyhis
in total. But since we are
adding the protein into 10 cm cell dish, this amount is still not large enough.

Still the result showed no obvious band comparing to the positive control
group
(which have 20.544 μg protein in total by BCA test) in the western blot analysis,
indicating two possibilities:

1. The scFv domain didn't bind to the HER2 protein.

2. The amount of scFv domain added to the cell plate is not enough to show the
band in the western blot analysis.

The result of the western blot analysis is shown below:

![Western Blot Analysis Result under Different Treatment](https://static.igem.wiki/teams/4702/wiki/results/results/wb.png)

**Figure 7.** Western Blot Analysis Result.

Future Plan

Though we failed to show the binding affinity of the scFv domain to the HER2
positive cell directly before the due day of this project, still we thought about
the method
to test the conjugation of the scFv domain to the HER2 positive cell.

As in our design, there have one type of scFv domain with a cysteine along
with a
His-tag attached to the end of the scFv domain, indicating that we could make use
of the thiol group
for conjugation with a fluorescent tag with maleimide. In this way we don't need
to use
the secondary and tertiary antibody here. Or we could use other common method used
to link a
fluorescent tag to a protein, such as directly link with the amine group on the
scFv domain,
using biotin-streptavidin system or click chemistry.

As for the improvement of the western blot analysis design, we could add more scFv
domain
and add less positive control scFv protein to improve the contrast. And more
control groups
like cells without treated with scFv domain should be added to show the
specificity of the binding.

Also, we could change the design for the protein synthesized in the bacteria to
the full length
anti-HER2 antibody, which would make it easier to find proper secondary antibody
to test the
biological activity of it. But this would increase the risk of facing the problem
of improper folding in bacteria. So we think the first or the second method
is more convenient for future examination.

# Cytotoxicity Examination of the Biologically Synthesized Nanoparticles and Chemically Synthesized Nanoparticle [[6]](#a1)

---

Method Description / Experimental Design

The Cell Counting Kit-8 (CCK-8) is commonly used to assess cell viability and cytotoxicity, in which WST-8
(a yellow, water-soluble tetrazolium salt) is added to the cell culture and this tetrazolium salt is taken
up by live cells.

Inside metabolically active cells, mitochondrial dehydrogenases convert WST-8 into a water-soluble
formazan dye. This conversion occurs in proportion to the metabolic activity of the cells. And the
formazan dye produced is soluble in the cell culture medium and has an orange color. The intensity of the
orange color is directly proportional to the number of living cells and their metabolic activity.

Therefore, it can be quantified by measuring the absorbance of the solution at a specific wavelength
using
a spectrophotometer or a microplate reader. In general, higher absorbance values indicate more viable and
metabolically active cells, while lower values suggest reduced cell viability.
Additionally, the calculation of the cell viability or cytotoxicity as a percentage use the following
formula:

Cell Viability (%) = [(OD of treated sample - OD of blank control) / (OD of control sample - OD of
blank
control)] x 100

Therefore, here we used the cck-8 kit to test the cytotoxicity of IONPS to detect its cancer cells
killing
capacity, which represents therapeutic effects. And also, since we designed antibodies targeting the HER2
domain of breast cancer cells (bccs) and linked them to the IONPS to equip IONPS with specificity of
killing bccs, the cytotoxicity differences between the IONPS linked to different antibodies and bare IONPS
can help to verify the success of linkage of our biosynthesized antibodies to the IONPS and targeting
effects of the antibodies.

We prepared samples of different concentrations in cell culture medium corresponding to the both the
HER2
positive breast cancer cells BT-474 and HER2 negative breast cancer cells MDA-MB-231. And after thawing
cells in 96-wells and the addition of the toxic substances (the samples of IONPs). The OD values were
measured in a 96-plate reader. The measured OD values were then calculated by the formula given in the
protocol of the cck-8 kit.

However, the first round of experiments indicated limited cytotoxicity of biosynthesized IONPs, which
might be caused by its low concentrations, while chemically synthesized IONPS showed relatively obvious
therapeutic effects. As a result, we conducted the following experiments mainly to verify therapeutic
effects of the latter and the targeting effects of our antibodies.

Result and Analysis

Generally, the results turned out that the cell viability showed negative correlation with increased
concentration of chemically synthesized IONPs (cIONPs) with antibodies and the corresponding bare IONPS.
But no evident cytotoxicity was shown for samples of bio-synthesized IONPs, which might be due to the low
concentration. Also, cIONPs linked with antibodies by T1 showed weak targeting specificity since there
shown limited differences between the cell viability of HER2 positive and HER2 negative cells. And cIONPs
linked with antibodies by T2 exhibited relatively evident targeting specificity on HER2 positive breast
cancer cells.

![cytotoxicity test for her-2 negative cell(MDA-MB-231)](https://static.igem.wiki/teams/4702/wiki/result/picture1.png)

**Figure 1.** cytotoxicity test for her-2 negative cell(MDA-MB-231).

![cytotoxicity test for her-2 negative cell(MDA-MB-231)](https://static.igem.wiki/teams/4702/wiki/result/picture2.png)

**Figure 2.** cytotoxicity test for her-2 negative cell(BT-474).

Note that Bio-AB represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2
antibody with poly-his tag attached at the end.

Chem-AB represents Chemically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody
with poly-his tag attached at the end.

Bio-CH represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2
antibody
with cystine-poly-his attached at the end.

CH represents scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.

AB represents scFv domain of anti HER2 antibody with poly-his tag attached at the end.

For treatment 1 nanoparticles, the first figure of the test with the her-2 negative cells
MDA-MB-231, all the groups exhibited obvious cytotoxicity for the cell viability of all groups showed
low. And there did not exist considerate difference between control, bio-unlinked. Bio-AB, and bio-CH,
as the cell viability were all relatively closed to 1.0.

And for her-2 positive cell BT-474 in the second figure, there did not show the obvious difference
except chem-AB and chem-CH. The cell viability of adding chem-AB and chem-CH were lower than other
groups significantly.

Compared two diagrams, there was no obvious change in cell viability in the two groups with the
addition
of chem-AB, which was convinced the cytotoxicity of the chem-AB. While the targeting effects of chem-CH
were exhibited, as there did exist a obvious difference between positive cell BT-474 and negative cell
MDA-MB-231 with cell viability in figure 1 was significantly higher than the figure 2.

![Treatment1 chem-synthesis nps cytotoxicity test(HER2 positive cells)](https://static.igem.wiki/teams/4702/wiki/result/picture3.png)

**Figure 3.** Treatment1 chem-synthesis nps cytotoxicity test(HER2 positive cells).

![Treatment1 chem-synthesis nps cytotoxicity test(HER2 negative cells)](https://static.igem.wiki/teams/4702/wiki/result/picture4.png)

**Figure 4.** Treatment1 chem-synthesis nps cytotoxicity test(HER2 negative cells).

Note that AB represents the anti HER2 antibody with poly his tag attached at the end and CH
represents anti HER2 antibody with poly-his tad attached at the end and here they were bound to some
of the nanoparticles with its scFv domain.

Chem\_nps\_1 and chem\_nps\_2 represent chemically synthesized nanoparticles with different batches.

For treatment 1 nanoparticles, the first figure of the test with the her2 positive cells BT-474,
all
the nps exhibited obvious cytotoxicity since the cell viability showed evident decline as the
concentration of nps increased, which indicates the cytotoxicity of all three samples in T1 to be
concentration-dependent. These nps showed a relevantly large cytotoxicity (close to 1) at low
concentrations. And there did not exist considerate differences between cytotoxicity of nps-2 linked
to antibodies and bare nps-2. Also, for her2 negative cells MDA-MB-231 in the second figure, all
three nps showed similar effects of lowering the cell viability with increased concentrations. These
results can be roughly concluded that the nps have great killing effects on these two breast cancer
cells and the linkage of antibodies by treatment 1 could have failed to equip our chemical
synthesized nps with expected specificity to her2 positive cells, which means the targeting effects
of the anti-her2 antibodies do not seem to function after linkage.

![Treatment2 chem-synthesis nps cytotoxicity test(HER2 positive cells)](https://static.igem.wiki/teams/4702/wiki/result/picture5.png)

**Figure 5.** Treatment2 chem-synthesis nps cytotoxicity test(HER2 positive cells).

![Treatment2 chem-synthesis nps cytotoxicity test(HER2 negative cells)](https://static.igem.wiki/teams/4702/wiki/result/picture6.png)

**Figure 6.** Treatment2 chem-synthesis nps cytotoxicity test(HER2 negative cells).

Note that AB here represents the same antibodies as in treatment 1 and CH represents anti HER2
antibody with poly-his tad attached at the end.

For treatment 2 nanoparticles, figure 1 shows the killing effects of nps on the her2 positive
cells
BT-474. The cell viability showed mild decline as the concentration of nps with antibodies AB and CH
increased, which indicates the cytotoxicity of all three samples in T2 to be concentration-dependent
as well. And the curve of cell viability of bare antibodies here does not show evident trend,
differing from nps linked to antibodies. Since the cytotoxicity of chemically synthesized nps has
been proved in previous experiments, this result indicates nps linked to antibodies successfully
targeted HER2 positive breast cancer cells. The subsequent tests conducted on HER2 negative breat
cancer cells MDA-MB-231 (in figure2) showed limited tendency of concentration-dependent cytotoxicity
of all three nps, which further justify that the linkage of treatment 2 successfully reserved the
targeting effects of the anti-HER2 antibody since the targeting specificity is brought in by the
HER-2 domain on cells. And the samples simply added by CH antibody showed its cell viability
relevantly consistent to 1, excluding the effects of antibodies that might harm cells. However, the
overall cell viability of HER2 negative cells are relevantly low, which means that there was
something wrong in the control, however, this error was not necessary and could be ignored.

![4h BT-474 Total](https://static.igem.wiki/teams/4702/wiki/result/picture7.png)

**Figure 7.** BT-474 cytotoxicity test result (HER2 positive cells).

![4h-Treatment1 chem-synthesis nps cytotoxicity test (HER2 positive cells)](https://static.igem.wiki/teams/4702/wiki/result/picture8.png)

**Figure 8.** 4h-Treatment1 chem-synthesis nps cytotoxicity test (HER2 positive cells).

![4h-Treatment2 chem-synthesis nps cytotoxicity test (HER2 positive cells)](https://static.igem.wiki/teams/4702/wiki/result/picture9.png)

**Figure 9.** 4h-Treatment2 chem-synthesis nps cytotoxicity test (HER2 positive cells).

For treatment 2 nanoparticles, the time period was changed into 4h. For the second figure, all the
nps exhibited obvious cytotoxicity since the cell viability showed evident decline as the
concentration of nps increased, which ndicates the cytotoxicity of all three samples in T2 to be
concentration-dependent. And in the third figure of HER2 positive cells, there did showed almost a
similar trend to the 2h-nps HER2 positive cells.

![2h Cytotoxicity of Biologically Synthesized Nanoparticle to BT-474](https://static.igem.wiki/teams/4702/wiki/result/picture10.png)

**Figure 10.** 2h Cytotoxicity of Biologically Synthesized Nanoparticle to BT-474

![2h Cytotoxicity of Biologically Synthesized Nanoparticle to MDA-MB-231](https://static.igem.wiki/teams/4702/wiki/result/picture10.png)

**Figure 11.** 2h Cytotoxicity of Biologically Synthesized Nanoparticle to MDA-MB-231

For treatment 2 nanoparticles, the first figure of the BT-474\_bio cells, the cell viability of all
the groups were relatively low.

And for the MDA-MB-231 in second figure, there did not exist obvious difference between bio+AB, cell
control, and CH(antibody), which were relatively closed to 1.0.

While the cell viability of the group bio-unlinked was significantly lower than other groups.
Compared two figures, the total average cell viability in MDA-MD-231 was relatively lower than the
BT-474. And there was no obvious evidence to show the relationship.

# His-tagged protein purification [[7]](#a1)

---

Method Description

In our vector design for scFv domain of anti-HER2 antibody, we added a 6 × His-tag
to the terminal of the scFv domain of the anti-HER2 antibody.

Therefore, we use the [HisTrap HP His tag protein purification column](https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/affinity-tagged-protein/histrap-hp-histidine-tagged-protein-purification-columns-p-00250) and the Union AKTA system to
purify the His-tagged protein from the cell lysate.

In our case, we are trying to express a eukaryotic gene in prokaryote
*E. coli*, therefore,
we chose the SHuffle strain for better disulfide bond formation in the protein
expressed to best maintain
the proper protein structure and avoid the formation of inclusionbody.

After the expression of the His-tagged protein, cell pellets were broken by
high pressure homogenizer and the cell lysate were collected.

Then, the cell lysate after dislodging the cell debris by centrifugation
were loaded into the
HisTrap HP His tag protein purification column and the His-tagged protein were
eluted by imidazole.

Results / Analysis

Basically, the His-tagged protein was successfully purified from the cell lysate,
including
the **scFv-polyhis**, **scFv-cys-polyhis** and **scFv-polyhis-cys**.

The purity of the His-tagged protein was acceptable for further use. And
the
concentrations of final product were in the range of 0.1-5 mg/ml, which are also
proper
for later use. By our method for protein expression and purification, when results
are stable,
we had a yield of 5 mg/ml with total volumn 100 μL from 150 mL culture, which is a
satisfying result.

The other thing we found is that, for scFv-cys-polyhis protein, the
induction under 18 ℃
shows higher final concentration result than that under 37 ℃. This may be due to
the fact that
lower temperature would leave enough time for the protein to fold properly.

The UV spectrum during purification process, the SDS-PAGE result and the
concentration
by BCA test could be found in the PDF file in the supplementary data.

Supplementary Data

The supplementary data including the UV280 absorption monitoring
information, SDS-PAGE result and BCA test result
for protein purification could be seen in the pdf file shown below:

His-tagged Protein Purification
Supplementary Data

# Author & Contribution

### Major Author

* Shuoyi HU:
  [[1]](#1)
  [[2]](#2)
  [[3]](#3)
  [[4]](#4)
  [[5]](#5)
  [[7]](#7)
* Yixiao XIAO:
  [[6]](#6)

### Contribution

Selective Electron Diffraction Result Interpretation: Ziqiao WANG

TEM Particle Size Counting: Yuqi SHEN

Cytotoxicity Result Interpretation: Yuqi SHEN

{% endblock %}