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:

Sited from BIO-RAD: pGLO Plasmid Map and Resources | Bio-Rad
So 50 ml 20% arabinose was prepared and saved at 4℃.

Recorded by Gehua WENG

Participant(s): Gehua WENG, Yuqi SHEN

Our first experimental BL21 are mentioned in the table below:

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

Recorded by Gehua WENG

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

Participant(s): Shuoyi HU, Gehua WENG:

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

LB low-salt
MH
        Refer the commercial protocols .

 

TSB (standard one)

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

 

Recorded by Gehua WENG

Participant(s): Yixiao XIAO:

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

Recorded by Gehua WENG

Dialysis Bag Activation

Participant(s): Shuoyi HU, Gehua WENG:

A reel of dialysis bag (Cat. No. FDM403-5m, Beyotime) was activated by strictly following the instruction manual provided by Beyotime and storing in 75% ethanol in 4ºC.

Instruction manual was provided by Beyotime

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

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

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

Ten tubes of SHuffle® Strain E. coli (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)

Recorded by Shuoyi HU

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

Ferric Ion Induced Nanoparticles Biosynthesis

Participant: Shuoyi HU

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

Culture medium: Mueller-Hinton Broth, 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:

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

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)

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.

Culture medium: Mueller-Hinton Broth, 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

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.

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

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:

Recorded by Shuoyi HU

Participant(s): Shuoyi HU, Songnan REN

5 mM Ferric Ion was used to induce IONPs synthesis.

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

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:

Recorded by Yixiao XIAO

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

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

Participant(s): Shuoyi HU

Vector scFv-pGLO was transformed into SHuffle strain E. coli.
(Named as SHuffle-antibody below)

Recorded by Shuoyi HU

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

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)

Recorded by Shuoyi HU

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

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

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

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. 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

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

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

Recorded by Shuoyi HU

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

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. 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.

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.

Recorded by Shuoyi HU

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

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 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 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.

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.

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

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:

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. 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. 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

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

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 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.

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.

Recorded by Songnan REN

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 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.

Recorded by Songnan REN

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

Participant(s): Yixiao XIAO

Culture medium was changed with another 10 ml medium with 20% of Fetal Bovine Serum to support the normal growth rate of BT-474 cells.

Cell condition under 10x microscope as below:

Cell Experiment Recorded by Yixiao XIAO

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

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:

Recorded by Yixiao XIAO

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:

Recorded by Yixiao XIAO

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

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.

Recorded by Yixiao XIAO

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.

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 (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. Standard Curve of BCA test was shown below:

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

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

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:

Recorded by Yixiao XIAO

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):

For treatment 2 (T2):

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:

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:

Recorded by Yixiao XIAO

Immunostaining, confocal and flow cytomotry examination

Participant: Shuoyi HU, Yixiao XIAO

Our experiment design was shown as below:

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

Ferric Ion Induced Iron Oxide Nanoparticle Synthesis

Participant: Shuoyi HU

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

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

Recorded by Shuoyi HU

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

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

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:

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:

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:

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:

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. 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.

Recorded by Shuoyi HU

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.
Standard Curve of BCA test was shown below:

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

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

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

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:

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:

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:
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:

Recorded by Yixiao XIAO

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:

Recorded by Yixiao XIAO

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.

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:

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.
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:

Recorded by Yixiao XIAO

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.

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.
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:

Recorded by Yixiao XIAO

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:

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

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

Immunostaining, Confocal and Flow Cytomotry Examination

Participant(s): Shuoyi HU, Shijie HE

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.

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:

Recorded by Yixiao XIAO

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:

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

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

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

Recorded by Shuoyi HU

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:

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

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:

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