iGEM 2019 — Team NJTech_China

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# Team:NJTech China/Notebook

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* [JUDGING FORM](https://igem.org/2019_Judging_Form?team=NJTech_China)
* Log

  [Notebook](https://2019.igem.org/Team%3ANJTech_China/Notebook)
  [SAFETY](https://2019.igem.org/Team%3ANJTech_China/Safety)
* TEAM

  [Team Members](https://2019.igem.org/Team%3ANJTech_China/Team)
  [Attributions](https://2019.igem.org/Team%3ANJTech_China/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ANJTech_China/Collaborations)
* Human Practices

  [Silver HP](https://2019.igem.org/Team%3ANJTech_China/Human_Practices)
  [Integrated HP](https://2019.igem.org/Team%3ANJTech_China/intergrated_hp)
  [Education & Public Engagement](https://2019.igem.org/Team%3ANJTech_China/Public_Engagement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ANJTech_China/Parts)
* PROJECT

  [Description](https://2019.igem.org/Team%3ANJTech_China/Description)
  [Design](https://2019.igem.org/Team%3ANJTech_China/Design)
  [Model](https://2019.igem.org/Team%3ANJTech_China/Model)
  [Results](https://2019.igem.org/Team%3ANJTech_China/Results)
  [Experiments](https://2019.igem.org/Team%3ANJTech_China/Experiments)

# NOTEBOOKS

#### Our notebooks are documented in PDF.

#### To check our notebooks, please click the below bottom.

[#### click here to view NOTEBOOKS](https://2019.igem.org/wiki/images/2/24/T--NJTech_China--notebook.pdf)

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[![](https://2019.igem.org/wiki/images/2/25/T--NJTech_China--genewiz.png)](https://www.genewiz.com.cn)
 [![](https://2019.igem.org/wiki/images/f/fd/T--NJTech_China--genescript.png)](https://www.genscript.com)
 [![](https://2019.igem.org/wiki/images/7/7a/T--NJTech_China--njtech.png)](http://www.njtech.edu.cn)
[![](https://2019.igem.org/wiki/images/3/37/T--NJTech_China--sg.png)](http://life-phar.njtech.edu.cn)
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### © 2019 NJTech\_China iGEM Team. All Rights Reserved.


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# Team:NJTech China/Experiments

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* [JUDGING FORM](https://igem.org/2019_Judging_Form?team=NJTech_China)
* Log

  [Notebook](https://2019.igem.org/Team%3ANJTech_China/Notebook)
  [SAFETY](https://2019.igem.org/Team%3ANJTech_China/Safety)
* TEAM

  [Team Members](https://2019.igem.org/Team%3ANJTech_China/Team)
  [Attributions](https://2019.igem.org/Team%3ANJTech_China/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ANJTech_China/Collaborations)
* Human Practices

  [Silver HP](https://2019.igem.org/Team%3ANJTech_China/Human_Practices)
  [Integrated HP](https://2019.igem.org/Team%3ANJTech_China/intergrated_hp)
  [Education & Public Engagement](https://2019.igem.org/Team%3ANJTech_China/Public_Engagement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ANJTech_China/Parts)
* PROJECT

  [Description](https://2019.igem.org/Team%3ANJTech_China/Description)
  [Design](https://2019.igem.org/Team%3ANJTech_China/Design)
  [Model](https://2019.igem.org/Team%3ANJTech_China/Model)
  [Results](https://2019.igem.org/Team%3ANJTech_China/Results)
  [Experiments](https://2019.igem.org/Team%3ANJTech_China/Experiments)

EXPERIMENTS

![center](https://static.igem.org/mediawiki/2019/7/75/T--NJTech_China--down.png)

[* top](#top)
[* Experiments](#Experiments)
[* Protocols](#Protocols)

#

# EXPERIMENTS

## 1. Production of mouse IL-2-RFP fusion protein

#### 1) Construction of plasmid pGAP-ss-mIL2-RFP

Plasmid pGAP-ss-mIL2-RFP was synthesized by GENEWIZ. The sequence of
mIL2-RFP
was optimized for Pichia pastoris and a 6xHis tag for
protein
purification was attached to the C-terminus of the sequence.

We added the DH5α E.coli containing
plasmid pGAP-mIL2-RFP synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and
incubated them at 37℃, 2000rpm overnight.

We extracted the plasmid from E.coli
with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by
microvolume
UV-Vis spectrophotometer. We recorded the concentration for further experiments.

#### 2) Transformation of Pichia

We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid
from
circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a
electrophoresis to ensure whether the digestion had been completed. If only one strip of the target
size
appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction
Kit. The concentration of linearized plasmid was measured for further experiments.

We prepared yeast competent cells according to the procedures in our
Protocol.
Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock.
After
kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L
G418
and incubated these plates at 30℃.

#### 3) Screening positive transformants

After 3 days’ culture, some colonies appeared on the screening plates. We
chose
some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of mIL2
and
KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of
transformation.
If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours
incubation
at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction
Kit
and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed
genome
as a template and ss-mIL2 F and ss-mIL2 R as primers, genomic PCR was performed to verify whether
our
target gene was successfully integrated into the yeast genome. After successful validation, we
submitted
the genomic samples to GENEWIZ for sequencing to further confirm the sequence.

#### 4) Fermentation

If the sequencing results match the designed sequence, we will take some
recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of
incubation,
we transformed some cultures into fermentation medium and incubated them in oscillating incubator at
200
rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the
fermentation solution at 4℃, waiting for product collection and purification.

#### 5) Product collection and purification

We dispersed the fermentation solution in order to get supernatant. We gave
the
supernatant a ultrafiltration. Since our produced protein is about 44kDa, we chose a 30kDa filter to
ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did
the
affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further
purified product. After each step of single processing, we used SDS-PAGE to detect the effect of
extraction and purification of the extracted and residual samples.

## 2. Production of mouse IL-2 protein

#### 1) Construction of plasmid pGAP-mIL2-RFP

We used the primers ss-mIL2 F and ss-mIL2 R to copy the fragment of mIL2
with
alpha signal sequence from plasmid pGAP-ss-mIL2-RFP by PCR. Since we had added these primers
homogenous
arm of XbaⅠregion in plasmid pGAP-KanR, so we could put our fragment into plasmid by one step
cloning
later. We used OMEGA Gel Extraction Kit to obtain fragments from PCR solution. We used FastDigest
Xba Ⅰ
to linearize the plasmid and extract linearized plasmid with OMEGA Gel Extraction Kit as well. Then
we
connected ss-mIL2 to plasmid using Vazyme ClonExpress II fast Cloning Kit. We used this cloning
solution
to transform competent DH5α E.coli cells according to the
procedures in our Protocol. Afterwards we screened the positive cells using LB medium plate
containning
0.1% Kana. We transformed some of colonies into 0.1% Kana LB liqiud medium. After incubated at 37℃
for
hours, these cultures were used for extraction of plasmid by AXYGEN AxyPrep Plasmid Miniprep Kit and
recombinant E.coli cells were stored in 30% glycerin YPD
solution at -20℃. We used primers to copy ss-mIL2 from extracted plasmid. We ran a electrophoresis
to
ensure that ss-mIL2 exsits in plasmid and sent PCR solution to GENEWIZ for sequencing.

According to the sequencing report supported by GENEWIZ, we knewn that we
had
gotten E.coli containing plasmid with correct ss-mIL2
sequence.
We added it into 1% LB liquid medium and extracted plasmid from cultures later. The concentration of
plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for
further
experiments.

#### 2) Transformation of Pichia

We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid
from
circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a
electrophoresis to ensure whether the digestion had been completed. If only one strip of the target
size
appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction
Kit. The concentration of linearized plasmid was measured for further experiments.

We prepared yeast competent cells according to the procedures in our
Protocol.
Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock.
After
kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L
G418
and incubated these plates at 30℃.

#### 3) Screening positive transformants

After 3 days’ culture, some colonies appeared on the screening plates. We
chose
some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of mIL2
and
KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of
transformation.
If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours
incubation
at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction
Kit
and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed
genome
as a template and ss-mIL2 F and ss-mIL2 R as primers, genomic PCR was performed to verify whether
our
target gene was successfully integrated into the yeast genome. After successful validation, we
submitted
the genomic samples to GENEWIZ for sequencing to further confirm the sequence.

#### 4) Fermentation

If the sequencing results match the designed sequence, we will take some
recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of
incubation,
we transformed some cultures into fermentation medium and incubated them in oscillating incubator at
200
rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the
fermentation solution at 4℃, waiting for product collection and purification.

#### 5) Product collection and purification

We dispersed the fermentation solution in order to get supernatant. We gave
the
supernatant a ultrafiltration. Since our produced protein is about 19kDa, we chose a 10kDa filter to
ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did
the
affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further
purified product. After each step of single processing, we used SDS-PAGE to detect the effect of
extraction and purification of the extracted and residual samples.

## 3. Production of human IL-2-RFP fusion protein

#### 1) Construction of plasmid pGAP-ss-hIL2-RFP

Plasmid pGAP-ss-hIL2-RFP was synthesized by GENEWIZ. The sequence of
hIL2-RFP
was optimized for Pichia pastoris and a 6xHis tag for
protein
purification was attached to the C-terminus of the sequence.

We added the DH5α E.coli containing
plasmid pGAP-hIL2-RFP synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and
incubated them at 37℃, 2000rpm overnight.

We extracted the plasmid from E.coli
with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by
microvolume
UV-Vis spectrophotometer. We recorded the concentration for further experiments.

#### 2) Transformation of Pichia

We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid
from
circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a
electrophoresis to ensure whether the digestion had been completed. If only one strip of the target
size
appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction
Kit. The concentration of linearized plasmid was measured for further experiments.

We prepared yeast competent cells according to the procedures in our
Protocol.
Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock.
After
kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L
G418
and incubated these plates at 30℃.

#### 3) Screening positive transformants

After 3 days’ culture, some colonies appeared on the screening plates. We
chose
some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of hIL2
and
KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of
transformation.
If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours
incubation
at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction
Kit
and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed
genome
as a template and ss-hIL2 F and ss-hIL2 R as primers, genomic PCR was performed to verify whether
our
target gene was successfully integrated into the yeast genome. After successful validation, we
submitted
the genomic samples to GENEWIZ for sequencing to further confirm the sequence.

#### 4) Fermentation

If the sequencing results match the designed sequence, we will take some
recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of
incubation,
we transformed some cultures into fermentation medium and incubated them in oscillating incubator at
200
rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the
fermentation solution at 4℃, waiting for product collection and purification.

#### 5) Product collection and purification

We dispersed the fermentation solution in order to get supernatant. We gave
the
supernatant a ultrafiltration. Since our produced protein is about 40kDa, we chose a 30kDa filter to
ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did
the
affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further
purified product. After each step of single processing, we used SDS-PAGE to detect the effect of
extraction and purification of the extracted and residual samples.

## 4. Production of TAT(rigid linker) fusion protein

#### 1) Construction of plasmid pGAP-TAT(rigid linker)

Plasmid pGAP-ss- TAT(rigid linker) was synthesized by GENEWIZ. The sequence
of
TAT(rigid linker) was optimized for Pichia pastoris and a
6xHis
tag for protein purification was attached to the C-terminus of the sequence.

We added the DH5α E.coli containing
plasmid pGAP- TAT(rigid linker) synthesized by GENEWIZ into LB liquid meidum containing 1%
kanamycin,
and incubated them at 37℃, 2000rpm overnight.

We extracted the plasmid from E.coli
with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by
microvolume
UV-Vis spectrophotometer. We recorded the concentration for further experiments.

#### 2) Transformation of Pichia

We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid
from
circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a
electrophoresis to ensure whether the digestion had been completed. If only one strip of the target
size
appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction
Kit. The concentration of linearized plasmid was measured for further experiments.

We prepared yeast competent cells according to the procedures in our
Protocol.
Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock.
After
kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L
G418
and incubated these plates at 30℃.

#### 3) Screening positive transformants

After 3 days’ culture, some colonies appeared on the screening plates. We
chose
some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of TAT and
KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of
transformation.
If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours
incubation
at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction
Kit
and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed
genome
as a template and ss-TAT F and ss-TAT R as primers, genomic PCR was performed to verify whether our
target gene was successfully integrated into the yeast genome. After successful validation, we
submitted
the genomic samples to GENEWIZ for sequencing to further confirm the sequence.

#### 4) Fermentation

If the sequencing results match the designed sequence, we will take some
recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of
incubation,
we transformed some cultures into fermentation medium and incubated them in oscillating incubator at
200
rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the
fermentation solution at 4℃, waiting for product collection and purification.

#### 5) Product collection and purification

We dispersed the fermentation solution in order to get supernatant. We gave
the
supernatant a ultrafiltration. Since our produced protein is about 55kDa, we chose a 30kDa filter to
ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did
the
affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further
purified product. After each step of single processing, we used SDS-PAGE to detect the effect of
extraction and purification of the extracted and residual samples.

## 5. Production of TAT(soft linker) fusion protein

#### 1) Construction of plasmid pGAP-TAT(soft linker)

Plasmid pGAP-ss- TAT(soft linker) was synthesized by GENEWIZ. The sequence
of
TAT(soft linker) was optimized for Pichia pastoris and a
6xHis
tag for protein purification was attached to the C-terminus of the sequence.

We added the DH5α E.coli containing
plasmid pGAP- TAT(soft linker) synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin,
and
incubated them at 37℃, 2000rpm overnight.

We extracted the plasmid from E.coli
with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by
microvolume
UV-Vis spectrophotometer. We recorded the concentration for further experiments.

#### 2) Transformation of Pichia

We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid
from
circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a
electrophoresis to ensure whether the digestion had been completed. If only one strip of the target
size
appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction
Kit. The concentration of linearized plasmid was measured for further experiments.

We prepared yeast competent cells according to the procedures in our
Protocol.
Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock.
After
kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L
G418
and incubated these plates at 30℃.

#### 3) Screening positive transformants

After 3 days’ culture, some colonies appeared on the screening plates. We
chose
some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of TAT and
KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of
transformation.
If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours
incubation
at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction
Kit
and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed
genome
as a template and ss-TAT F and ss-TAT R as primers, genomic PCR was performed to verify whether our
target gene was successfully integrated into the yeast genome. After successful validation, we
submitted
the genomic samples to GENEWIZ for sequencing to further confirm the sequence.

#### 4) Fermentation

If the sequencing results match the designed sequence, we will take some
recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of
incubation,
we transformed some cultures into fermentation medium and incubated them in oscillating incubator at
200
rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the
fermentation solution at 4℃, waiting for product collection and purification.

#### 5) Product collection and purification

We dispersed the fermentation solution in order to get supernatant. We gave
the
supernatant a ultrafiltration. Since our produced protein is about 55kDa, we chose a 30kDa filter to
ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did
the
affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further
purified product. After each step of single processing, we used SDS-PAGE to detect the effect of
extraction and purification of the extracted and residual samples.

## Validation

### 1. Proliferation effect on lymphocyte

#### (1) CCK8 proliferation assay

The proliferation effect of IL-2 on CD3+ T cell is evaluated by CCK8 assay,
where 5×104 cells/100 µl/well is seeded in 96-well plate. Next, 100µl of complete media
(RPMI1640
which is supplemented with FBS 10% and ConA 2.5 µg/ml) containing various concentrations of IL-2
(0.3, 1, 3, 10, 30, 100 and 300 nM), as a positive control, is added and incubated for 72h in 37 °C.
Afterwards, 20µl CCK8 is added to each well and incubate for 4h in 37 °C. Optical density is
measured at 450 nm on a Microplate reader.

#### (2) CFSE staining proliferation assay

CFSE staining proliferation assay is performed using carboxyfluorescein
succinimidyl ester (CFSE) (BD bioscience, USA) staining. CD3+ T cell are stained by 10 µM CFSE and
incubated for 20 min at 37℃. After washing by RPMI 1640 containing 10%FBS (Gibco, Brazil), cells are
seeded into the 24-well plate at 1.0×106 cells/ml in the fresh media containing ConA 2.5
µg/ml, with
different concentrations of IL-2 (0，0.3, 1, 3, 10, 30, 100 and 300 nM) for incubation at 37℃. After
10 days, the cells are analyzed by FACS Calibur Flowcytometry (BD Biosciences, USA), and data are
analyzed by FlowJo 7.6.2 software (FlowJo, LLC, CA). To determine cell proliferation, lymphocytes
are gated according to their forward and side scatters (SSC). To compare the rounds of proliferation
in various culture conditions, cell populations with different levels of CFSE intensity (dilutions)
are considered as a generation.

### 2. specifically binding of NJT0401 and PD-L1 on tumor cells

MDA - MB - 231 cells are seeded into 3 24-well plates at 2.0×104 cells/ml.
Then cells are treated with 10ng/ml INF-γ for 24h. 500µl of complete media (RPMI1640 which is
supplemented with FBS 10%) containing various concentrations of fusion protein (0.01nM, 0.1Nm, 1nM,
10nM, 100Nm, 1000nM), is separately added and incubated in 37℃. For the second plate, 500µl of
complete media (RPMI1640 which is supplemented with FBS 10% ) containing various concentrations of
anti-PD-L1(0.01nM, 0.1Nm, 1nM, 10nM, 100Nm, 1000nM), as a positive control, is separately added and
incubated in 37℃. For the third plate, 500µl of complete media(RPMI1640 which is supplemented with
FBS 10% ) , as a blank control, is added and incubated in 37℃.After 24h incubation avoiding light,
the cells are washed with PBS twice and test with FACS Calibur Flowcytometer (BD Biosciences, USA).
(rabbit anti-human pd-l1 monoclonal antibody (E1L3N) bound with phycoglobin (PE) is used to prepare
samples. As a control, the cell samples are stained for 30 minutes with rabbit IgG monoclonal
antibody (DA1E) conjugated with PE. The cell count of each sample is >105. The data is analysed by
FlowJo 7.6.2 software (FlowJo, LLC, CA)

#### 3. Sustained drug releasing model：

BSA is used as a model protein drug to study the drug release behavior.
Analyze the hydrogels drug release kinetics of bovine serum protein (BSA) with different
crosslinking degrees in vitro, and explore whether hydrogels loaded with drugs can achieve the
effect of slow release.

Proliferation of T cells is determined  in
vitro using a BrdU proliferation
ELISA kit (Roche). To investigate cell
proliferation in vivo , TAT are injected into the left rear
foot pad of tumor bearing mice .3 d
later,tumor-bearing mice received an injection of 50 mg BrdU/kg of body weight. Animals are killed
18 h after
the last injection. CD8+ T cells are isolated from the thymus, spleen by MACS as described above.
The
percentages of BrdU+ CD8+T cells are determined by flow cytometry.

An in situ polymerization method is used to incorporate BSA molecules into
hydrogel.Briefly, BSA is
initially dissolved in PBS to get a 0.02 wt% solution, and then HACHO and γ-PGA-ADH are dissolved in
0.02 wt% BSA solution, respectively. After that, these two solutions are mixed to prepare the
BSA-loaded hydrogels at room temperature. The drug release tests are performed by immersing
BSA-loaded hydrogels in 80 mL PBS, then placed in a circular mold and sealed with a sealing membrane
,which occasionally shaking at 37℃. At every 4 hours, the release medium (0.2 mL) is taken out and
replaced with an equal volume of PBS, the tests lasted 72 hours. The BCA protein concentration assay
kit is used to determine the total amount of BSA by color reaction.

All release experiments are performed in triplicate.

### 4. Anti-tumor effect and long-term immune memory effect of fusion protein (a perspective).

Mice are inoculated subcutaneously with 5\*105 4T1 cells and randomly divided
into four groups. After inoculation, they are individually treated with (1) PBS, (2) γGlu (3) γGlu
and fusion protein, (4) fusion protein and then the primary tumors are surgically resected on day
28. Four weeks later, these mice are re-challenged with 4T1 cells in the right flank. The distant
tumor size and survival are measured every 2 days, and the experimental end point is defined as
either death or a tumor size greater than 1000 mm3. The primary and distant tumors are imaged by
IVIS Spectrum (PerkinElmer, USA) after intraperitoneal injection with d-Luciferin sodium salt at a
dose of 3 mg/mouse.

To investigate cell proliferation in vivo, NJT0401 are injected into the left rear foot pad of tumor bearing mice. Three days later, tumor-bearing mice received an injection of 50 mg BrdU/kg of body weight. Animals are performed euthanasia 18 hours after the last injection. CD8+ T cells are isolated from the thymus, spleen by MACS as described above. The percentages of BrdU+ CD8+T cells are determined by flow cytometry.

2.5\*105 4T1 tumor cells are orthotopically implanted into Balb/c mice. When tumor volumes reach to 50-100mm3, mice are randomly divided into 4 groups, (1)PBS, (2)γGlu, (3)γGlu and fusion protein, (4)γGlu+anti-PDL1 (n=8). Treat i.p.at days 10, 12, 14 and 16, respectively. Twenty-four hours after the last treatment, carry out intratumoral analysis of surface PD-L1 accumulation.

# \*

# .........................................

# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

# PROTOCOLS

#### [click here to view protocol details](https://static.igem.org/mediawiki/2019/4/4f/T--NJTech_China--protocol.pdf)

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### © 2019 NJTech\_China iGEM Team. All Rights Reserved.

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# Team:NJTech China/Results

[![](https://static.igem.org/mediawiki/2019/5/5e/T--NJTech_China--white_logo.png)](https://2019.igem.org/Team%3ANJTech_China)

* [JUDGING FORM](https://igem.org/2019_Judging_Form?team=NJTech_China)
* Log

  [Notebook](https://2019.igem.org/Team%3ANJTech_China/Notebook)
  [SAFETY](https://2019.igem.org/Team%3ANJTech_China/Safety)
* TEAM

  [Team Members](https://2019.igem.org/Team%3ANJTech_China/Team)
  [Attributions](https://2019.igem.org/Team%3ANJTech_China/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ANJTech_China/Collaborations)
* Human Practices

  [Silver HP](https://2019.igem.org/Team%3ANJTech_China/Human_Practices)
  [Integrated HP](https://2019.igem.org/Team%3ANJTech_China/intergrated_hp)
  [Education & Public Engagement](https://2019.igem.org/Team%3ANJTech_China/Public_Engagement)
* PARTS

  [Parts Overview](https://2019.igem.org/Team%3ANJTech_China/Parts)
* PROJECT

  [Description](https://2019.igem.org/Team%3ANJTech_China/Description)
  [Design](https://2019.igem.org/Team%3ANJTech_China/Design)
  [Model](https://2019.igem.org/Team%3ANJTech_China/Model)
  [Results](https://2019.igem.org/Team%3ANJTech_China/Results)
  [Experiments](https://2019.igem.org/Team%3ANJTech_China/Experiments)

RESULTS

## 1. Construction and validation of murine IL-2-RFP fusion protein and IL-2 gene expression cassette

![](https://static.igem.org/mediawiki/2019/1/11/T--NJTech_China--T--NJTech_China--gr1.png)

The plasmid pGAP-ss-IL-2-RFP was extracted using AXYGEN AxyPrep
Plasmid Miniprep Kit, and the extracted
plasmid was digested by BamHⅠ single digestion. The enzyme digestion result was verified by agarose gel
electrophoresis.

![](https://static.igem.org/mediawiki/2019/c/ca/T--NJTech_China--gr2.png)

The coding gene of the signal peptide and interleukin 2 was
amplified by using the recombinant plasmid
pGAP-ss-IL-2-RFP as template and ss-IL2 F and ss-IL2 R as primers.

![](https://static.igem.org/mediawiki/2019/8/8d/T--NJTech_China--gr3.png)

The plasmid pGAP-KanR was extracted using AXYGEN AxyPrep
Plasmid
Miniprep Kit, and the extracted plasmid
was digested by XbaⅠ single digestion. The enzyme digestion result was verified by agarose gel
electrophoresis.

![](https://static.igem.org/mediawiki/2019/4/4e/T--NJTech_China--gr4.png)

## 2. Transformation and verification of murine IL-2-RFP fusion protein and IL-2 gene expression cassette

The recombinant plasmid pGAP-ss-IL-2 was linearized by BamHⅠ
single digestion. The enzyme digestion
result was verified by agarose gel electrophoresis.

![](https://static.igem.org/mediawiki/2019/e/eb/T--NJTech_China--gr5.png)

Pick a single colony growing on the resistant plate and
transfer to
a new YPD plate. Colonies growing on
the transfer point plate were picked for colony PCR to select positive transformants.

![](https://static.igem.org/mediawiki/2019/6/6e/T--NJTech_China--gr6.png)

For the positive clones identified by colony PCR, the yeast
genome
was extracted using the Solarbio Yeast
Genomic DNA Extraction Kit. Using the proposed genome as a template and ss-IL2 F and ss-IL2 R as
primers,
genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast
genome.

![](https://static.igem.org/mediawiki/2019/5/59/T--NJTech_China--gr7.png)

## 3. Expression, collection and purification of our target proteins

![](https://static.igem.org/mediawiki/2019/b/b3/T--NJTech_China--gr8.png)

![](https://static.igem.org/mediawiki/2019/d/d0/T--NJTech_China--gr9.png)
![](https://static.igem.org/mediawiki/2019/b/b9/T--NJTech_China--gr10.png)

Four days' microscopic examination results showed that all the
fermentation broths were not contaminated
with bacteria during our expression process.

![](https://static.igem.org/mediawiki/2019/f/fa/T--NJTech_China--gr11.png)
![](https://static.igem.org/mediawiki/2019/1/16/T--NJTech_China--gr12.png)

![](https://static.igem.org/mediawiki/2019/8/82/T--NJTech_China--gr13.png)
![](https://static.igem.org/mediawiki/2019/c/c8/T--NJTech_China--gr14.png)

The '1, 2, 3, 4' in the legend represents four flasks for
fermentation. It can be
seen from the line graph that at the beginning of fermentation, with the prolongation of expression
time,
the OD600 value shows a gradually increasing trend. When the value stabilizes, we conclude that the
expression process is over. The collection and purification of the product can be carried out in the
next
step.

![](https://static.igem.org/mediawiki/2019/b/b4/T--NJTech_China--gr15.png)

Red fluorescent spots were observed under fluorescent
illumination
at a wavelength
of 515-520 nm. However, yeast cells cannot be found in the visible field of view.

### We further confirmed the presence of the target protein by SDS-PAGE.

![](https://static.igem.org/mediawiki/2019/d/de/T--NJTech_China--gr16.png)

### Interpretation of experimental results:

In the black box line is the protein gel of the 100-fold
diluted
sample. The samples from left to right
were centrifuged supernatant, ultrafiltration supernatant, and ultrafiltration. No protein bands
appeared in the undiluted sample and the 100-fold diluted sample, except that the 100-fold diluted
ultrafiltration showed a protein band of approximately 40 kDa.

### Analysis：

In theory, the centrifugation supernatant (i.e. the first
strip)
should have multiple strips, and at
least one strip should appear in the ultrafiltration supernatant or ultrafiltration. Moreover, since the
membrane used for ultrafiltration is 10 kDa, ultrafiltration should not reveal a faint 40 kDa protein
band. Therefore, the above results have yet to be verified.

![](https://static.igem.org/mediawiki/2019/3/32/T--NJTech_China--gr17.png)

After reprocessing the sample, we performed SDS-PAGE again. In
order to rule out the impact of
non-standardized individual experimentation on the results, we invited a graduate student in the lab to
help us with this experimental operation. The results showed that there were still no bands in the three
samples. The picture above shows the photo of the SDS-PAGE gel. Among them, the left side is the protein
marker lane, the middle is the sample lane, and the far right is the sample strip of a graduate student
in the laboratory that day who shared the gel with us that day.

### Analysis：

Combined with the previous experimental results, we speculated
that the protein was not dissolved in the
fermentation broth, so no bands appeared on the SDS-PAGE gel these days. And that faint strip is most
likely a hybrid protein.

In summary, there may be two cases: 1 protein may be
precipitated
and mixed with yeast cells in the form of
a precipitate during centrifugation. 2 The protein may still be intracellular, which also explains the
results of fluorescence microscopy. The red spot that is seen may be the hybrid protein released by the
broken yeast.

# Validation Results

![](https://static.igem.org/mediawiki/2019/2/28/T--NJTech_China--guchuny.png)

Figure 1. In vitro release profiles of BSA with different concentrations HA/γ-Glu hydrogels at 37 °C.
A indicates 12% (ωt) γ-Glu and 12% (ωt) HA cross-linking hydrogels. B indicates 10% (ωt) γ-Glu and
10% (ωt) HA cross-linking hydrogels.

Fig. 1 shows the BSA-loaded hydrogels release kinetics. The
release curves were ﬁtted by the software Origin 8.0 according to several kinetic models. The drug
showed a continuous release state in two kinds of cross-linking hydrogels. Compared with the release
time of drugs from injectable HA hydrogels prepared by physical mixing (about one hours), the
release of BSA from HA/γ-PGA hydrogels with various compositions showed sustained release behavior
(>80 h). The crosslinking density is one of the factors that aﬀect drug release, so the factors
inﬂuencing the hydrogel crosslinking degree also aﬀect the drug release. Next, we load different
concentrations BSA and extend the measurement time to see an obvious drug releasing trend.

## In summary

After all the above experiments were completed, we discussed
all
the problems that occurred and carefully
recalled the entire process of designing the experiment. Finally, the designer of the
sequence found that an extra signal peptide was hidden in the front of our IL-2 sequence, which may be
used
to mediate the secretion of IL-2 by T cells. However, this signal peptide is superfluous to our yeast
and
may affect the conformation of the expressed protein, which in turn affects the function of the protein.

During this time, our previous animal model team members
received a
response from the iGEM Safety Committee
on animal experiment applications. Since it was the first time for our team members to design their own
animal experiments, many points were not perfect enough. Although the experimental ethics approval and
3R
report have been obtained, the feedback from the Safety Committee indicates that there is still much
room
for improvement in the experimental design.

As students majoring in Bioengineering and Pharmacy, we all
understand the many ethical issues that animal
experiments face and the inevitable harm to those animals. And iGEM, as an international synthetic
biology
academic event, does not want us to do unnecessary damage to life. Finally, we decided to give up the
animal experiment and design more cytology
experiments, trying to prove our experiment results in another way.

[![](https://static.igem.org/mediawiki/2019/2/2d/T--NJTech_China--snapgene.png)](https://www.snapgene.com)
[![](https://static.igem.org/mediawiki/2019/2/25/T--NJTech_China--genewiz.png)](https://www.genewiz.com.cn)
 [![](https://static.igem.org/mediawiki/2019/f/fd/T--NJTech_China--genescript.png)](https://www.genscript.com)
 [![](https://static.igem.org/mediawiki/2019/7/7a/T--NJTech_China--njtech.png)](http://www.njtech.edu.cn)
[![](https://static.igem.org/mediawiki/2019/3/37/T--NJTech_China--sg.png)](http://life-phar.njtech.edu.cn)
[![](https://static.igem.org/mediawiki/2019/f/f8/T--NJTech_China--yaoxy.png)](http://pharm.njtech.edu.cn)

### © 2019 NJTech\_China iGEM Team. All Rights Reserved.