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Notebook
Work record.

Brief Calendar

Apr-19

Administrative Work
Brain storms were hold to identify the project. The project was confirmed to be the detection and therapy of breast cancer based on synthetic receptor.

Laboratory Work
Some preliminary experiments were performed.

Human Practice
Went to Henan to provide medical services and popularize the concept of synthetic biology.

Mar-19

Administrative Work
Started to have regular meetings every week. Completed team registration for iGEM 2018.

Laboratory Work
Multiple plasmids were under construction.

Human Practice
Articles to popularize and explain synthetic biology was published on Wechat public accounts of SMMU-China.

Jun-19

Administrative Work
Attended the iGEM Seminar hold by the Third Military Medical University in Chongqing.

Laboratory Work
IL-15 expression by E.coli and HEK-293 were performed.
EGFR and HER2 plasmids were transfected into MCF-7 cells.

Human Practice
Explained synthetic biology to elderly people.
Met with Lu Jian, the chief of the department of pathology, Changhai Hospital.

Aug-19

Administrative Work
Attended the 5th CCIC(Conference of China iGEMer Community).

Laboratory Work
IL-15 proteins with 2x, 4x and 6xsuntag were largely produced by E.coli.

Human Practice
Promoted synthetic biology and iGEM to high school students.
Went to Henan Province as volunteer teachers again.

Sep-19

Administrative Work
Started to write Wiki.

Laboratory Work
The function of synNotch receptor was confirmed.

Oct-19

Laboratory Work
The function of GEMS receptor was confirmed.

Timeline

    • The first group meeting of all members was hold. Group members were encouraged to raise ideas about the project this year. After discussion of students and instructors, the theme of project this year was identified: the detection and therapy of breast cancer based on synthetic receptor. Web group members including Wang Chuqi, Zhu Mengmei, Song Juan, Yang Qiliang, Qian Kewen, Zhang Zheng started to design the experiments under the instruction of Pro.Hu. And dry group members began to design logo, software and modeling.

    • All members of wet group were introduced to the lab. The specific scheme of the experiments was not confirmed yet. All members of wet group were assigned to read literature and propose plans. At the same time, some preliminary experiments were performed to make group members familiar with the basic experimental procedures.

    • Yang Qiliang, Song Juan, Wang Chuqi and Liu Shuowu carried out the experiment of total RNA extraction, cDNA synthesis and PCR. The protocols were listed here.

    • Several meetings and multiple discussion was hold between students and instructors. On the third group meeting of all members, Wang Chuqi proposed that our project this year should be composed of two main parts. One is core device with the function of killing cancer cells and transmission signals. Another is external device that could accept upstream signals to achieve multiple functions such as dynamic monitoring.

    • Meanwhile, wet group members continued doing preliminary experiments. Zhu Mengmei performed bacterial transformation. Liu Shuowu and Wang Chuqi carried out plasmid isolation and agarose gel electrophoresis.

    • After several rounds of brain storms, the receptor used in core device was determined as SynNotch and CAR (Chimeric Antigen Receptor) receptor which is able to detect antigens on the surface of tumor cells. And the receptor in external device was GEMS receptor, which could identify the soluble antigens. And the plasmids needed was listed and assigned to every single member. In the next period, construction and isolation of multiple plasmids listed here was our primary mission.

    • At this time, the wet group was further divided into two subgroup. Subgroup one included Wang Chuqi, Song Juan, Yang Qiliang, which were mainly responsible for experiments of artificial promoters synNotch receptor and CAR receptor. Subgroup two consisted of Liu Shuowu, Zhu Mengmei, Qian Kewen and Zhang Zheng, which performed experiments about GEMS receptor, IL-15 expression and reporting gene expression.

      •

    • At this period, all members of wet groups were constructing the plasmids. Because some of our members were freshmen in our university, they were not familiar with the molecular biological techniques. Therefore, at first, experimental progress was out of scheme. But after about 3 weeks, our members have been very skilled at doing basic experiments. And half plasmids of our experiments have been constructed at this point.

    • The construction of blue chromoprotein expressing plasmid was finished by Qian Kewen, Zhu Mengmei and Yang Qiliang. And the sequencing was confirmed right. Then the transforming was performed. To explore the best expression condition of blue pigment protein in E.coli. The concentration of IPTG was set to 0, 0.5, 1.0, 1.5, 2.0 mmol/ul, then shaking the bacteria for 3.5 hours. Then 2ml of bacteria culture was added into an EP tube and centrifuged to precipitate bacteria. Then we observed that only culturing with IPTG of 2.0 mmol/ul enabled the expression of blue chromoprotein.

    • Wang Chuqi, Liu Shuowu and Zhang Zheng started to culture NK92 and MCF-7 cells. MCF-7 cells were easy to culture, but NK92 need α-MEM adding IL-2 medium. We are still exploring the proper ways to culture NK92 cells.

      •

    • To further confirm the expression of blue chromoprotein in E.coli, Qian Kewen, Zhangzheng and Zhu Mengmei performed the SDS-PAGE protein electrophoresis. The protein sample was prepared using bacteria cultured with different concentrations of IPTG. The results was listed here, and we could see the expression of blue chromoprotein was maximal under the IPTG of 2.0 mmol/ul.

    • Others were still working on the construction of plasmids. Wang Chuqi succeeded to construct the CMV-eGFP and CMV-mcherry plasmids. The construction of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) plasmids have been finished by Wang Chuqi, Liu Shuowu and Song Juan. And they transfected the plasmids into MCF-7 cells respectively.

      •

    • Wang Chuqi and Zhang Zheng started to transfected CMV-eGFP and CMV-mcherry plasmids into HEK-293 cells to see if HEK-293 cells could express fluorescence signal. The results were listed here. We observed that obvious protein expression was seen under microscope. And if we digested and centrifuged the transfected cells into cell mass, it can transmit different fluorescence under different excited light.

    • The CAR receptor plasmids were transfected into NK92 cells by Qian Kewen, Zhu Mengmei and Song Juan. Then we used flow cytometry to measure the expression level of CAR on NK92 cells’ surface.

      •

    • Cell lines with various HER-2 expression levels were determined by flow cytometry by Zhang Zheng and . These cells will be co-cultured with core device, to see if engineered cells with synNotch receptors could detect cells with different HER-2 levels. Six cell lines including MCF-7, CHO, HEK-293, A549, SK-BR3 and H1975. And from the results, we could concluded that HEK-293 have little HER-2, assuring it could be a tool cell line. SK-BR3 have highest HER-2 expression while MCF-7, CHO and H1975 cell lines have middle HER-2 expression level.

    • Wang Chuqi, Liu Shuowu and Song Juan cultured sorted synNotch NK cells, and CAR-NK cells with MCF-7 EGFR, MCF-7 HER2 to test recognition of different antigen expressing tumor cells. Both CTZ.CAR-NK92 cells and TTZ.CAR-NK92 cells effectively killed EGFR+ or HER2+ cancer cells in 4 and 24 h co-cultures. By contrast, synNotch NK-92 cells recognized cancer cells expressing EGFR or HER2, and comparable lytic activity to CAR-NK-92 cells required 24 h of co-culture, consistent with previous reports showing that 12–24 h is required to fully upregulate CAR expression after engagement of the synNotch receptor.

      •

    • This week, Wang Chuqi, Liu Shuowu and Zhang Zheng performed multiple experiments in order to check the functions of GEMS receptor. First, we transfected CMV-IL-15-suntag into HEK-293 cells. Then we collected the culture medium 48 hours after transfection. And meanwhile CMV-anti IL-15-GEMS and pSTAT-eGFP plasmid were co-transfected into HEK-293 cells. Then we added the culture medium containing IL-15-suntag into co-transfected HEK-293 cells. However, we did not get the positive results.

    • In fact, there are several problems in the experiments we performed this week. On the one hand, we cannot confirm the expression of IL-15-suntag in the culture medium. On the other hand, we were not able to make sure that GEMS receptor was expressed on the surface of transfected cells. So we would repeat the experiments step by step.

    • The transfection of IL-15-suntag was both performed on HEK-293 cells and bacteria. It is a pity that the IL-15 expression of transfected HEK-293 cells was too little to be detected (if there were any). So Yang Qiliang, Song Juan and Zhu Mengmei chose to use E.coli to manufacture the IL-15 protein with suntag. We transfected IL-15-suntag and Blue chromoprotein plasmid into E.coli. Nevertheless, we did not see any protein expression on SDS-PAGE gel.

    • In addition, the presence of antigen was sufficient to induce secretion of the IL-2-Tag protein at 24h, which is an indicator of the medical conditions and is a signal molecule to trigger the response of external device cells(Figure 5b).

    • This week we were still working on the expression of IL-15-suntag in E.coli. Yang Qiliang, Qian Kewen, Zhu Mengmei and Song Juan decided to use blue chromoprotein to explore the conditions that enabled E.coli to produce large amount of protein. From the SDS-PAGE gel, we could see that shake culturing for 3.5 hours and 4.5 hours were suitable for E.coli to express blue chromoprotein. And then we transfected IL-15-suntag in E.coli under this condition, then we could see obvious production of IL-15-suntag protein.

    • Wang chuqi and Qian Kewen constructed the anti-IL-15-GEMS-eGFP plasmid.

      •

    • This week, Wang Chuqi, Zhang Zheng and Zhu Mengmei decided to explore that if GEMS receptor could be expressed on the surface of HEK-293 cells. So we would like to transfected the anti-IL-15-GEMS-eGFP plasmid into HEK-293 cells. After 24 and 48 hours, we will observe the fluorescence intensity of the transfected cells. However, 24 hours after transfection, we found that cells were polluted by bacteria. So we had to stop the experiments. We decided to repeat experiments in the next few weeks.

    • To further explore the best conditions of E.coli to express of the IL-15 protein with 2x, 4x, 6x and 8x suntag, we have cultured the transfected bacteria under inducing temperature of 37°C and 16°C with IPTG concentration of 1mM. Then we could see that 37°C was better for protein expression. Until this time, we have completed learned the best conditions of E.coli expression IL-15 proteins.

      •

    • Yang Qiliang, Song Juan and Qian Kewen started to manufacture IL-15 protein with 2x、4x、6x suntag. Then the protein was purified and measured by SDS-PAGE and ELISA. From the ELISA, we could see that IL-15-suntag protein could be produced largely by E.coli. Thus, we collected the products of the transfected bacteria. These proteins will be added into GEMS transfected HEK-293 cells to test anti-IL-15 and anti-suntag GEMS receptors.

    • Anti-IL-15-GEMS-eGFP and Anti-suntag-GEMS-eGFP plasmids were transfected into HEK-293 cells by Wang Chuqi and Zhang Zheng. We could see fluorescence under microscope, which meant that GEMS receptor did express on the surface of HEK-293 cells. Then we transfected Anti-IL-15-GEMS, Anti-suntag-GEMS and pSTAT-eGFP into HEK-293 cells. Unluckily, we could not observe fluorescence this time. Maybe the GEMS receptors we constructed had some problems.

    • At the same time, Zhu Mengmei, Qian Kewen and Yang Qiliang were trying to use HEK-293 to express IL-15 proteins. We transfected IL-15 with 2x, 4x, 6x suntag into HEK-293 cells. Then we collected the culture medium and tested IL-15 concentration by ELISA. The concentration was very low, about 50 pg/ml, which could not be detected by GEMS receptor.

    • This week, Wang Chuqi, Zhu Mengmei and Song Juan planned to test the function of synNotch receptors. Anti-HER2-synNotch-Gal4 with 5xUAS-mCherry, Anti-HER2-synNotch-PIP-8 with 8xPIR-mCherry, Anti-HER2-synNotch-ZF21-16 with 8xZF21-16-mCherry and Anti-HER2-synNotch-ZF43-8 with 8xZF43-8-mCherry were transfected into HEK-293 cells. Then transfected HEK-293 cells was co-cultured with SK-BR3 cells. We could see fluorescence under microscope, which meant that synNotch receptor could detect the expression of HER2.

    • CMV-5xUAS-mCherry, CMV-8xPIR-mCherry, CMV-8xZF21-16-mCherry and CMV-8xZF43-8-mCherry was transfected into HEK-293 to measure the expression of transcription factors using fluorescence.

    • SynNotch transfected HEK-293 cells were co-cultured with SKBR3、231 and MCF7 cell lines by Wang Chuqi, Zhu Mengmei and Song Juan. We expected that the fluorescence of transfected HEK-293 cells were different. Nevertheless, the fluorescence under three co-culture conditions were similar. The reasons were still at investigation.

    • We started working on the Wikis. And the left experiments were finished in this period.

Protocols

Total RNA extraction, cDNA synthesis

1. The spleen of a mouse was separated and pulverized. Then digestive ferment was added to separate the tissue into single cells.

2. 48 h post-transfection, the total cellular RNA is extracted using a Total RNA Extraction Kit, according to the manufacturer’s protocol stated as below.

3. Wash cells three times with the PBS. Strip cells with cell scraper, collect cells in a nuclease-free EP tube by centrifugation at 1000 rpm for 5 minutes, and then discard the supernatant.

4. Add 100 μl of RNA lysate to the cell pellet, resuspend the cells with a pipette, transfer the lysate into a 1.5 ml nuclease-free EP tube, then add 100 μl of the dilution, and mix the mixture by inverting four times.

5. Incubate the lysate at 70°C for 3 minutes to increase the RNA yield.

6. Centrifuge at 14,000 x g for 5 minutes. Carefully aspirate the supernatant into a 1.5 ml nuclease-free EP tube.

7. Add 0.5 times the supernatant volume of anhydrous ethanol, mix 4 times with a pipette.

8. Remove the centrifuge column/collection tube, and transfer the mixture to the centrifuge column. If the volume of the mixture is greater than 750 μl, add the mixture in batches.

9. Centrifuge at 14,000 x g for 1 minutes, discard the filtrate, and put the centrifuge column back into the collection tube.

10. Add 600 μl wash buffer to the column, centrifuge at 14,000 x g for 45 seconds, and discard the filtrate.

11. Take a nuclease-free tube, add 5 μl of 10 x DNase I buffer, 5 μl of DNase I, and 40 μl of nuclease-free water, and then gently mix them. The amount for extraction of a tube of RNA required by DNase I incubation is 50 μl.

12. Add 50 μl of freshly prepared DNase I incubation solution to the centrifuge tube and incubate at room temperature for 15 minutes.

13. Add 600 μl RNA wash buffer to the column, centrifuge at 14,000 x g for 45 seconds, and discard the filtrate.

14. Repeat step 13 once. And then centrifuge at 14,000 x g for 2 minutes.

15. Transfer the centrifuge column into the elution tube, add 50 μl of nuclease-free water to the spin-off membrane, incubate at room temperature for 2 minutes, and centrifuge at 14,000 x g for 1 minute.

16. Add the elution solution to the center of the column, incubate at room temperature for 2 minutes, and centrifuge at 14,000 x g for 1 minute to elute RNA again.

17. Determine the concentration of RNA by measuring the absorbance at 260 nm.

18. Prepare the reverse transcription reaction system (20 μl) as follows to synthesize cDNA:


5 x RT Master Mix (TaKaRa, Japan) RNA RNase-free ddH2O
4 μl 2 μg add up to 20 μl

Plasmid Isolation

1. Each student labels two 1.5ml Centrifuge tubes with his or her name.

2. Transfer 1.5ml overnight culture into labeled 1.5ml centrifuge tube.

3. Centrifuge at high speed for 3-5 minutes to pellet bacteria. Pour off the supernatant.

4. Add 200μl Cell Suspension Solution and mix the contents by flicking the tube or pipetting several times. This solution contains Tris (pH 7.5), and EDTA (ethylenediaminetetraacetic acid). The basic pH helps to denature the DNA and the metal ion chelator, EDTA, stabilizes the cell membrane by binding the divalent cations of Mg2+ and Ca2+. RNase can also be added at this stage to degrade the RNA when the cells are lysed.

5. Incubate the vial on ice for 15 minutes.

6. Add 200μl Lysis Buffer and mix the contents by gently inverting the tube 4-5 times. This solution contains sodium hydroxide and SDS (sodium dodecyl sulfate). The sodium hydroxide denatures the plasmid and chromosomal DNA into single strands. SDS, an ionic (charged) detergent dissolves the phospholipids in the membrane causing lysis and release of the bacteria contents, including the DNA, into the solution.

7. Add 200μl Neutralization Buffer and mix the contents by inverting the tube 4-5 times. This is a potassium acetate solution. The potassium acetate causes the precipitation of a SDS-protein complex as a white precipitate, consisting of SDS, lipids and proteins. In addition, the potassium acetate neutralizes the solution allowing the renaturation of the DNA. The large chromosomal DNA is captured in the precipitate, whereas the small plasmid DNA remains in solution.

8. The tube is then centrifuged for 10 minutes at high speed (>5,000xg).

9. Transfer the clear liquid, or supernatant, to a fresh labeled 1.5ml tube. This can be stored for up to a week at 4°C.

10. Alcohol Precipitation: To precipitate the plasmid DNA, add 480μl (0.8 volumes) Precipitation Solution (isopropanol) to the supernatants from step 9. Ethanol can be used instead of isopropanol and should be used at 2.5 volumes.

11. Mix the tubes by inverting 5 times. Place the tubes at -20°C for 10 minutes.

12. To pellet the plasmid DNA centrifuge at full speed for 15 minutes.

13. After centrifugation, examine the tubes for a small white pellet of plasmid DNA. Pour off the supernatants.

14. Add 300μl DNA Wash (70% isopropanol) to the pellets to wash away any excess salt. Centrifuge the tubes at full speed for 5 minutes.

15. Carefully remove the supernatants with a pipette and leave the tubes open on your bench to allow all residual alcohol to evaporate.

16. Once all alcohol has evaporated, add 50μl TE buffer to the pellets. Wait 2 minutes then vortex to resuspend the DNA. This is your purified plasmid DNA.

17. The DNA can be run on an agarose gel to visualize the DNA or can be subjected to restriction digestion analysis and then agarose electrophoresis to check the plasmids.



Transformation

1. Put the tube of E. coli cells on ice until the last crystals disappear. And add 1-5 ul containing 1-100ng plasmid DNA to 50 ul of cells in a transformation tube on ice.

2. Place the mixture on ice for 30 minutes and never shake it.

3. heat shock at exactly 42°C fors 90 seconds. Then pipette 500 ul LB into the mixture and put the tube at 37°C for 60 minutes and shake vigorously.

4. warm the plates containing Ampicin to 37°C. Then spread the mixture onto the plate and incubate at 37°C for 10-12 hours.

5. Picking up monoclonal colonies into 4ml LB broth containing Ampicin and put the tube at 37°C for 8-10 hours and shake vigorously.

6. send the LB broth to bio-company to sequence the DNA plasmid. If the sequence is right, we can extract the plasmid from bacterial for next experiments.



Agarose Gel electrophoresis

1. Preparing the agarose gel

  • Measure 1.0g of agarose powder and add 100 mL of TAE 1X;
  • Melt in a micro oven until solution becomes clear;
  • Let it cool;
  • Add 1µL Gelred;
  • Pour the melted agarose solution into the casting tray and let cool until it is solid.

2. Loading the gel

  • Add enough TAE 0.5 buffer so that there is about 2-3 mm of buffer over the gel;
  • Add 10X loading buffer to each PCR reaction;
  • Pipette 30µL each sample into separate wells in the gel;
  • Pipette 5µL of DNA ladder standard into at least one well.

3. Running the gel

  • Turn on the power supply to about 140 volts let it run for about 20 minutes.

4. Revelation

  • Put the gel under UV;
  • Make a copy of the image;
  • Get the gel containing the DNA we need.


Preparation of LB Broth

1. Add 10g Trytone;

2. Add 5g yeast extract;

3. Add 10g sodium chloride;

4. Add 1L purified water;

5. Use autoclave to sterilize.



PCR

1. Add the following component as listed below

Component prime STAR dNTP 10 µM Forward primer 10 µM Reverse primer DNA Template (10 ng/µL) ddH2O
50 µL reaction 10 4 2 2 Less than 200ng Up to 50

Assemble all reaction components on ice and quickly transfer the reactions to thermocycle preheated to 94 °C and begin thermocycling.


2. Thermocycling conditions for a routine PCR.

Cycling step Denaturation Denaturation Annealing Extension Final extension Hold
Temperature 98°C 98°C X°C 72°C 72°C 16°C

Restriction Digest of Plasmid DNA

Component Target DNA Restriction enzyme I Restriction enzyme I ddH2O
Volume Variable (~50 ng) 1 μL 1 μL Add to 10 μL

1. Add all the components listed in the table above to a microcentrifuge tube.

2. Incubate the ligation mixture at 37°C overnight.



Ligation

Component Vector DNA Insert DNA 10 × T4 DNA ligase buffer T4 DNA Ligase ddH2O
Volume Variable (~50 ng) Variable (molar ratio of vector:insert=1:3) 2 μL 1 μL Add to 20 μL

1. Add all the components listed in the table above to a microcentrifuge tube.

2. Incubate the ligation mixture at 16°C for 1h.



Cell culture

All of the procedure about cells must be carried out under sterile conditions.

1. Resuscitation cell. Put the tube of frozen cells into 37°C water within 1-2 minutes. Then transfer the cells into 15 ml falcon containing 10 ml of DMEM and centrifuge cells at 180g for 5 minutes at room temperature. Pour off the supernatant and use 8ml fresh DMEM (10% fetal bovine serum ) resuspend the cells. Transfer the cells to T75 flask and put the flask in a 37°C incubator at 5% CO2.

2. Passage of the cells. Monitor the cell density daily. When culture reaches 80% confluence, cells should be passaged.

Prewarm the DMEM and trypsin-EDTA solution at room temperature. Remove the medium from the flask and wash cells once with 5ml of DMEM (with no FBS). Add 1 ml trypsin-EDTA into the flask. After 1-1.5 minutes, add 5ml of DMEM (with 10% FBS) and resuspend softly. Then transfer the cells into 15ml falcon and centrifuge at 150g for 5 minutes. Pour off the supernatant and use 14ml fresh DMEM (10% fetal bovine serum ) resuspend the cells. Transfer the cells to two T75 flasks and put the flasks in a 37°C incubator at 5% CO2.



Plasmid transfection

1. Dilute DNA in 250 μl of Opti-MEM I Reduced Serum Medium without serum (or other medium without serum). Mix gently.

2. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in 250 μl of Opti-MEM I Medium (or other medium without serum). Mix gently and incubate for 5 minutes at room temperature.

3. After 5 minutes incubation, combine the diluted DNA with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-Lipofectamine 2000 complexes to form.

4. Add the 500 μl of DNA-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.

5. Incubate the cells at 37°C in a CO2 incubator for 24-72 hours until they are ready to assay for transgene expression. It is not necessary to remove the complexes or change the medium; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.



Flow cytometry

1. Spin down cell suspension at 1000 RPM for 5 minutes and decant supernatant. Resuspend the pellet in 1X PBS. Count the cells with a hemocytometer. Add the total desired number of cells to a flow tube. Wash the cells by adding ~1 ml (or more if many samples) of 1X PBS to the flow tube. Spin down cell suspension at 1000 RPM for 5 minutes and decant supernatant. Gently tap the tube to loosen the cell pellet. Add an appropriate volume of staining buffer (generally 50 ul per 1 x 106cells). Add 1 x 106 cells (generally 50 ul) to the desired number of flow tubes.

2. Add the full amount of antibody to 50 ul of staining buffer and add this to the 50 ul of cell suspension, pipetting up and down to mix. Unlabeled primary antibody: Incubate on ice for 30-60 min.

3. Add ~1 ml of staining buffer and spin down cells at 1000 RPM for 5 min. Decant supernatant and wash cells twice with 1-2 mls of staining buffer. Resuspend each cell pellet with 100 ul of secondary antibody solution. Incubate on ice for 30 min; protect from light during incubation.

4. Wash cells twice with 1-2 ml of staining buffer. After the final decanting, resuspend stained cells in an appropriate amount of staining buffer. Acquire data on a flow cytometer following manufacturer`s recommendations.


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