Team:Tongji Shanghai/Notebook
Notebook
Part1 synthesis of UCNPs:
- Prepare mixture I and mixture II. Mix I is the mixture of MnCl2(0.5mL, 0.5M), Yb(NO3)3(0.9mL,0.2M), Er(NO3)3(0.1mL, 0.2M), Y(NO3)3(1.1mL, 0.5M) and the mix II is the mixture of NaOH(0.3g), deionized water (1.5 mL), oleicacid (5 mL) and ethanol (10 mL).
- Then, 2.0mL of deionized water contained 4 mmol of NaF was dropwise added to the mixture.
- After vigorous stirring at room temperature for 15 min, the colloidal solution weretransferred into a 25 mL Teflon-lined autoclave, sealed and heated at 200 °C for 8h.
- The systems were then allowed to naturally cool to room temperature.
- The finalproducts were collected by means of centrifugation, washed with ethanol anddeionized water for several times to remove any possible remnants, and then dried invacuum at 70 °C to obtain the dried UCNPs powder.
Part2 synthesis of GQDs:
- sodium copper chlorop(SCC)Use deionized water to prepare a concentration of 0.2M of SCC and heat it at 180℃ for 12h.
- Centrifugate(room temperature, 7000rpm, 10min) and remove the supernanant.
- Scrub the precipitate by deionized water 3 times.
- Add 20mLaqua regia and condensing reflux at 90℃ for 1h. After that, the precipitate would turn yellow.
- Scrub the precipitate by deionized water 3 times.
- Add 5.0mL hydrazine hydrate and stir. Then, the precipitate would back to black.
- Scrub the precipitate by deionized water 3 times.
- Store it with 2.0mL ddH2O at room temperature.
Part3 synthesis of UCNPs with SiO2 and GQDs
- Mix Co-520(0.1mL), cyclohexane(6.0mL) and UCNPs(4.0mL, 0.01M dissolved in cyclohexane) and stir for 10min.
- Add Co-520(0.4mL) and ammonia(0.08mL, 30%).
- Then obturate vessel and sonicate for 20min.
- Add absolute ethyl alcohol(2.0mL), GQDs(100μL), TEOS(0.04mL) and stir at 600rpm for 2 days.
- Add acetone until the solution becomes cloudy and centrifugate at 6000rpm for 10min.
- Scrub the precipitate by the mixture of deionized water and ethanol(volume ratio is 1:1) 2 times and store in deionized water.
Part4 photothermal property of integrative material
- We test the photothermal property of UCNPs, UCNPs with SiO2 and GQDs, UCNPs with SCC, GQDs, SCCat irradiation of 670nm and 980nm laser separately to confirm that the thermal effect is mainly caused by the SCC absorbing 670nm laser converted via UCNPs absorbing 980nm laser.
Part 1. Synthesis of AuNRs
- GNRs are synthesized through the seedless growth route. The growth solution is made from a stepwise mixing CTAB (cetyl trimethyl ammonium bromide) aqueous solution and HAuCl4 aqueous solution (1mM,5ml). Then add AgNO3 aqueous solution slowly with stirring, followed by AA aqueous solution, and stop stirring when the solution is transparent. Finally the growth of AuNRs is induced by injecting freshly-prepared ice-cold NaBH4 aqueous solution. The resultant solution needs to be kept undisturbed at room temperature for 6 hours.[2]
- The concentration and material volume are listed as following:
- CTAB(cetyl trimethyl ammonium bromide): 0.2M 5ml
- HAuCl4(chloroauric acid):1mM5ml
- NaBH4(sodium borohydrate):0.01M 15μl
- AA(ascorbic acid):78.8mM 70μl
- AgNO3(silvernitrate)：4mM 300μl
Part 2. Au concentration measurements
- The as-synthesized AuNRs is purified by three cycles of centrifugation to remove excessive Au ions. Afterwards, the precipitates are dissolved with aqua regia and diluted by deionized water. An ICP-OES (inductively coupled plasma optical emission spectrometer) is applied for measuring the ratio of reacted HAuCl4.
Part 3. Photothermal application of GNRs
- First, we test UV–vis-NIR extinction spectra of the AuNRs.
- Second, we test the photothermal effect of the AuNRs. We prepare a 200μl aqueous suspension of AuNRs with different concentration in anadiabatic box. Then irradiate with laser for 10 minutes. The laser we use is an 808nm laser generator. We test the AuNRs at different power density.
- Third, we test the photothermal stability of the AuNRs. We irradiate the GNRs solution from an ambient temperature to the top temperature for consecutive four cycles, testing whether the top temperature will change or not.
- We record the temperature every 1 minute.
Part 4. Surface modification
- In our experiment, we use poly ethylene glycol (PEG) to minimized dose-related side effects of single AuNRs.
- The treatment is mixing prepared AuNRs with PEG and leaving undisturbed at room temperature for 24h. The final concentration of PEG is 0.8mg/ml.[3]
Part 5. Material toxicity test
Results:
- We get AuNRs with an aspect ratio of~3.9(41.9 × 10.6 nm), the transmission electron microscopy(TEM) image is shown as figure1(a),the TEM image clearly indicates that the products are single crystals and the shape is quite even.
- As to the AuNRs with the aspect ratio of 3.9, two LSPR peaks at 546 and 770 nm are observed (figure 1(b)).The former arises from the transverse resonant oscillation, while the latter results from the resonant oscillation[1], the LSPR range of the AuNRs is wide in near infrared region.
- The temperature variation of the GNRs is revealed by irradiating a 200μl aqueous solution using an 808 nm laser. The temperature increases obviously(figure 1(c)) as the increase of the GNRs concentration. In addition, as the laser power density increases, the temperature increment is higher, because more energy is absorbed by the solution (figure 1(d)).Figure 1(e) shows that the top temperature is almost unchanged after four cycles, which indicates the photothermal stability of GNRs. These results indicate that the GNRs synthesized by us possess good photothermal property and stability.
Biosafety:
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The photothermal stability test result indicates that the AuNRs exhibits excellent photothermal stability. So it can be irradiated by laser many times with its properties unchanged. The cyto-toxicity of CTAB-GNRs is independent of their aspect ratio[4]. And the cell toxicity experiment shows that the toxicity of the AuNRs is dose-related.When the AuNRs concentration is low, it doesn’t show obvious cell toxicity, but it exhibits cell toxicity with the concentration increase. The cell toxicity is the result of the AuNRs caninduce cell apoptosis and autophagy by damaging mitochondria and activating intracellular reactive oxygen species (ROS).[4]Surface modifications have been extensively used to improve the biocompatibility and stability of gold nanorods. Our results show that with the surface modification of PEG, the toxicity of the AuNRs decreased dramatically. In a word, all of the results testify that our material have well-controlled biosafety both in material stability and its cell toxicity.
Figure1a
Figure1b
Figure1c
Figure1d
Figure1e
Figure1.(a)The TEM image of the AuNRs. (b)The UV–vis-IR absorption spectrum.(c)The temperature incrementwith different concentration of AuNRs.The power density of the 808 nm laser is fixed at 2.80W/cm2.(d)The temperature increment with three different laser power density. The concentration of the AuNRs is 100 μg/ml. (e)The temperature variation of 19.2 μg/ml AuNRs suspension as irradiated by the 1.80 W/cm2 808 nm laser for four cycles.
Plasmid part
2015.10.12
Reading papers to find appropriate genes.
2015.10.30
Find the ideal gene p53 ,promoters of hTert and HSP.
2015.11.5
Design the PCR primers of P53.
2015.11.14
Synthesis of promoter of hTert and HSP.
2015.11.20
The amplification of p53 gene (use taq PCR protocol)
AGE ( agarose gel electrophoresis )
Gel extraction of p53 gene.
Transformation of p53 gene in E•coli DH5α.
Transformation of vector (pGL3)
2015.11.21
Select a single clone from the plate (pGL3) .Put the E.coli in 4ml LB medium with Ampicillin and culture for one night at 37℃.
Plasmid extraction of E.coli DH5α with pGL3 in it.
enzyme cleavage the pGL3 using BamHI and EcoRI.(digestion protocol)
AGE ( agarose gel electrophoresis ) of digested vector---pGL3.
Gel extraction of pGL3
purification of p53 witch has been digested with BamHI and EcoRI.
2015.11.22
Ligation of pGL3, HSP promoter and p53. (ligation protocol)
Transformation of ligation product: HSP-p53 (in pGL3).
2015.11.23
Select a single clone of plate. (HSP-p53, pGL3) .Put the E.coli in 4ml LB medium and culture for 18h at 37℃.
Plasmid extraction of HSP-P53(pGL3).
2016.1.7
PCR using the new plasmid as a template
AGE ( agarose gel electrophoresis ) of the product of PCR
(it worked! Our first part is done!)
2016.1.10
Transformation of HSP-P53(pGL3) in order to get more plasmid.
2016.1.20
Transformation of vector (FUGW)
2016.1.21
Select a single clone from the plate (FUGW) .Put the E.coli in 4ml LB medium with Ampicillin and culture for one
night at 37℃.
Plasmid extraction of E.coli DH5α with FUGW in it.
enzyme cleavage the FUGW using BamHI and EcoRI.(digestion protocol)
AGE ( agarose gel electrophoresis ) of digested vector---FUGW.
Gel extraction of FUGW
purification of p53 and GFP gene witch has been digested with BamHI and EcoRI.
2016.1.28
Ligation of FUGW, hTert promoter and p53 or GFP. (ligation protocol)
Transformation of ligation product: hTert-p53 and hTert-GFP (in FUGW).
2016.1.29
Select a single clone of plate. (hTert-p53, FUGW; hTert-GFP, FUGW) .Put the E.coli in 4ml LB medium and culture for 18h at 37℃.
Plasmid extraction of hTert-P53 and hTert-GFP(FUGW).
2016.1.30
PCR using the new plasmid as a template
AGE ( agarose gel electrophoresis ) of the product of PCR
Transformation of hTert-P53 and hTert-GFP (FUGW) in order to get more plasmid.
2016.3.5
Design the primer of luciferase
2016.3.20
The amplification of luciferase gene (use taq PCR protocol)
AGE ( agarose gel electrophoresis )
vGel extraction of luciferase
2016.3.22
Ligation of pGL3, HSP promoter, p53 and luciferase. (ligation protocol)
Transformation of ligation product: HSP-p53-luciferase (in pGL3).
2016.3.23
Select a single clone of plate. (HSP-p53-luciferase, pGL3) .Put the E.coli in 4ml LB medium and culture for 18h at 37℃.
Plasmid extraction of HSP-p53-luciferase (pGL3).
2016.4.1
PCR using the new plasmid as a template
AGE ( agarose gel electrophoresis ) of the product of PCR
2016.4.3
Transformation of HSP-p53-luciferase (pGL3) in order to get more plasmid.
2016.5.10
Transfect the plasmid hsp70-p53 into hcc1937 cell by PEI(cotransfected GFP)
2016.5.12
Confirm the efficiency
2016.5.13
Heat shock the cell
2016.5.14
Extract the cell for western blot
2016.5.15
Western blot
2016.6.20
Transfect the plasmid hsp70-p53 into 293T cell by PEI(cotransfected GFP)
2016.6.23
Confirm the efficiency
2016.6.24
Heat shock the cell
2016.6.25
Extract the cell for western blot
2016.6.27
Western blot
2016.7.10
Deliver the plasmid hTert-p53 and hTert-GFP into hcc1937,hela and myeloma cell by lentivirus packaging
2016.7.13
Observe the cell at fluorescent microscope
2016.8.17
Deliver the plasmid hTert-p53 and hTert-GFP into hcc1937,hela and myeloma cell by lentivirus packaging again for the efficiency is not high
2016.8.19
Observe the cell at fluorescent microscope
(As our expectancy, GFP was observed in hcc 1937 and hela but not the negative control, which means the promoter
can help the specific promotion of certain gene in most cancer cells.
2016.9.10
Make Backbone, transformation of backbone. Culture at 37℃ for 16h. (according by protocol offered by iGEM)
Digest HSP-p53, HSP-p53-luciferase with XbaI and Spel.(digestion protocol)
2016.9.11
Plasmid Extraction(backbone)
2016.9.12
Digest backbone with XbaI and Spel
2016.9.13
Ligation of HSP-p53, HSP-p53-luciferase and backbone (ligation protocol)
2016.9.14
Transformation of HSP-p53, HSP-p53-luciferase, Culture at 37℃ for 16h.
2016.9.15
Select single clone from culture plate (HSP-p53, HSP-p53-luciferase,).And culture for 12h.
2016.9.16
Select single clone from culture plate.(Backbone)
Plasmid Extraction (HSP-p53, HSP-p53-luciferase, hTert-p53, hTert-GFP).
Plasmid Extraction (Backbone).
Digest of hTert-p53, hTert-GFP using XbaI and Spel.(digestion protocol)
2016.9.17
Make sure that the gene had been successfully ligated into the plasmids.
Digest of backbone with XbaI and Spel.(digestion protocol)
Make sure our backbone is made in the right way.
Material part
2015.10.10
Read papers about AuNRs
2016.3.15
Synthesize the AuNRs through a seedless route
2016.3.16
Purify the AuNRs
Measure the reacted HAuCl4
016.4.10-2016.4.20
Material toxicity test(cell experiment)
2016.5.1-2016.5.8
Test the rise of temperature at different power density( the amount is ranged from 1 to 100ug/ml)
2016.7.9-2016.7.20
Test the photothermal stability of the AuNRs
2016.8.10
PEG modification of the material
Cell experiment
In vitro cytotoxicity test
2016.8.20
Seed the 293T cells 2016.8.21
Add different amout of PEG-AuNRs to the cell
Incubate for 12h
2016.8.22
Test the cell viability
2016.9.1
Seed the hcc1937 cells
2016.9.2
Add different amout of PEG-AuNRs to the cell
Incubate for 12h
2016.9.3
Test the cell viability
2016.9.4
in vitro photo thermal effect
Seed the 293T cells
2016.9.5
Change the medium
Incubate for 12h
Test the cell viability
2016.9.20
Seed the 293T cells
2016.9.21
Change the medium(with or without AuNRs)
Incubate for 2h
2016.9.22
Test the cell viability
2016.9.28
Seed the 293T cells(infected with plasmids or with control)
2016.9.29
Change the medium(with or without AuNRs)
Incubate for 2h
2016.9.30
Test the cell viability
2016.10.1
Seed the hcc1937 and hela cells(infected with plasmids or with control)
2016.10.2
Change the medium(with or without AuNRs)
Incubate for 2h
Test the cell viability
2016.10.3
Seed the hcc1937 and hela cells again (infected with plasmids or with control)
2016.10.4
Change the medium(with or without AuNRs)
Incubate for 2h
2016.10.5
Test the cell viability
Mouse experiment
2016.9.18
Breed the mice of similar situation for 2 weeks
2016.9.28
Tumor transplants
2016.10.3
Measure the size of tumor
2016.10.5
Measure the size of tumor
2016.10.7
Measure the size of tumor
2016.10.11
Measure the size of tumor
2016.10.12
Measure the size of tumor
Inject the plasmids or PBS
2016.10.13
Inject the AuNRS
2016.10.14
Measure the size of tumor
2016.10.16
Measure the size of tumor
2016.10.18
Measure the size of tumor
Timeline
2015.11Read papers; brain storm;
2015.12
12.27 visit Shanghai Jiaotong University.
2016.1
Find out the project; search for the ideal gene p53 and the promoter hsp; begin to make out the plasmid.
2016.2
Keep working on build our plasmid; read papers and begin to make our next plasmid: hTert-P53.
2016.3
Build the material AuNRs. Make another two plasmids hsp-p53-luciferase and hTert-GFP to show the efficiency.
2016.4
Cell experiment with material.
2016.6
6.19 Visit New York University Shanghai.
2016.7
Cell experiment with plasmids hsp-p53 and material.
2016.8
Cell experiment with plasmids hTert-p53 and material.
8.6 Participate in Taipei Workshop in National Yang-Ming University; 8.8-8.14 participate in the 4th iGEM Asia-Pacific Conference 2016 at National Cheng Kung University; 8.16 participate in a biology-educational exhibition in Shanghai Science and Technology Museum
2016.9
Mice experiment Preparation.
9.30 Hold a biology-educational exhibition for No.2 Middle School Affiliated to Tongji University
2016.10
10.10 Hold a simple project-introducing exhibition in Tongji Hospital Affiliated to Tongji University; Mice experiment. Analyze our data. Write the result on wiki. Send our biobricks to IGEM.
