iGEM 2023 — Team guangxi-u-china

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{% block title %}Description{% endblock %}
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![](https://static.igem.wiki/teams/4581/wiki/left7.png)

* [Genetic engineering of bacteria](#div1)
* [Metabolomic and proteomic analysis of lactic acid-adaptive ecn](#div2)
* [3D modeling of tnbc tumor spheroids.](#div3)
* [Chemical modification of bacteria with MnO2@AuNPs nanoparticles](#div4)

Notebook

1. Genetic engineering of bacteria for the expression of SIRPα that can specifically recognize CD47 protein on
   the surfaces of tumor cells.
2. Metabolomic and proteomic analysis of Escherichia coli Nissle 1917 (EcN) that live on lactic acid.
3. 3D modeling of TNBC tumor spheroids.
4. Chemical modification of bacteria with MnO2@AuNPs nanoparticles.

Subject 1: Genetic Engineering of Bacteria

1.Apparatus and reagents

|  |  |
| --- | --- |
| 实验耗材  Apparatus | 厂商  Manufacturer |
| T25/T75 cell culture flask | Corning |
| FBS | Gibco |
| 2mL RNase free EP | Eppendorf |
| DMEM | Gibco |
| PBS | Gibco |
| HS | Gibco |
| P/S | Gibco |
| 0.25% Trypsin-EDTA | Beijing Solarbao Technology Co., Ltd. |
| Total RNA Extractor (Trizol) | Sangon Biotech (Shanghai) Co., Ltd. |
| MightyScript First Strand cDNA Synthesis Master Mix | Sangon Biotech (Shanghai) Co., Ltd. |
| LB | Sangon Biotech (Shanghai) Co., Ltd. |
| PCR Kit | Sangon Biotech (Shanghai) Co., Ltd. |
| Tris-HCL | Sangon Biotech (Shanghai) Co., Ltd. |
| L-Cys | Beijing Solarbao Technology Co., Ltd. |
| DTNB | Macklin |
| Na2HPO4（pH＝7.0） | Sangon Biotech (Shanghai) Co., Ltd. |

|  |  |
| --- | --- |
| 仪器名称  Instrument | 厂商  Manufacturer |
| Ultrasonic cell breaker | Ningbo Scientz Biotechnology Co.,Ltd |
| High-speed cryogenic centrifuge | Hunan Xiang Yi Laboratory Instrument Development Co., Ltd. |
| MILI-Q Water Purifier | Shanghai Canrex Analytic Instrument Co.,Ltd Pudong Shanghai China |
| Ultrasonic cleaner | Ningbo Scientz Biotechnology Co.,Ltd |
| Orbital Shaker | Kylin-Bell Lab Instruments Co.,Ltd. |

2. Experimental protocol for the extraction of RNA

1. Take a bottle of cells, open the cap and empty the medium, and wash twice with pbs solution with 1 mL of PBS
   solution.
2. Add 2 mL of transzol reagent to the culture bottle, blow it repeatedly with the gun until the cells are
   fully integrated into the solution, suck out the solution and put it into two 1.5 mL centrifuge tubes.
3. Stand at room temperature for 15 min.
4. For each 1 mL transzol solution, 0.2 mL RNA extraction was added, violently shaken for 15 s, and incubated
   at room temperature for 3 min.
5. 10000 Xg was centrifuged at 4°C for 15 min. At this time, the sample is divided into three layers, with the
   colorless water phase in the upper and middle layers, and the pink organic phase in the lower layer. The RNA
   is mainly in the aqueous phase, whose volume is about 60% of the transzol reagent used.
6. Transfer the colorless water phase to a centrifuge tube, using 1 mL of transzol reagent to add 0.5 mL of
   isopropanol, mix well and enrich for ten minutes at room temperature.
7. 10000 Xg was centrifuged at 4°C for 10 minutes and remove the supernatant. A gelatinous precipitate is
   formed at the tube side and bottom.
8. Add 1 mL of 75% ethanol (prepared with DEPC water) to a vigorous vortex.
9. 7500 Xg was centrifuged at 4 °C for 5 minutes.
10. Discard the supernatant and let dry at room temperature for about 10 minutes and dissolve in 75 μL of RNA
    dissolution solution.
11. 57°C for 10 min and samples were stored to -80°C refrigerator for long-term use.
12. RNA Purity and concentration detection: the concentration of RNA was measured by agarose gel electrophoresis

3.Experimental protocol for reverse transcription and PCR amplification

(1) After residual genomic DNA was removed, configuration mixture (15 μL), and incubated for 10 min in a 42°C
water bath.

|  |  |
| --- | --- |
| constituents | application amount |
| template DNA | 2μL |
| 5×gDNA digester Mix | 3μL |
| RNase free ddH2O | 10μL |

(2) Configure the reverse recording reaction system.

|  |  |
| --- | --- |
| constituents | application amount |
| Rereaction solution for the first step | 15μL |
| 4×III MLVRTMix | 5μL |

(3) Reverse transcription using a PCR instrument according to the following conditions.

|  |  |
| --- | --- |
| temperature | hour |
| 25°C | 5 min |
| 55°C | 15 min |
| 85°C | 5 min |

(4) Configuration of PCR system (50 μL)

|  |  |
| --- | --- |
| constituents | application amount |
| ddH2O | 25 μL |
| 10\*Buffer(PCR) | 10 μL |
| Mix(2mM) | 10 μL |
| CDNA | 2 μL |
| primer（F/R） | 1+1 μL |
| enzyme（polymerase） | 1μL |

(5) Using a PCR instrument, amplification.

|  |  |  |
| --- | --- | --- |
| temperature | hour | recurring number |
| 95°C | 3 min | 1 |
| 98°C | 30 s | 30 |
| 64/66/68°C | 30 s |  |
| 72°C | 90 s |  |
| 72°C | 10 min | 1 |
| 4°C | limitless | 1 |

4.Experimental protocol for the recovery of target genes

1. Rubber blocks containing the target fragments were cut from the agarose gel and weighed.
2. Add 4 times Buffer B2 and 50 C water bath for 5 minutes.
3. The sol solution was moved into the adsorption column and centrifuged at 8000 Xg for 30s. Remove the liquid
   from the collection tube.
4. Add 500 μL Wash Solution and centrifuge at 9000 Xg for 30 seconds. Remove the liquid from the collection tube.
5. Repeat Step 4 once.
6. The empty adsorption column was centrifuged at 9000 Xg for 1 minute.
7. Put the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL Elution Buffer in the center of the
   adsorption membrane, stand at room temperature for 1 minute, centrifuge for 1 minute, and save the DNA
   solution in the tube

5. Experimental protocol for the construction of recombinant plasmids

(1) Three bottles of 100 mL of LB liquid medium were prepared and sterilized.

(2) 1 mL of bacterial broth was added to each vial of medium and incubated for 3 hours at constant temperature.

(3) Measuring OD values

|  |  |  |  |
| --- | --- | --- | --- |
| The first test | 1.381 | 1.284 | 1.390 |
| The first test | 1.361 | 1.359 | 1.394 |
| The first test | 1.35 | 1.394 | 1.3769 |

Do sensory cells

（4）Plasmid enzyme was cut, and ligated

|  |  |
| --- | --- |
| constituents | application amount |
| Pet-28 plasmid | 1 μL |
| Hind 3 | 1 μL |
| Ecrol | 1 μL |
| 10\*m Buffer | 2 μL |
| DEPC water | 15 μL |

As for the 37 C water bath for 1 hour

6. Experimental protocol for plasmid transformation

(1) Add 4 μL of reaction solution to the prepared Nissle1917 sensory cells, flick several times, and incubate on
ice for 30 min.

(2) Heat-excite the cells in a 42°C water bath for 90 seconds and then quickly put them on ice for 5 minutes.

(3) Add 500 μl of SOC liquid medium and incubate at 37°C for 45-60 minutes. d. Centrifuge at 4000 rpm for 3
minutes.

(4) Centrifuge at 4000 rpm for 3 minutes to collect the organisms and spread a certain amount of organisms
evenly on the antibiotic-containing plate as needed.

7.Experimental protocol for bacterial culture to induce expression

(1) Pick a single colony and inoculate it into 100mL of LB medium containing 50ug/mL kanamycin, incubate it at
37°C, 220 rpm for 12-16h, and then incubate it for 12-16h.

(2) When the OD600 of the bacterial solution is about 0.4-0.6, add IPTG at a final concentration of 1 mM in an
ultra-clean bench, add 50 μg/mL kanamycin, and incubate at 28°C, 160 rpm for 20h to induce large amounts of
protein expression.

Subject 2: Metabolomic and Proteomic Analysis of Lactic Acid-Adaptive EcN

1.Growth curve

(1) Table 1 Absorbance of EcN at different time

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Time (hour) | Absorbance 1 | Absorbance 2 | Absorbance 3 | Average absorbance |
| 0 | 0.885 | 0.884 | 0.888 | 0.886 |
| 1 | 0.894 | 0.898 | 0.896 | 0.896 |
| 2 | 0.893 | 0.898 | 0.895 | 0.895 |
| 3 | 0.892 | 0.896 | 0.892 | 0.893 |
| 4 | 0.909 | 0.906 | 0.901 | 0.905 |
| 5 | 0.941 | 0.930 | 0.969 | 0.947 |
| 6 | 1.115 | 1.111 | 1.105 | 1.110 |
| 7 | 1.412 | 1.410 | 1.421 | 1.414 |
| 8 | 1.462 | 1.467 | 1.473 | 1.467 |
| 9 | 1.509 | 1.512 | 1.520 | 1.514 |
| 10 | 1.543 | 1.547 | 1.554 | 1.548 |

2. Gradient screening of EcN strains that adapt to high lactic acid concentrations

(1) Prepar LB solid medium with lactic acid.

(2) Put EcN on LB solid media and grown at 37℃

(3) Gradual increase in lactic acid concentration to eventually obtain a strain (EcN-2.5) that grew stably at LB
solid media with 2.5g/L lactic acid.

(4) Prepare LB liquid medium with 2.5g/L lactic acid, Expanded culture of EcN-2.5.

(5) Gradual increase in lactic acid concentration to eventually obtain a strain (EcN-3.0) that grew stably at LB
liquid media with 3.0g/L lactic acid.

3. Sample preparation for LC-MS

(1) Collect the bacterial solution, centrifuge it at 4000 xg for 10 min, collect the bacterial body in 2 mL
centrifugal tube (weigh it in advance and calculate the bacterial weight).

(2) Wash three times with pre-cooling 0.01M (1X) PBS (pH 7.4) to remove supernatant.

(3) Add four times the volume of methanol to the precipitate

(4) fully oscillate and mix (about 5min)

(5) Centrifuge at 14,000 rpm for 15 min at 4 ℃ and collect the supernatant.

4. LC-MS analysis.

LC conditions:

(1) Using a ACQUITY ULPC®HSS T3 1.8 μm(2.1X100 mm) chromatography column.

(2) The temperature of the autosampler is set to 10℃, with gradient elution at 0.1 mL/min flow rate, 40℃, column
temperature and 2 μ L of injection.

(3) The mobile phase is positive ion: 0.1% formic acid water-0.1% formic acid acetonitrile; negative ion: 0.1%
formic acid water-0.1% formic acid acetonitrile.

(4) The gradient elution procedure setting

0-1
min
2％B

1-9
min
2％-50％B

9-12
min
50％-98％B

12-13.5
min     98％B

13.5-14
min     98％-2％B

14-17min       2％B

Mass spectrometry conditions:

The instrument uses an electrospray ion source (ESI), positive and negative ion ionization mode, positive ion
spray voltage is 3.50 kV, negative ion spray voltage is 2.50 kV, sheath gas is 30 arb, auxiliary gas is 10 arb.
The capillary temperature is 325 ℃, the full scan is performed at a resolution of 70,000, the scanning range is
81-1000, and the HCD is used for secondary cracking, the collision voltage is 30 eV, and the unnecessary MS/MS
information is removed by dynamic exclusion.

Subject 3: 3D Modeling of TNBC Tumor Spheroids.

1. Experimental protocol for 3D tumor spheroid culture

(1) Cultivate MCF-7 in T25 cell culture flasks to 70%-80% adherence.

![](https://static.igem.wiki/teams/4581/wiki/notebook.png)

Subject 4: Chemical Modification of Bacteria with MnO2@AuNPs Nanoparticles

1. Experimental protocols for the preparation of standard curves

(1) Tris-HCL buffer (0.25 M): after accurate preparation by DDW, adjust pH 8.3 with hydrochloric acid.

(2) Cysteine standard solution (1 mM): accurately weigh 0.017563 g of L- cysteine (175.63), dissolve with 1 mL
of formic acid, and then volume to 100 mL with DDW;

(3) DTNB (molecular weight: 396.35) standard solution (10 mM): accurately weigh 0.198175 g DTNB with 50 mM
Na2HPO4 (pH = 7.0) to make 50 mL of solution, stored in a brown bottle in the dark at low
temperature;

(4) DTNB analytical solution (0.1 mM): prepared from 1 volume of 10 mM DTNB standard solution plus 99 volume of
0.25 M Tris buffer, ready to use.

2. Experimental protocol for the preparation of thiolated Escherichia coli Nissle 1917

(1) Remove E. coli cultured for 12h, centrifuge at 5000xg for 10min, remove the supernatant. wash twice with PBS
and resuspend with 987.5μL ice PBS. Add 12.5 μL 2-iminothiophane solution (2.5 μg/mL) to the suspension and
incubate for 90min at room temperature.

(2) Thiolated bacteria (EcN@SH) were washed twice with PBS to eliminate residual 2-iminothiolane solution.

(3) Determination of the number of thiol groups. The number of thiol groups on the bacteria was determined after
quantitative modification with DTNB reagent (Ellman's reagent). Bacteria were resuspended in 3 × 108 cfu /mL of
PBS, and DTNB was added to a final working concentration of 0.1 mM, and the reaction was performed at room
temperature for 2 h. After centrifugation to remove the precipitated bacteria (6000 × g, 5 min), 200 µL of the
supernatant was transferred to a microtitre plate, and the absorbance was measured immediately at 412 nm. The
amount of thiol fraction was calculated from the standard curve obtained with increasing concentration of L
-cysteine hydrochloride hydrate.

3. Experimental protocol for EcN@SH - Tumor spheroid co-culture

(1) Prepare co-culture medium (without penicillin-streptomycin and containing 1.5 μg/mL gentamicin).

(2) Centrifuge the bacterial culture, determine the optical density OD600 value of the bacterial culture in a
Nano Drop spectrophotometer, dilute the OD600 of the bacterial suspension with PBS to ~0.5. 2µL of bacterial
solution was added to each well.

(3) At 4 h, 8 h and 12 h of co-culture, the co-culture plate was removed, and iodoacetamide solution was added
to 1 well respectively, which reacted with the sulfhydryl group on the bacterial surface, and was excited to
emit green fluorescence under UV irradiation at 335 nm, and the detection wavelength was 493 nm. According to
the three fluorescence micrographs of the prolonged fluorescence observation with the time of co-cultivation,
the fluorescence signals of the bacteria can be seen infiltrating into the center of the growing spheroplast and
colonizing in the necrotic region. necrotic regions were colonized. (Iodoacetamide (IAM) decomposes easily in
the presence of light, store at 4℃ in a brown bottle and use it now.)

(4) After 2d of co-culture, change the solution. Aspirate 60µL of sphere medium (tilt the tip of the gun at 45
degrees to prevent aspirating too many spheres), add 160µL of PBS to each well (the spheres will swell, add the
liquid gently), aspirate 160µL of medium, and aspirate as much residual PBS as possible. 200µL of co-culture
medium is added to remove bacteria from outside the spheres and to limit the growth of bacteria in the anoxic
area of the tumor spheres.

4. Experimental protocol for the synthesis of manganese dioxide and gold composite nanoparticles

(1) Preparation of raw material. Weigh 0.0115g of tetrachloroauric acid solid trihydrate in a 1.5 mL EP tube,
add 600 microliters of ultrapure water and mix by blowing with a pipette gun. Weigh another 0.0061 g of sodium
citrate dihydrate in a 1.5 mL EP tube, add 600 microliters of ultrapure water and mix by blowing with a pipette
gun. Then 100 µl of 1 mg/mL of hollow mesoporous manganese dioxide nanoparticles and 3700 µl of ultrapure water
were aspirated.

(2) Premixing. A premixed solution of 600 microliters of tetrachloroauric acid solution and 600 microliters of
sodium citrate solution will be added sequentially to 3800 microliters of hollow mesoporous manganese dioxide
nanoparticle solution.

(3) Construction of reaction conditions. 95 mL of ultrapure water was taken in a conical flask to which a
magnetic stirrer was added and heated to boiling.

(4) The pre-mixed reaction solution was rapidly poured into the above conical flask, the heating gear was turned
on to the highest setting, while the magnetic stirrer was turned on to the fifth setting, and the reaction was
carried out with vigorous stirring for 60 minutes.

(5) Purification. The solution after 60 min of reaction was cooled at room temperature, transferred to a 50 mL
EP tube, 12,000 rpm, centrifuged for 20 min, and the supernatant was decanted to leave a precipitate.

(6) Dissolution by sonication. Add 10 mL of ultrapure water to the above 50 mL EP tube and sonicate at 40 kHz
for 15 minutes to dissolve almost all of the precipitate.

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MnO2

{% extends "layout.html" %}
{% block title %}Genetic Engineering{% endblock %}
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![](https://static.igem.wiki/teams/4581/wiki/left7.png)

* [Overall framework](#div1)
* [Design](#div2)
* [Engineering process](#div3)
* [Quality control and assurance](#div4)
* [References](#div5)

Genetic Engineering

On this page, we describe the genetic engineering design consisting of a few subprojects: RNA extraction, reverse transcription, PCR amplification, recovery of target gene, construction of recombinant plasmid, plasmid transformation, screening and expansion, and expression of target gene.

Overall framework

The designed robot tumor targeting bacteria (TuTaBa, 兔大巴) are engineered from E. coli nissle 1917 strain. The TuTaBa has five distinguished features as an immunotherapeutic robot. (1) Its intrinsic facultative anaerobic characteristics makes it move towards the hypoxic areas of tumor cells with self-propelling flagella. (2) It can also induce innate or adaptive immune responses of the host, secret toxins and form biofilms that are helpful for the treatment of tumors.1 (3) With the expression of receptor signal-regulatory protein alpha (SIRPα) gene, it should recognize CD 47 proteins present on surfaces of tumor cells. Then it can specifically move towards and attack on tumor cells. (4) CD 47 can also function as the engulfment signal and dominant macrophage immune checkpoint that represent a signal “do not eat me” to the immune system. The interaction of CD 47 with SIRPα can cause the suppression of phagocytic activities. The expression of SIRPα on bacteria should compete with the SIRPα on macrophage to interact with CD 47 on TNBC tumor cells. Then the phagocytic and cytotoxic activities of macrophages against TNBC tumor cells would be enhanced. (5) It has intrinsic MCT 1 expressed on surfaces that can deplete lactic acid in tumor microenvironment (TME). Figure 1 shows the major parts and functions of the robot TuTaBa.

![](https://static.igem.wiki/teams/4581/wiki/project1.png)

Figure 1. Genetic engineering of E coli Nissle 1917.

Design

pET-28a (+) plasmid was chosen as the expression vector. The pET-28a (+) plasmid is a prokaryotic expression vector that contains a His tag at the C-terminal end and a His tag, thrombin cleavage site, and T7 tag at the N-terminal end.2It contains several commonly used enzymatic cleavage sites, which facilitates the cloning of different genes. Unique sites are shown on the circle map in Figure 2 (A). We inserted the SIRPα gene in the MCS region of the pET-28a (+) plasmid as shown in Figure 2 (B).

![](https://static.igem.wiki/teams/4581/wiki/engneering-3.png)

Figure 2. pET-28a (+) plasmid mapping. (A) Without SIRPα gene. (B) With SIRPα gene.

The secondary structure of the protein is predicted by using SOPMA as shown in Figure 3 (A). SOPMA is the abbreviation of Self-Optimized Prediction Method with Alignment that is a tool to predict the secondary structure of a protein.3It is shown that the protein contains 48.12% random coil, 7.90% beta turn, 31.11% extended strand and 12.88% alpha helix. Random coils represent the regions of the protein chain that do not form regular secondary structure and are not characterized by any regular hydrogen bonding pattern. Such regions are usually found in terminal arms and loops. The transmembrane topology is predicted by using DeepTMHMM as shown in Figure 3 (B). DeepTMHMM is a deep learning protein language model-based algorithm that can detect and predict the topology of both alpha helical and beta barrels proteins with unprecedented accuracy.4 The target SIRPα gene sequence and primers are listed as follows.

![](https://static.igem.wiki/teams/4581/wiki/project4.png)

Figure 3. Prediction of secondary structure and transmembrane. (A) SOPMA. (B) DeepTMHMM.

Engineering process

Figure 4 shows the procedures used for the genetic engineering of E. coli Nissle 1917. MCF 7 cells have been used for the extraction of full-length RNA samples that are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning.

![](https://static.igem.wiki/teams/4581/wiki/engneering-2.png)

Figure 4. Engineering processes starting from the full-length RNA extraction from cells.

1. RNA extraction and cDNA synthesis

Most eukaryotic genes contain introns and we usually don't know how they are spliced without their mRNA. For this is the reason you need to extract the full-length mRNA and generate cDNA for gene cloning and expression. Cultured MCF 7 cells were used for the RNA extraction.

![](https://static.igem.wiki/teams/4581/wiki/project5.png)

Figure 5. Gel electrophoresis of extracted RNA.

2. Cloning of target gene

Primers were designed according to the gene sequence and PCR was performed with the following PCR system.Primers used in the experiment is summarized in Table 1 and gel electrophoresis of DNA is shown in Figure 6.

| Table 1. Primers used for cloning of target gene SIRPα | |
| --- | --- |
| Primers | Sequences |
| F- SIRPa | ATGGGTCGCGGATCCGAATTCATGGAGCCCGCCGGCCCGGC |
| R-SIRPa | TCGAGTGCGGCCGCAAGCTTTCAGTGGTGATGGTGATGATGCTTCCTCGGGACCTGGACGCTGGCGTACT |

![](https://static.igem.wiki/teams/4581/wiki/project6.png)

Figure 6. Gel electrophoresis of DNA.

3. Bacterial culture and expression

Gel pieces containing intended DNA were excised and subjected to further experiments for the recovery of DNA from gel slices with a gel dissolution method. Then conduct a DNA ligation to fuse the insert to the recipient plasmid. Competent cells DH5alpha are used for the transformation. Figure 7 shows bacterial clones on the plate.

![](https://static.igem.wiki/teams/4581/wiki/project-7.png)

Figure 7. Engineered E coli Nissle 1917 bacterial clones.

Quality control and assurance

Quality control (QC) refers to the process through which the team can ensure the quality of the whole experiments and activities to be under control. We have established experimental and safety protocols that help standardize both experiments and behaviors.

✓ Carefully-designed experiments. In accordance with national and institutional safety regulations and guidelines, all experiments are carefully designed so as to minimal risk of dangerous operations and exposures. Team members can obtain immediate help from PIs, instructors and advisors who have different educational backgrounds in chemistry, microbiology and medicine.

✓ Trained personnel. Before the starting of the project, team members must be trained not only in techniques but also in general label safety, biosafety, ethics, and norms.

✓ Standardized experimental protocols. Various experimental procedures including step-by-step reactions, instrumental operations are optimized and finalized as written protocols that can be shared in lab.

✓ Characterization of intermediate and final products. Starting from the extraction of full-length RNA from tumor cells, intermediate products and final products should be characterized with electrophoretic, chromatographic and mass spectrometric analysis.

References

1. Gurbatri, C. R., Arpaia, N., Danino, T., Engineering bacteria as interactive cancer therapies. Science 2022, 378, 858-864.
2. Tham, H. Y., Song, A. A., Yusoff, K., & Tan, G. H. (2020). Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin. Biotechniques, 69(3), 161-170.
3. Geourjon, C., Deleage, G. (1995). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinformatics, 11(6), 681-684.
4. Hallgren, J., Tsirigos, K. D., Pedersen, M. D., Almagro Armenteros, J. J., Marcatili, P., Nielsen, H., Winther, O. (2022). DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. BioRxiv, 2022-04.

{% endblock %}

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

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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