iGEM 2023 — Team hangzhou-sdg

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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![](https://framerusercontent.com/images/p8sitktbGQByv1Jn2xsR3nXEcY.png?scale-down-to=2048)

Save our earth: No more silent spring

Engineering Success

**Abstract**

In this comprehensive project, we engaged in an in-depth exploration
of bio-genetically engineered pesticides. Our goal is to create an
effective and safe fungal biopesticide. We successfully introduced the
insect-specific toxin, LqhIT2, to *Metarhizium anisopliae*
ACCC30104, and it significantly improved the virulence of the fungus
towards pests. To find the best suicide switch, we tested different
combinations of Pmcl1/Pmcl1(short) and SuperNova/KillerRed. The
results showed that Pmcl1(short)-SuperNova was the most effective
suicide switch combination.

1. **Design**

**1.1 The fungus:** ***Metarhizium anisopliae***

Starting with fungal biopesticide selection, our choice is *Metarhizium anisopliae*. This fungus is a well-documented pathogen of insects with a broad
host range, capable of parasitizing over 200 distinct insect species.
Its safety profile was particularly interesting: it poses no harm to
humans, other mammals, or the environment at large (Kim et al., 2020).
This fungus naturally occurs in soils across the world and has been
previously utilized as a biopesticide due to its strong insecticidal
properties. Specifically, we planned to focus on *M. anisopliae*
ACCC30104, which exhibited strong pathogenicity.

![](https://static.igem.wiki/teams/4882/wiki/success/1.png)

Figure 1. *M. anisopliae* ACCC30104 on PDA plate

**1.2 Plasmid selection**

Resistance of *M. anisopliae*
ACCC30104 to several antifungal agents was tested to determine the
selection marker to use.

**Problem**

According to Inglis et al., 2000, *M. anisopliae* should be susceptible to glufosinate. So we planned to use
glufosinate as its selection marker and chose to use pBARGPE1 as the
backbone. However, when we tested *M. anisopliae*
ACCC30104 on PDA plates containing 100 µg/mL and 300 µg/mL
glufosinate, it grew well. As a result, we had to test more antifungal
agents and change our plasmid choice.

**Problem solved**

We prepared PDA plates containing 100 µg/mL, and 300 µg/mL G418, 100
µg/mL, and 300 µg/mL glufosinate, 10 µg/mL, 100 µg/mL, and 300 µg/mL
benomyl (dissolved in ethanol), 0.1%, 0.2%, and 2% ethanol (control of
benomyl group). *M. anisopliae*
spores were transferred to the plates and cultured at 30 ℃. The
diameters of colonies were measured on days 0, 3, 5, and 10.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng1.png)

Figure 2. A-F. *M. anisopliae*
ACCC30104 growth on PDA plates on day 5. A. PDA control; B. PDA
containing 100 µg/mL G418; C. PDA containing 100 µg/mL glufosinate; D.
PDA containing 10 µg/mL benomyl; E. PDA containing 100 µg/mL benomyl;
F. PDA containing 300 µg/mL benomyl; G. Growth curve of *M. anisopliae*
ACCC30104 on G418 plates; H. Growth curve of *M. anisopliae*
ACCC30104 on glufosinate plates; I. Growth curve of *M. anisopliae* ACCC30104 on benomyl plates.

Results showed that our *M. anisopliae*
strain ACCC30104 is only susceptible to benomyl, so we chose to use
pBARGPE1-EGFP-BenA as our backbone. pBARGPE1 is a classic filamentous
fungus-*Escherichia coli*
shuttle expression vector. Compared to the original pBARGPE1 vector,
the pBARGPE1-EGFP-BenA changes the bialaphos resistance gene (BarR) to
the benomyl resistance gene (BenA), which is suitable for our strain.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng2.png)

Figure 3. Plasmid map of pBARGPE1-EGFP-BenA.

**1.3 The toxin: LqhIT2**

To improve the efficiency and effectiveness of *M. anisopliae*
as a pesticide, one strategy is to introduce a safe toxin. We selected
LqhIT2, a 61-amino acid-long depressant toxin derived from the
scorpion species *Leiurus quinquestriatus hebraeus*. This toxin displays a high affinity for voltage-gated sodium
channels in insects, contributing to its potent insecticidal activity
(Karbat et al., 2007). Importantly, LqhIT2 is non-toxic to humans and
other mammals, making it a safe choice for our purpose. It is worth
noting, however, that the biochemical metabolism of this toxin within
crops remains an active area of research, and further exploration will
be part of our project scope.

**Problem**

When submitting the check-in forms, the iGEM Safety and Security Committee raised concerns about LqhIT2. We needed to prove the safety of LqhIT2.

**Problem solved**

We sought instruction from The Center of Biosafety Research and Strategy at Tianjin University. Prof. Wang from the Center helped to assess the biosafety of LqhIT2. To check the potential of a mutated LqhIT2 becoming toxic to humans, we searched similar sequences of LqhIT2 by running Protein Blast on NCBI. Results showed that genes share a similar sequence with LqhIT2 all encode for insect-specific toxins, which showed the safety of LqhIT2 in our project. See [Safety](https://2023.igem.wiki/hangzhou-sdg/safety) for detailed results. We showed the results to Prof. Wang, and he agreed with us.

To promote the secretion of the toxin, a *Metarhizium*
collagen-like promoter signal sequence (Mcl1ss) should be linked at
the 5’ end of the LqhIT2 gene (Peng et al., 2014).

The Pmcl1 is a strictly regulated and hemolymph-inducible promoter
from *M. anisopliae*. Compared to the full-length Pmcl1 (2764bp), a truncated, shorter
version of Pmcl1 (1586bp) can lead to a twofold increase in downstream
gene expression (Kanjo et al., 2019). This strong hemolymph-inducible
promoter was the perfect promoter to be used in our project as it only
turns on when hyphae enter the insect body. We planned to put
Mcl1ss-LqhIT2 downstream of Pmcl1 (short). This design limited the
LqhIT2 strictly inside the insect body and improved the biosafety of
our product.

Our next step involved designing the expression vector for LqhIT2. We
planned to use EcoRI and NdeI to remove the unwanted EGFP and gpaA
promoter of pBARGPE1-EGFP-BenA. Mcl1ss-LqhIT2 would be inserted
between the two restriction sites, forming
pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng3.png)

Figure 4. A. Map of Mcl1ss-LqhIT2; B. Plasmid map of
pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA.

**1.4 The suicide switches**

Following the preparation of the fungal pathogen and toxin, our
attention turned towards the exploration of a suicide switch. This
term refers to the introduction of certain genes into target cells,
leading to the death of the gene-carrying recipient cells. The primary
aim of this strategy was to regulate the reproductive efficiency of
specific cells, thereby improving controllability and safety. In
particular, we planned to incorporate a light-induced suicide switch
into our genetically modified strain of *M. anisopliae*
to prevent its unintended spread into the environment. This switch
involves a phototoxic protein, SuperNova, which produces lethal
reactive oxygen species (ROS) upon exposure to light. To improve the
effectiveness of SuperNova and ROS, a nuclear localization signal
(NLS) derived from the SV40 T antigen should be connected to the 3’
end of the SuperNova sequence (Lu et al., 2021). The SV40 NLS should
guide the protein to be transported into the cell nucleus, and let ROS
attack the most vulnerable genomic DNA (Paardekooper et al., 2019).

The 2016 NYMU\_Taipei iGEM Team ligated a KillerRed gene after a
full-length Pmcl1 promoter. As described above, Pmcl1 (short) should
be three times stronger than the full-length Pmcl1. And Onukwufor et
al. have proven that SuperNova can produce three times as much ROS as
KillerRed. As a result, theoretically, a Pmcl1 (short) combined with
SuperNova should be a stronger suicide switch than the 2016
NYMU\_Taipei iGEM Team’s. To verify our hypothesis and to find the
strongest suicide switch, we planned to test Pmcl1-SuperNova,
Pmcl1(short)-SuperNova, Pmcl1-KillerRed, and Pmcl1(short)-KillerRed.

In terms of designing the expression vector for the suicide switches,
we planned to use EcoRI and NdeI to remove the unwanted EGFP and gpaA
promoter of pBARGPE1-EGFP-BenA. Suicide switches would be inserted
between the two restriction sites, forming
pBARGPE1-Pmcl1-SuperNova-SV40-BenA,
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA,
pBARGPE1-Pmcl1-KillerRed-SV40-BenA,
pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng4.png)

Figure 5. A. Map of SuperNova-SV40; B. Map of
pBARGPE1-Pmcl1-SuperNova-SV40-BenA; C. Map of
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA; D. Map of
pBARGPE1-Pmcl1-KillerRed-SV40-BenA; E. Map of
pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA.

2. **Build**

**2.1 Vector construction: LqhIT2**

Mcl1ss-LqhIT2 (246 bp) was chemically synthesized by GENEWIS (Suzhou,
Jiangsu, China). Pmcl1 (short, 1586 bp) was acquired by PCR, with the
genomic DNA of *M. anisopliae* ACCC30104 as the template (Phusion High-Fidelity PCR Master Mix,
Thermo Fisher, Waltham, MA, USA). pBARGPE1-EGFP-BenA was cleaved by
EcoRI and NdeI (FastDigest, Thermo Fisher, Waltham, MA, USA).

Pmcl1 (short), Mcl1ss-LqhIT2, and the cleaved pBARGPE1-EGFP-BenA
vector were connected by Gibson Assembly (ClonExpress Ultra One Step
Cloning Kit, Vazyme, Nanjing, Jiangsu, China), forming
pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA. The recombinant vector was
transformed into *M. anisopliae*
ACCC30104 by protoplast transformation.

![](https://static.igem.wiki/teams/4882/wiki/success/6.png)

Figure 6. Gel electrophoresis of colony PCR products for verification
of correct transformation of plasmid
pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA into *E .coli* DH5α.

**2.2 Vector construction: the suicide switches**

SuperNova-SV40 NLS (747 bp) and KillerRed-SV40 NLS (747 bp) were
chemically synthesized by GENEWIS (Suzhou, Jiangsu, China).
Full-length Pmcl1 (2764 bp) was acquired by PCR (Phusion High-Fidelity
PCR Master Mix, Thermo Fisher, Waltham, MA, USA), with the genomic DNA
of *M. anisopliae* ACCC30104 as the template. pBARGPE1-EGFP-BenA was cleaved by EcoRI
and NdeI (FastDigest, Thermo Fisher, Waltham, MA, USA).

To test the effect of Pmcl1, Pmcl1(short), KillerRed, and SuperNova
in suicide switch and find the best combination, we planned to
construct four recombinant vectors by Gibson Assembly (ClonExpress
Ultra One Step Cloning Kit, Vazyme, Nanjing, Jiangsu, China):
pBARGPE1-Pmcl1-SuperNova-SV40-BenA,
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA,
pBARGPE1-Pmcl1-KillerRed-SV40-BenA,
pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA.

**Problem**

Although we succeeded in constructing plasmids containing
Pmcl1(short), we could not construct plasmids containing full-length
Pmcl1 by Gibson Assembly as expected. The long length of Pmcl1 (2764
bp) might lower the efficiency of assembly. As a result, we had to try
another method.

**Problem solved**

We designed two primers at the 5’ end of the Pmcl1(short, 1586 bp)
and linearized the pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA and
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA by PCR.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng5.png)

Figure 7. A. Map of linearized
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA; B. Gel electrophoresis of
colony PCR products for verification of correct transformation of
plasmid pBARGPE1-Pmcl1-KillerRed-SV40-BenA (1-4) and
pBARGPE1-Pmcl1-SuperNova-SV40-BenA (5-8) into *E .coli* DH5α.

Then, the truncated part of Pmcl1 (1181 bp) was amplified by PCR and
connected to the linearized plasmids to restore the full-length Pmcl1.
As a result, pBARGPE1-Pmcl1-KillerRed-SV40-BenA and
pBARGPE1-Pmcl1-SuperNova-SV40-BenA were constructed based on
pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA and
pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA.

Each recombinant vector was then transformed into *M. anisopliae*
ACCC30104 by protoplast transformation.

3. **Test**

**3.1 Virulence tests**

We tested the virulence of the engineered *M. anisopliae*
on the larvae of the greater wax moth (*Galleria mellonella*). Spore suspensions from the original strain, the strain expressing
LqhIT2, and the strain containing Pmcl1(short)-SuperNova were
prepared. Different concentrations of spore suspensions (1 × 107, 1 × 106, 1 × 105
spores/mL) were inoculated to different groups. The larvae were
divided into ten groups (15 larvae/group) based on different
treatments:

a) Group I: uninoculated control larvae;

b) Group II: inoculate larvae with 1 × 107 WT *M. anisopliae* spores;

c) Group III: inoculate larvae with 1 × 106 WT *M. anisopliae* spores;

d) Group IV: inoculate larvae with 1 × 105 WT *M. anisopliae* spores;

e) Group V: inoculate larvae with 1 × 107 LqhIT2 *M. anisopliae* spores;

f) Group VI: inoculate larvae with 1 × 106 LqhIT2 *M. anisopliae* spores;

g) Group VII: inoculate larvae with 1 × 105 LqhIT2 *M. anisopliae* spores;

h) Group VIII: inoculate larvae with 1 × 107 Pmcl1(short)-SuperNova *M. anisopliae* spores;

i) Group IX: inoculate larvae with 1 × 106 Pmcl1(short)-SuperNova *M. anisopliae* spores;

j) Group X: inoculate larvae with 1 × 105 Pmcl1(short)-SuperNova *M. anisopliae* spores;

The larvae and fungi were incubated at 30 °C, and the death numbers
of each group were recorded every morning for 15 days.

![](https://static.igem.wiki/teams/4882/wiki/success/8.png)

Figure 8: The larvae of *G. mellonella* infected by LqhIT2 *M. anisopliae.*
A. Alive on day 1; B. Dead on day 4; C. Hyphae appeared on the surface
on day 7; D. Hyphae covered the body on day 10.

![](https://static.igem.wiki/teams/4882/wiki/yuqi-eng6.png)

Figure 9: A. The survival rate curve of WT groups; B. The survival
rate curve of LqhIT2 groups; C. The survival rate curve of
Pmcl1(short)-SuperNova groups; D. The survival rate curve of WT,
LqhIT2, and Pmcl1(short)-SuperNova groups inoculated with 1 × 107 spores/mL.

The median lethal cell density (LC50) and the median time to death
(LT50) were used to evaluate the virulence of each strain. The LqhIT2
strain and the Pmcl1(short)-SuperNova strain had similar LC50 and
LT50, indicating that the suicide switch did not influence the
virulence of *M. anisopliae.*
On the other hand, the LC50 of the LqhIT2 strain is 25.26-fold lower
than that of the WT strain, showing a remarkable increase in
virulence. The LT50 of the LqhIT2 strain was also significantly lower
than that of the WT strain (p < 0.05). To conclude, the toxin,
LqhIT2, significantly increased the virulence of *M. anisopliae*, and made it more efficient as a fungal biopesticide.

|  |  |  |  |
| --- | --- | --- | --- |
|  | WT | LqhIT2 | Pmcl1(short)-SuperNova |
| LC50 (day 5) | 2.20 × 106 | 8.71 × 104 | 2.24 × 106 |
| LT50 (1 × 107)95% CI | 4.429 (3.965-4.861) | 3.414 (3.024-3.760) | 4.4 (3.894-4.864) |

Table 1. The median lethal cell density (LC50, spores/mL) on day 5
and the median time to death (LT50, day) under 1 × 107 spores/mL of each strain, calculated using SPSS 26.0.0.2.

**3.2 Find the best suicide switch**

We tested the suicide switches based on the amounts of spore
formation and the spore germination rates. 1 × 107 spores of WT, Pmcl1(short)-LqhIT2, Pmcl1-SuperNova,
Pmcl1(short)-SuperNova, Pmcl1-KillerRed, and Pmcl1(short)-KillerRed
strains were inoculated to the larvae of the greater wax moth. The
Petri dishes containing the pest corpses were placed under sunshine
for days until spores formed outside the insect body. Spores were
collected by vertexing and the concentrations of spore suspensions
were checked under a microscope.

The result showed that the expression of LqhIT2 did not impact spore
formation. All the suicide switches worked but with different
efficiency. The suicide switch combinations from the strongest to the
weakest were Pmcl1(short)-SuperNova, Pmcl1-SuperNova,
Pmcl1(short)-KillerRed, and Pmcl1-KillerRed. The Pmcl1(short)
generally worked better than Pmcl1, and the SuperNova was stronger
than KillerRed. The Pmcl1(short)-SuperNova (p < 0.001),
Pmcl1-SuperNova (p < 0.01), and Pmcl1(short)-KillerRed (p <
0.01) strains formed significantly fewer spores than the WT strain.
Among all strains, the combination of Pmcl1(short)-SuperNova was the
best suicide switch, with a 56.9% decrease in the spore amount.
However, this suicide switch is not close to perfect, and it requires
further improvement. Melanin synthesized by *M. anisopliae* absorbs light, and it might help the cells to survive.

![](https://static.igem.wiki/teams/4882/wiki/success/10.png)

Figure 10. A. The amount of spore formation by each strain on *G. mellonella*
corpses; B. The spore germination rate of each strain in liquid PDB
media after 10h. \*: p < 0.01; \*\*: p < 0.001.

After being collected, the spores from each strain were resuspended
in liquid PDB media at the concentration of 5 × 105
spores/mL. The suspensions were incubated at 30 °C, 180 rpm for 10h to
facilitate spore germination.

The spore germination rates of different strains were all high (~92%)
and nearly identical. None of the groups showed significant
differences (all p > 0.4). The results indicated that our
light-induced suicide switches could not influence spore viability
after spore formation.

4. **Learn**

We proved that LqhIT2 could increase the virulence of *M. anisopliae*
and that Pmcl1(short)-SuperNova was an acceptable suicide switch. The
results showed that the toxin and the suicide switch worked
independently.

**Future plans**

Our first plan is to incorporate the toxin and the suicide switch into one plasmid, placing them downstream of the same Pmcl1(short) promoter and dividing them by a 2A peptide sequence. Then, the virulence of the fungal biopesticide can be further improved by incorporating other toxins.

In terms of biosafety, Prof. Wang mentioned the concerns about our engineered fungus impacting the environment and gave us suggestions about biosafety tests. In order to stop the uncontrolled spread, improvements should be made to the suicide switch. Prof. Wang suggested that the selection of the control switch should not be overly simplistic, and we should consider multi-level regulatory approaches. First of all, we plan to increase the sensitivity of the light-inducible suicide switch by downregulating the melanin synthases of *M. anisopliae*. Secondly, we plan to disrupt the RNA-binding proteins that allow it to adapt to the cold. Research has shown that Crp1 and Crp2 protected cells against freezing, and disrupting them would cause the engineered fungi to be killed in winter (Fang & St Leger, 2009).

We plan to test our improved suicide switch systems under various light (high, medium, low), humidity, and temperature conditions. All the tests are planned to be carried out in two stages. Stage one happens in artificial climate chambers. We will introduce soil, *Arabidopsis thaliana*, and larvae of *G. mellonella* into the chambers. Stage two happens in experimental fields. We will measure each factor and evaluate the biosafety.

**References**

Inglis PW, Aragão FJ, Frazão H, Magalhães BP, Valadares-Inglis MC.
Biolistic co-transformation of Metarhizium anisopliae var. acridum
strain CG423 with a green fluorescent protein and resistance to
glufosinate ammonium. FEMS Microbiol Lett. 2000 Oct 15;191(2):249-54.
doi: 10.1111/j.1574-6968.2000.tb09347.x. PMID: 11024271.

Fang W, St Leger RJ. RNA binding proteins mediate the ability of a fungus to adapt to the cold. Environ Microbiol. 2010 Mar;12(3):810-20. doi: 10.1111/j.1462-2920.2009.02127.x. Epub 2009 Dec 27. PMID: 20050869.

Kanjo, K.; Surin, S. I.; Gupta, T.; Dhanasingh, M.; Singh, B.; Saini,
G. K. Truncated, Strong Inducible Promoter PMCL1 from Metarhizium
Anisopliae. *3 Biotech* 2019, *9*
(3). DOI:10.1007/s13205-019-1610-2.

Karbat, I.; Turkov, M.; Cohen, L.; Kahn, R.; Gordon, D.; Gurevitz,
M.; Frolow, F. X-Ray Structure and Mutagenesis of the Scorpion
Depressant Toxin LqhIT2 Reveals Key Determinants Crucial for Activity
and Anti-Insect Selectivity. *Journal of Molecular Biology* 2007, *366*
(2), 586–601. DOI:10.1016/j.jmb.2006.10.085.

Kim HM, Jeong SG, Choi IS, Yang JE, Lee KH, Kim J, Kim JC, Kim JS,
Park HW. Mechanisms of Insecticidal Action of *Metarhizium anisopliae*
on Adult Japanese Pine Sawyer Beetles (*Monochamus alternatus*). ACS Omega. 2020 Sep 24;5(39):25312-25318. doi:
10.1021/acsomega.0c03585. PMID: 33043210; PMCID: PMC7542838.

Lu J, Wu T, Zhang B, Liu S, Song W, Qiao J, Ruan H. Types of nuclear
localization signals and mechanisms of protein import into the
nucleus. Cell Commun Signal. 2021 May 22;19(1):60. doi:
10.1186/s12964-021-00741-y. PMID: 34022911; PMCID: PMC8140498.

Onukwufor, J. O.; Trewin, A. J.; Baran, T. M.; Almast, A.; Foster, T.
H.; Wojtovich, A. P. Quantification of Reactive Oxygen Species
Production by the Red Fluorescent Proteins KillerRed, Supernova, and
mCherry. *Free Radical Biology and Medicine* 2020, *147*, 1–7. DOI:10.1016/j.freeradbiomed.2019.12.008.

Paardekooper LM, van Vroonhoven E, Ter Beest M, van den Bogaart G.
Radical Stress Is More Cytotoxic in the Nucleus than in Other
Organelles. Int J Mol Sci. 2019 Aug 25;20(17):4147. doi:
10.3390/ijms20174147. PMID: 31450682; PMCID: PMC6747261.

Peng G, Xia Y. Expression of scorpion toxin LqhIT2 increases the
virulence of Metarhizium acridum towards Locusta migratoria
manilensis. J Ind Microbiol Biotechnol. 2014 Nov;41(11):1659-66. doi:
10.1007/s10295-014-1497-1. Epub 2014 Aug 29. PMID: 25168679.

{% 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)

{% endblock %}