iGEM 2023 — Team nanjing-sdg

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{% block title %}Notebook{% endblock %}
{% block lead %}Nanjing-SDG{% endblock %}
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### 7.1-7.9 Conducting laboratory safety training and safety assessments

all wet team members

### 7.10-7.14 Construstion of plasmid pMTL-Pthl-adhE2-dac

1. Fragment dac was amplified by PCR on the genome of Clostridium tyrobutyricum L319.

2. The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.

3. Plasmid pMTL-Pthl-adhE2-dac was obtained by Gibson assembly

(Experimenter: Xiaojing Tong, Zilai Chen, Yinong Fu, Yuyang Fu, Mingzhe Zhao)

#### Experiment records：

7.10：Preparation of media (solid & liquid), making gels; PCR amplification of fragments Dac.The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template；

7.11：The gel was recovered to obtain the target DNA; the vector system was configured and the vector was ligated to the fragment:pMTL-Pthl-adhE2-Dac;

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig1.png)
![](https://static.igem.wiki/teams/4885/wiki/nbook-fig2.png)

7.12：Transformation, 12 hours culture at 37 degrees Celsius;

7.13：Colony PCR validation, #2,4,5 were successful;

7.14：Purity testing; SDS-page; Bacterial preservation；

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig3.png)
![](https://static.igem.wiki/teams/4885/wiki/nbook-fig4.png)

### 7.17-7.21 Construstion of plasmid pMTL-Pthl-adhE2-Pcat1-cat1

1. The fragment cat1 and the cat1 promoter was amplified by PCR on the genome of Clostridium tyrobutyricum L319.

2. The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.

3. Plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained by Gibson assembly

(Experimenter: Xiaojing Tong, Zilai Chen, Yinong Fu, Yuyang Fu, Mingzhe Zhao, Tianyunran Huang)

7.17-7.18: The fragment cat1, cat1 promoter and the vector pMTL-Pthl-adhE2 were obtained by PCR。The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig5.png)
![](https://static.igem.wiki/teams/4885/wiki/nbook-fig6.png)

7.19: Fragment and vector ligation；Transformation

7.20: Colony PCR validation

7.21: SDS-page; Bacterial preservation;

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig7.png)
![](https://static.igem.wiki/teams/4885/wiki/nbook-fig8.png)

### 7.24-7.28: Construstion of plasmid pMTL-Pthl-adhE2-bcd-crt

1. The linearized vector Vp-adhE2 was amplified by PCR using Pthl-Pthl-adhE2(BBa\_K4408008) as the template.

2. The fragment bcd, crt was amplified by PCR on the genome of Clostridium tyrobutyricum L319;then bcd gene fragment and crt gene fragment were fused into bcd-crt fragment by fusion PCR.

3.Gibson assembly method was used to link bcd-crt gene fragment to Vp-adhE2 linearized vector

(Experimenter:Yangyang Liu, Minmo Zhu, Hongchang Lv, Lingyi Chen, Kewei Li, Jian Zhou)

Experiment records：

7.24 The fragment bcd was amplified by PCR

7.25 The fragment crt was amplified by PCR; Linear pMTL-Pthl-adhE2

7.26 The target plasmid verified by colony PCR was pMTL-Pthl-adhE2-bcd

7.27 The template plasmid was pMTL-Pthl-adhE2-bcd

7.28 The target plasmid verified by colony PCR was pMTL-Pthl-adhE2-bcd-crt；SDS-page; Bacterial preservation;

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig9.png)

### 7.31-8.4: Promoter engineering: Replace the promoter in Pthl-adhE2 to build Ptkt-adhE2

(Experimenter:Yangyang Liu, Minmo Zhu,Kewei Li,Yuyang Fu,Yinong Fu)

1. pMTL-Pthl-adhE2 plasmid (BBa\_K4408008) was used as a template, and the linearized vector Vtkt was amplified.

2. Clostridium tyrobutyricum genome was used as template to amplify the fragment tkt.

3. Gibson Assembly ligated the linearized vector Vtkt with the fragment tkt to obtain the recombinant plasmid pMTL-Ptkt-adhE2;

4. Colony PCR validation; SDS-PAGE assay.

5. Bacterial preservation;

![](https://static.igem.wiki/teams/4885/wiki/nbook-fig11.png)

### 8.6-8.18:Product determination

（Experimenter:Yangyang Liu, Minmo Zhu, Hongchang Lv）

8.6 All of the preserved strains were recovered.

8.7 After 12 hours of cultivation, add CALB lipase and hexadecane to the culture medium. And then continue to cultivate for 203 hours.

8.17-8.18 HPLC analysis of acetate, butyrate and butanol; GC analysis of butryl butyrate. Data analysis.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Nanjing-SDG{% endblock %}
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### Abstract:

Our project aims to efficiently and massively synthesize the biofuel butyl butyrate through microbial
fermentation, addressing energy-related issues on a global scale. To achieve this, we genetically engineered
Clostridium tyrobutyricum (C. tyrobutyricum) to enable efficient and large-scale synthesis of butyrate and
butanol. We employed a method of lipase catalyzed esterification of butyrate and butanol into butyl butyrate in
which reaction the optimal butyrate-to-butanol molar ratio is 1:1. In the process of constructing C.
tyrobutyricum, we used the method developed by Worldshaper-NJBIOX team last year which utilized the strain’s
natural pathway for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. We further
engineered C. tyrobutyricum to efficiently produce butyrate and butanol for the successive esterification into
butyl butyrate through two strategies, adjusting the product molar ratio closer to 1:1 (butyrate:butanol) and
co-enhancing the two products yield. The first strategy was achieved by enhancing the expression of deacetylase
(Dac) in the synthesis pathway of butyrate and butanol, overexpressing CoA transferase (cat1) to inhibit the
competing pathway of the byproduct, acetate, and using a weaker promoter Ptkt to express adhE2. The second
strategy was fulfilled by overexpressing rate-limiting enzymes (bcd and crt) in the synthesis pathway. This
allowed us to develop a C. tyrobutyricum strain with a high metabolic flux for the synthesis of butyrate and
butanol and a more optimal product ratio, which can be used for efficient large-scale microbial fermentation for
butyl butyrate.

We successfully constructed 8 parts to engineer C. tyrobutyricum. We overexpressed several enzymes to enhance
the synthesis pathway of butyrate and butanol. BBa\_K4885000 and BBa\_K4885002 were constructed to enhance the
expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa\_K4885001,
BBa\_K4885005 and BBa\_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt).
BBa\_K4885003-BBa\_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of
the byproduct, acetate. BBa\_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol
synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and
indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.

## Table 1 Part list

| NO. | Name | Type | Description | Length |
| --- | --- | --- | --- | --- |
| 1 | BBa\_K4885000 | basic | Dac, NAD-dependent deacetylase | 723bp |
| 2 | BBa\_K4885001 | basic | bcd, butyryl-CoA dehydrogenase coding sequence | 1140bp |
| 3 | BBa\_K4885002 | composite | Pthl-adhE2-Dac | 3973bp |
| 4 | BBa\_K4885003 | composite | Pcat1-Cat1 | 1720bp |
| 5 | BBa\_K4885004 | composite | Pthl-adhE2-Pcat1-Cat1 | 4858bp |
| 6 | BBa\_K4885005 | composite | Pthl-bcd-crt | 2593bp |
| 7 | BBa\_K4885006 | composite | Pthl-adhE2-bcd-crt | 5112bp |
| 8 | BBa\_K4885012 | composite | Ptkt-adhE2 | 2919bp |

The general design of our project is illustrated in Figure 1. In our project, we achieved the following five
targets to construct a C. tyrobutyricum strain that produced butanol and butyrate with a favorable product ratio
for the synthesis of butyl butyrate by lipase catalyzed esterification.

![](https://static.igem.wiki/teams/4885/wiki/des-fig1.png)

*Figure 1 Flow chart of our project to synthesize butyl butyrate using microbial fermentation and CBD
bound lipases*

### Target 1: Construction of a butanol synthesis pathway (dehydrogenase, adhE2)

2022 Worldshaper-NJBIOX team has engineered C. tyrobutyricum to express adhE2 and produce butanol. Inspired by their
project, our team used the same part Pthl-adhE2 (BBa\_K4408008) (plasmid pMTL-Pthl-adhE2) to express adhE2 in C. tyrobutyricum
L319 to construct a butanol synthesis pathway (Figure 2). adhE2 gene was derived from Clostridium acetobutylicum
ATCC 834. Pthl is a strong transcriptional promoter from C. tyrobutyricum L319. C. tyrobutyricum transfected
with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2).

![](https://static.igem.wiki/teams/4885/wiki/es-f2.png)

*Figure 2 Pthl-adhE2*

### Target 2: Direct enhancement of butyrate and butanol synthesis (deacetylase, Dac) Build: Construction of pMTL-Pthl-adhE2-Dac plasmid

We constructed pMTL-Pthl-adhE2-Dac plasmid responsible for the expression of adhE2 and Dac with Pthl promotor in
C. tyrobutyricum L319.

Using the recombinant plasmid Pthl-adhE2 constructed by BBa\_K4408008 in the database as template, VAD-F and
VDAC-R as primers, Vdac vector (8004 bp) was amplified by PCR. Using C. tyrobutyricum genome as template and
DAC-F and DAC-R as primers, Dac gene fragment (767 bp) was amplified. Gibson assembly method was used to link
Dac gene fragment to Vdac vector linearized vector. Colony PCR was performed on the transformed colonies (1050
bp) with DAC-PF and Pb-PR as primers. The positive colonies were transferred and plasmid was extracted. After
gene sequencing verification, recombinant plasmid pMTL-Pthl-adhE2-Dac was obtained.

![](https://static.igem.wiki/teams/4885/wiki/es-f3.png)

*(a)Genetic circuit of pMTL-Pthl-adhE2-Dac;

(b) Verification of pMTL-Pthl-adhE2-Dac plasmid by colony PCR

Figure 3 Construction of pMTL-Pthl-adhE2-Dac plasmid*

### Test: Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Dac

Ct(adhE2::Dac) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Dac using E. coli CA434
as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.

SDS-PAGE confirmed the overexpression of Dac protein (26 kDa) in Ct(adhE2::Dac) (Figure 4).

Growth performance analysis showed that overexpressing Dac in C. tyrobutyricum improved the growth of the
strain, with the maximum OD600 increased by 33% (Figure 5).

HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2),

overexpressing Dac in C. tyrobutyricum improved the yield of butyrate (1.73 g/L) by 50% while did not change the
butanol yield (5 g/L) (Figure 6-7), increasing the butyrate-to-butanol molar ratio from 0.19 to 0.29.

Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used
to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to
determine the yield. The yield of butyl butyrate in Ct(adhE2::Dac) increased 27% compared with that in Ct(adhE2)
(Figure 8).

![](https://static.igem.wiki/teams/4885/wiki/es-f4.png)

*Figure 4 Verification of Dac protein overexpression in Ct(adhE2::Dac) by SDS-PAGE*

![](https://static.igem.wiki/teams/4885/wiki/es-f5.png)

*Figure 5 Growth performance of Ct(adhE2::Dac)*

![](https://static.igem.wiki/teams/4885/wiki/es-f6.png)

*Figure 6 Butyrate fermentation performance of Ct(adhE2::Dac)*

![](https://static.igem.wiki/teams/4885/wiki/es-f7.png)

*Figure 7 Butanol fermentation performance of Ct(adhE2::Dac)*

![](https://static.igem.wiki/teams/4885/wiki/es-f8.png)

*Figure 8 Production of butyl butyrate by Ct(adhE2::Dac)*

### Target 3: Inhibition of the competing pathway: acetate synthesis (CoA transferase, cat1) Build: Construction of pMTL-Pthl-adhE2-Pcat1-cat1 plasmid

We constructed pMTL-Pthl-adhE2-Pcat1-cat1 recombinant plasmid to inhibit the synthesis of a by-product, acetic acid,
so that the synthesis of butanol and butyrate was enhanced indirectly.

Using the recombinant plasmid Pthl-adhE2 constructed by BBa\_K4408008 in the database as template and Vad-f and
Vad-r as primers, the Vpthl-adhE2 vector (7985 bp) was amplified. Using C. tyrobutyricum genome as template and
Cat-f and Cat-r as primers, cat1 gene fragment (1378 bp) was amplified. ,and Pcat-f and Pcat-r primers were used to amplify Pcat1 fragment. Gibson assembly method was used to link
the cat1 gene fragment Pcat1 fragment to the Vpthl-adhE2 linearized vector. Colony PCR (1333 bp) was performed on the
transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transferred and plasmid was
extracted. After gene sequencing verification, the recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained.

![](https://static.igem.wiki/teams/4885/wiki/design-fig51.jpg)

*Figure 9 Genetic circuit of pMTL-Pthl-adhE2-Pcat1-cat1*

### Test: Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Pcat1-cat1

Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 using E. coli
CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.

SDS-PAGE confirmed the overexpression of cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure10).

HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 11), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.

![](https://static.igem.wiki/teams/4885/wiki/es-f10.png)

*Figure 10 Verification of cat1 protein overexpression in Ct(adhE2::cat1) by SDS-PAGE*

![](https://static.igem.wiki/teams/4885/wiki/es-f11.png)

*Figure 11 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::cat1)*

### Target 4: Direct enhancement of butyrate and butanol synthesis pathway (rate-limiting enzymes, crt and bcd) --- Build : Construction of pMTL-Pthl-adhE2-bcd-crt plasmid

Using the recombinant plasmid Pthl-adhE2 constructed by BBa\_K4408008 in the database as template and VEAD-F and
veAD-r as primers, Vp-adhE2 vector (8001 bp) was amplified. Using C. tyrobutyricum genome as template, bcd gene
fragment (1196 bp) was amplified with bcd-f and BCD-R as primers, and crt gene fragment (813 bp) was amplified
with CRT-F and crt-R as primers. bcd gene fragment and crt gene fragment were fused into bcd-crt fragment by
fusion PCR. Gibson assembly method was used to link bcd-crt gene fragment to Vp-adhE2 linearized vector. Colony
PCR (1249 bp) was performed on the transformed colonies with primers bcd-PF and Pb-PR. The positive colonies
were transferred and the plasmid was extracted. After gene sequencing verification, the recombinant plasmid was
obtained: pMTL-Pthl-adhE2-bcd-crt.

![](https://static.igem.wiki/teams/4885/wiki/es-f12.png)

*Figure 12 Genetic circuit of pMTL-Pthl-adhE2-bcd-crt*

### Test : Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-bcd-crt

Ct(adhE2::bcd::crt) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-bcd-crt, using E.
coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.

SDS-PAGE confirmed the overexpression of bcd and crt proteins in Ct(adhE2::bcd::crt) (Figure13).

The growth performance of Ct(adhE2::bcd::crt) was significantly better than Ct(adhE2), with the maximum OD600
reaching 10.2, an increase of 58% (Figure 14).

HPLC experiment showed that after fermentation for 215 hours, compared to the yields in Ct(adhE2),
overexpressing bcd and crt increased the yield of butyrate by 20% and the yield of butanol by 40% (Figure 15).

Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used
to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to
determine the yield. 620 mg/L butyl butyrate was produced by Ct(adhE2::bcd::crt), which was 59% higher than the
390 mg/L yield by Ct(adhE2)(Figure 16).

![](https://static.igem.wiki/teams/4885/wiki/es-f13.png)

*Figure 13 Validation of the overexpression of bcd and crt in Ct(adhE2::bcd::crt) by SDS-PAGE*

![](https://static.igem.wiki/teams/4885/wiki/es-f14.png)

*Figure 14 Growth performance of Ct(adhE2::bcd::crt)*

![](https://static.igem.wiki/teams/4885/wiki/es-f15.png)

*Figure 15 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::bcd::crt)*

![](https://static.igem.wiki/teams/4885/wiki/es-f16.png)

*Figure 16 Production of butyl butyrate by Ct(adhE2::bcd::crt)*

### Target 5: Use Ptkt promoter for adhE2 expression to decrease butanol synthesis and indirectly increase butyrate synthesis ---

During our project, we were inspired by a new part Ptkt developed by the Nanjing-BioX team this year. Ptkt is a native promoter that drives the expression of transketolase (tkt) gene in C. tyrobutyricum. Ptkt promoter was found to have weaker transcriptional strength than Pthl promoter. adhE2 expression driven by Pthl in Ct(adhE2) showed too much exceeding synthesis of butanol to butyrate. So we replaced Pthl with the weaker promoter Ptkt to lower the production of butanol and increase the yield of butyrate in order to reach a more optimal butyrate-to-butanol product molar ratio (closer to 1:1) in our strain for the esterification of butyl butyrate.

### Build : Construction of pMTL-Ptkt-adhE2 plasmid

Using pMTL-Pthl-adhE2 plasmid as the template and Vtkt-F and Vtkt-R as the primers, a linearized vector Vtkt was amplified (7825 bp). Using the C. tyrobutyricum genome as the template and TKT-F and TKT-R as the primers, a tkt fragment was amplified (300 bp). The linearized vector Vtkt and the tkt fragment were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies (300 bp) with TKT-F and TKT-R as the primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Ptkt-adhE2 was obtained.

![](https://static.igem.wiki/teams/4885/wiki/es-171.png)

*Figure 17 Genetic circuit of recombinant plasmid pMTL-Ptkt-adhE2*

![](https://static.igem.wiki/teams/4885/wiki/es-181.png)

*Figure 18 Colony PCR verification of pMTL-Ptkt-adhE2 recombinant plasmid*

### Test: Construction of C. tyrobutyricum L319 with pMTL-Ptkt-adhE2 ---

Ct(Ptkt-adhE2) strain was obtained by conjugation of recombinant plasmid pMTL-Ptkt-adhE2 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2), also notated as Ct(Pthl-adhE2 in the figure, was used as the control.
HPLC showed that after fermentation for 215 hours (Figure 19), compared with Ct(Pthl-adhE2), Ct(Ptkt-adhE2) had increased the yield of butyrate by 66.5% and decreased the yield of butanol by 24.3%. Therefore, by using the weaker promoter Ptkt instead of Pthl, we raised the butyrate-to-butanol molar ratio from 0.29 to 0.63, much closer to the optimal ratio of 1:1. In addition, the production of acetate was reduced in Ct(Ptkt-adhE2) which was in accordance with the increased synthesis of butyrate.

![](https://static.igem.wiki/teams/4885/wiki/es-191.png)

*Figure 19 Butyrate, butanol and acetate fermentation performance of Ct(Ptkt-adhE2)*

### Conclusion and future work

We successfully modified C. tyrobutyricum via genetic engineering to have enhanced pathways for butanol and
butyrate synthesis which are the precursors for producing butyl butyrate. Using the strains developed by our
project, efficient and large-scale butyl butyrate production can be achieved via biofermentation.
For the esterification of butyl butyrate, the best product molar ratio of the precursors butyrate and butanol is
1:1. By introducing adhE2 into C. tyrobutyricum, the yield of butanol was much more than the butyrate yield. We
found three approaches to successfully adjust the product ratio of butyrate and butanol to be as close to 1:1 as
possible. We firstly enhanced Dac expression in the strain, which successfully increased butyrate yield while
keeping butanol yield unchanged. We also overexpressed Cat1, which effectively increased butyrate production and
decreased butanol synthesis. We further optimized the promoter by using Ptkt to drive the expression of adhE2 so
that butyrate production was enhanced while butanol synthesis was lowered. In addition to adjusting the product
ratio, we enhanced the yields of both butyrate and butanol by overexpression of bcd and crt. Due to time limit,
we were not able to combine those approaches in one strain which would be the goal for future work. In addition,
in the future, we can further improve the efficiency of the synthesis of butyl butyrate by lipase immobilization
which can speed up the lipase catalyzed esterification reaction of butanol and butyrate. Lipase immobilization
can be done by combining chitin binding domain (CBD) to lipase via genetic engineering.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Nanjing-SDG{% endblock %}
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## 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)

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Nanjing-SDG{% endblock %}
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## 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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