iGEM 2023 — Team thinker-shanghai

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{% block detail\_title\_a1 %}Experimental preparation for week 1{% endblock %}
{% block detail\_content2 %}Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain
Experimental procedure:
1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack, Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.
2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2 boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it was cleaned up properly afterwards.
3. Activated strains: 1 tube of Escherichia coli DH5α/ empty vector 1 and 1 tube of DH5α/ carrying MFS gene. One tube of Escherichia coli DH5α/ empty vector 2, one tube of DH5α/ carrying ismA gene, and one tube of DH5α/ carrying MFS gene
galU gene, turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the tube orifice and tube stopper, put 100 μL preservation bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the tube orifice and tube stopper again. The test tube was sealed, bundled, and incubated on a shaker overnight (150 rpm, 37℃).
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{% block detail\_title\_a2 %}2 weeks to extract the templates, began to construct the plasmid{% endblock %}
{% block detail\_content3 %}Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR
Experimental procedure:
1. Plasmid extraction: Four kinds of plasmids DH5α/ empty vector 1,2,MFS,ismA,galU were extracted according to the instruction of plasmid extraction kit, and the bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added and resuspended, p2 was mixed upside down, p3 was mixed upside down, and then centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.
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2. Diluted primer
The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water was added to dilute the primer to the appropriate concentration
3.PCR reaction:
(1) Target genes pheP, PAL and two kinds of vector DNA fragments were amplified by high fidelity enzyme Pfu. The reaction system is as follows:
Pfu 0.5 μL
10xPfu buffer 5 μL
Primer 1 1 μL
Primer 2 1 μL
Template (plasmid extracted above) : 1μL
25μL dNTP Mixture
20 μL of sterilized water
(2) Perform the PCR amplification procedure
At 98 ° C for 5 min
The following 3-step reaction was performed for 30 cycles
98℃ 10s
55℃ 5s
72℃ 1min/kb
72℃ 8 min
The plasmids, primers and products required for PCR are shown in the table below:
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{% block detail\_title\_a3 %}3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling{% endblock %}
{% block detail\_content5 %}Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental procedure:
1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled.
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2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit
Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min, and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA concentration was determined using Nanodrop.
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3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:
1 μg of DNA
1 μL of EcoRI
1 μL XhoI
4 μL 10xY buffer
H2O up to 20μL
The enzyme was digested at 37 ° C for 4h
4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.
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{% block detail\_title\_a4 %}At week 4, competent cells were prepared{% endblock %}
{% block detail\_content9 %}Experimental plan: Activation of E. coli DH5α and Rosetta to prepare competent cells
Experimental procedure:
1. Activated strains: including Escherichia coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper, connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube stopper again. After sealing the tube, the tube was bound and incubated on a shaker overnight (220 rpm, 37℃). The OD value of the bacteria solution was measured, and the competent state was prepared when the OD value was about 0.5.
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2. Preparation of competent cells:
(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a refrigerator at 4 ° C.
(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard, and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again (4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.
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{% block detail\_title\_a5 %}At week 5, the cells were connected and transformed{% endblock %}
{% block detail\_content11 %}Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform ligation products
Experimental procedure:
1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).
2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment volume ratio can also be calculated according to the instructions) :
T4 DNA ligase 1 μL
2 μL of 10 x ligation buffer
1 μL of Linear plasmid
Insert 6 μL of DNA
Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was stored at -20 ° C.
3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1 mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100 μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.
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{% block detail\_title\_a6 %}At the 6th week, the two successfully constructed plasmids were extracted and expanded{% endblock %}
{% block detail\_content13 %}Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation
Experimental procedure:
1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as templates for colony PCR using Taq enzyme. The primers are TF, TR. The reaction system and procedures were carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed
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2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp antibiotic was added to expand the culture.
3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.
4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.
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{% block detail\_title\_a7 %}Effect of oleic acid on bacterial growth at week 7{% endblock %}
{% block detail\_content15 %}Various concentrations of oleic acid were added to LB medium and E.coli Rosetta was inoculated, and the OD600 values of E.coli Rosetta were measured using a spectrophotometer. The results showed that 5 mM oleic acid almost did not affect the growth of bacteria, while 10 mM oleic acid inhibited the growth of bacteria.
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{% block detail\_title\_a8 %}Response of the oleic acid promoter at week 8 {% endblock %}
{% block detail\_content17 %}pOleic acid-mRFP was constructed in pSB1A3. The recombinant plasmid was transformed into E.coli Rosetta last. Various concentrations of oleic acid were added to LB medium and E.coli Rosetta/pOleic acid-mrFP was inoculated. Adjust the initial OD600 to 0.1. After 12 hours, OD600 values and mRFP fluorescence intensity were measured using a microplate reader. The results showed that the oleic acid promoter induced about 4.5-fold change in the engineered bacteria under the induction of 0mM and 5 mM oleic acid.
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{% block detail\_title\_a9 %}Determination of cholesterol transporter at week 9{% endblock %}
{% block detail\_content19 %}Each bacterial culture was grown in LB medium as previously described and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanolcholesterol stock solution was added to LB medium to achieve a final concentration of 80 μg/mL. Cholesterol quantification kit (mlbio, ml094955) was used to measure the cholesterol concentration in each medium. OD600 and cholesterol concentrations were measured again after 12 h of culture. To normalize the data, we calculated Δcholesterol/ΔOD600, which represents the uptake of cholesterol by bacteria during growth. All values were determined based on the mean of all repeated measurements.
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{% block detail\_title\_a10 %}Week 11 ismA degradation cholesterol test{% endblock %}
{% block detail\_content22 %}The ismA gene was cloned into the pET28a plasmid, validated and transformed into Escherichia coli Rosetta. The recombinant strain Rosetta was then incubated in LB medium containing 0.5 mM IPTG for 12 h at 16˚C. Ten mL of the bacterial culture was collected, the medium supernatant was discarded by centrifugation, the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.0), the crude enzyme solution was collected by pre-sonication on ice, and the protein concentration was determined by the Bradford method. The 5 mg/mL crude enzyme solution was incubated with 200 μM cholesterol for 12 h. Cholesterol concentration was subsequently determined based on fluorescence emission using the Amplex™Red Cholesterol Assay Kit (Sigma, A12216).
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ismA expression was controlled by the oleic acid promoter at week 12
The ismA gene was cloned downstream of pOleic acid and validated before transformation into Escherichia coli Rosetta. The recombinant strain Rosetta was then placed in LB medium containing various concentrations of oleic acid. After 12 hours, the crude enzyme was extracted as described previously. And 5 mg/mL of the crude enzyme solution was incubated with 200 μM cholesterol for 12 h. Cholesterol concentration was subsequently determined according to fluorescence emission using the Amplex™Red Cholesterol Assay Kit (Sigma, A12216).
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{% block detail\_title\_a11 %}At week 13, cholesterol adsorption was enhanced by enhancing EPS content{% endblock %}
{% block detail\_content25 %}Overexpression of galU enhanced EPS production in E. coli Rosetta.
Each bacterial culture was grown in LB medium as previously described and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanolcholesterol stock solution was added to LB medium to achieve a final concentration of 80 μg/mL. Cholesterol quantification kit (mlbio, ml094955) was used to measure the cholesterol concentration in each medium. OD600 and cholesterol concentrations were measured again after 12 h of culture. Δcholesterol/ΔOD600 was calculated, which indicates how well the bacteria adsorb cholesterol during growth
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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4988/wiki/cover/engineering.jpg{% endblock %}
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{% block detail\_title\_a1 %}Overview{% endblock %}
{% block detail\_content2 %}Synthetic biology is an interdisciplinary field that combines biology, genomics, engineering, informatics, and other fields. It integrates methods of observation and analysis from life sciences with the design thinking of engineering, enabling humans to engineer, modify, and even synthesize biological systems with specific functions. In recent years, synthetic biology and its applications have had a profound impact on the development of fields such as chemical engineering, food, consumer goods, energy, healthcare, and agriculture, creating significant social and economic value.
The main objective of our project is to construct a gene circuit that enables our strain to effectively degrade cholesterol in food. We encountered some issues during the design and testing phase, but we made adjustments and designed the final version of our gene circuit, testing its effectiveness. The gene circuit largely aligns with our expectations and goals.
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{% block detail\_title\_a2 %}Cycle 1: Transport and degradation of cholesterol{% endblock %}
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{% block detail\_title\_b1 %}Design{% endblock %}
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{% block detail\_img1 %}https://static.igem.wiki/teams/4988/wiki/engi/img1.png{% endblock %}
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Figure 1 Experimental design schematic diagram

Our initial gene circuit includes genes encoding cholesterol transport proteins and two genes that can degrade cholesterol (ACS and IsmA).
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{% block detail\_title\_b2 %}Build{% endblock %}
{% block detail\_content6 %}First, the T7 promoter expresses a large amount of transport enzyme that expresses cholesterol transport proteins, which will transport cholesterol into the bacterial cell. Then, IsmA and ACS will degrade it to achieve a reduction in cholesterol absorption.
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{% block detail\_img2 %}https://static.igem.wiki/teams/4988/wiki/engi/img2.png{% endblock %}
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Figure 2 Schematic diagram of gene components

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{% block detail\_title\_b3 %}Test{% endblock %}
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Figure 3 Determination of cholesterol transporters（MFS）

While maintaining identical experimental conditions, we establish trials to measure cholesterol concentrations in untreated plasmid E. coli Rosetta and plasmid E. coli Rosetta treated with protein pT7-MFS after twelve hours of cultivation conducted in LB medium. As illustrated by Fig. B and Fig. C, with identical initial cholesterol concentrations of 80 µg/mL, the untreated plasmid displayed an ultimate cholesterol concentration of roughly 72.16 µg/mL, while the cholesterol concentration plasmid treated with protein pT7-MFS dropped to approximately 55.83 µg/mL. Comparatively, the cholesterol concentrations reduced were approximately 7.84 µg/mL and 24.17 µg/mL, respectively for untreated and protein-treated plasmid samples, as shown in Fig. E. In this case, precise calculations reveal that the concentration of cholesterol is reduced by roughly 9.8% in untreated plasmid E. coli Rosetta, while in the protein-treated plasmid E. coli Rosetta, the concentration is reduced by approximately 30.21%.
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Figure 4 The expression of ismA .

The ismA gene was cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta. The recombinant Rosetta bacteria were then cultured in LB medium containing 0.5 mM IPTG and incubated at 16°C for 12 hours. A 10 mL bacterial culture was taken, centrifuged to remove the supernatant, and the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.0). The cells were then subjected to ice-cold sonication to collect the crude enzyme solution, and the protein concentration was determined using the Bradford assay. The crude enzyme solution was mixed with 5 mg/mL cholesterol and incubated for 12 hours. Subsequently, the cholesterol concentration was determined by measuring the fluorescence emission using the Amplex™Red Cholesterol Assay Kit. The results, as shown in the figure, indicate that the engineered strain significantly degraded cholesterol compared to the wild-type.
The ACS was also cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta. However, the experimental results were not satisfactory, and ACS did not play a significant role.
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{% block detail\_title\_b4 %}Learn{% endblock %}
{% block detail\_content11 %}Preliminary results have been obtained, but there is still room for improvement.We plan to collaborate with other teams to introduce EPS to enhance the adsorption of cholesterol.
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{% block detail\_title\_a3 %}Cycle 2 Constructing EPS for efficient cholesterol degradation.{% endblock %}
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{% block detail\_title\_b5 %}Design{% endblock %}
{% block detail\_content13 %}Considering that cholesterol may not be completely removed by the Isma gene, we decided to produce EPS, which interferes with the absorption of cholesterol in the intestines by binding to cholesterol and finally eliminating it from the body.In this way we can effectively remove the residual cholesterol.
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{% block detail\_title\_b6 %}Build{% endblock %}
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Figure5 Schematic diagram of gene components

We use promoter to express the galU gene, and the constructed plasmid is then introduced into E. coli Rosetta.
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{% block detail\_title\_b7 %}Test{% endblock %}
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Figure6 Images of experimental results

GalU was cloned into pET28a vector and transformed into E.coli Rosetta. Inoculated into LB medium, cultured OD600=0.4 at 37℃ and 180 rpm, and induced 16h with 0.5 mM IPTG. Adjust OD600 to 1. Take 10 mL of bacterial solution and use anthrone sulfuric acid method (see: EPS measurement method) to detect EPS yield. The EPS yield of blank strain was 46 mM, and the EPS yield of engineering strain under 0.5 mM IPTG induction was 105mM. The results of this study clearly indicate that EPS production in E. coli can be enhanced by overexpression of galU in figure A.
Each bacterial culture was cultured in LB medium and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanol cholesterol solution was added into LB medium to make the final concentration reach 80 μg/mL. The cholesterol concentration in each group's medium was detected with a quantitative cholesterol kit (mlbio, ml094955). OD600 and cholesterol concentrations were detected again after 12 h of culture. Δcholesterol/ΔOD600, which represents the absorption of cholesterol by bacteria during growth, was calculatedin figure B.Figure C,D,E and F are the effect of increased EPS content on adsorption of cholesterol.
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{% block detail\_title\_b8 %}Learn{% endblock %}
{% block detail\_content18 %}After introducing the gene galU, the effect is significantly improved. However, in order to initiate gene expression more efficiently, we would like to design a switch to make gene expression controllable.
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{% block detail\_title\_a4 %}Cycle 3: Constructing an oleic acid promoter to better initiate gene expression{% endblock %}
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{% block detail\_title\_b9 %}Design{% endblock %}
{% block detail\_content20 %}We interviewed a nutrition expert and learned that oleic acid has the effect of reducing cholesterol. Inspired by this, we hope to combine our project with oleic acid, so we designed an oleic acid promoter. We hope that the addition of oleic acid promoter can activate the expression of downstream genes, while the intake of oleic acid can also degrade cholesterol. Moreover, during the fermentation stage in the factory, because there is no oleic acid in the factory, it will not be produced and the downstream genes will not be expressed, reducing the pressure on the bacteria. Therefore, we chose to use the oleic acid promoter as our promoter.
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{% block detail\_title\_b10 %}Build{% endblock %}
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Figure7 Schematic diagram of gene components

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{% block detail\_title\_b11 %}Test{% endblock %}
{% block detail\_content23 %}The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta. The OD600 value of E.coli Rosetta was measured by spectrophotometer. The results showed that 5 mM oleic acid had little effect on bacterial growth, while 10 mM oleic acid inhibited bacterial growth.
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Figure8 Effect of oleic acid on bacterial growth

The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta/pOleic ACID-MRFP. Adjust the initial OD600 to 0.1. After 12 hours, the OD600 value and mRFP fluorescence intensity were measured using an microplate reader. The results showed that under the induction of oleic acid at 0mM and 5 mM, oleic acid promoter mediated the engineering bacteria to produce about 4.5 Fold change under the induction of 5mM oleic acid comparing to the result produced by omM oleic acid.
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Figure9 Response of oleic acid promoter

The ismA gene was cloned into the downstream of pOleic acid and transformed into Escherichia coli Rosetta after verification.The recombinant strain Rosetta was then placed in LB medium containing different concentrations of oleic acid. After 12 hours, the crude enzyme was extracted according to the above method. 5 mg/mL crude enzyme solution was incubated with 200 μM cholesterol for 12 h. The Amplex™Red cholesterol test kit (Sigma, A12216) was then used to determine cholesterol concentrations based on fluorescence emission. Under the induction of 5mM of oleic acid , the cholesterol concentration goes through a dramatic decrease due to the expression of IsmA gene, comparing to the concentration when omM oleic acid is present. The result shows oleic acid can stimulate the oleic acid promoter to express IsmA gene which can then lower cholesterol concentration.
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{% block detail\_img10 %}https://static.igem.wiki/teams/4988/wiki/engi/img10.png{% endblock %}
{% block detail\_content26 %}

Figure10 The expression of ismA is controlled by oleic acid promoter

{% endblock %}
{% block detail\_title\_b12 %}Learn{% endblock %}
{% block detail\_content27 %}The gene design is largely consistent with our expectations and goals, and it has shown good results.
{% endblock %}
{% block detail\_title\_a5 %}Conclusion{% endblock %}
{% block detail\_content28 %}After three rounds of iterative design, we have constructed a complete gene circuit that aligns with our expectations and is also practically meaningful. In the factory, our engineered probiotics will be added into high cholesterol food such as egg and cheese. Besides, oils that are rich in oleic acid, such as sunflower oil will also be added during the process to help stimulate oleic acid promoter. The oleic acid promoter can sense the presence of oleic acid and initiates the expression of downstream genes. Cholesterol transport proteins will transport cholesterol into bacterial cells, where it will be degraded by IsmA and ACS to reduce cholesterol absorption.
{% endblock %}
{% block page\_content %}

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[{{self.detail\_title\_a5()}}](#a5)

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4988/wiki/cover/experiments.jpg{% endblock %}
{% block page\_content %}{% 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 %}