iGEM 2023 — Team squirrel-chn

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{% block title %}Notebook{% endblock %}
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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 E. coli DH5α/ empty vector 1 and 1 tube of DH5α/ pheP gene. One tube of E. coli DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube orifice and plug, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the test tube orifice and plug again. After sealing the tube, the tube was 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 and one tube DH5α/ pheP gene were extracted according to the instruction of plasmid extraction kit. One tube of E. coli DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. The bacterial solution was centrifuged for 1min and then the precipitate was removed. 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.
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{% block detail\_content4 %}Strains and plasmid sample white floc after drip into the P3
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The experimental work
2. Dilute the primers
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
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Centrifuge dry powder primer manipulation
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\_content7 %}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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Plot of gel electrophoresis results
2. Gel recovery: The PCR product DNA gel was recovered according to the gel recovery kit instructions
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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Completed rubber recycling product concentration determination of DNA
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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The researchers cut gel
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Photo of the experimenter at work
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{% block detail\_title\_a4 %}Competent cells were prepared at week 4{% endblock %}
{% block detail\_content13 %}Experimental plan: E. coli DH5α and Rosetta were activated 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. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).
Experimental procedure:
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 packed 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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1. Pre-activated aliquots of DH5α and Rosetta bacterial samples
2. Bacterial samples in an ice bath
3 Add the CaCl2 solution
4. After secondary centrifugation of the bacterial sample, white precipitate was produced
5 Add glycerol CaCl2 mixing buffer
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{% block detail\_title\_a5 %}Week 5 Connect and transform{% endblock %}
{% block detail\_content16 %}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).
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Pour the liquid LB medium into the disposable Petri dish
2 Ligation reaction: The vector was ligated by double enzyme digestion of the target fragment. 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
Connections were made using a PCR apparatus at 16 ° C for 1h. 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\_content18 %}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
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 %}The ability of cells to metabolize phe was measured at the 7th week{% endblock %}
{% block detail\_content19 %}Experimental plan:
Experimental procedure:
1 Incubation, ELISA: To measure the capacity of whole cells to metabolization of phe, the PAL engineered strain was resuspended to an OD600 of 0.1 in 1 mL of Phe assay buffer (M9 0.5% glucose containing 1 mM millimoles per liter of Phe) and incubated in microtubes. The amount of Phe was then determined according to the phenylalanine ELISA kit. , the results are shown in FIG. A below.
2. The strains were resuspended in 1 mL Phe experiment buffer (M9 0.5% glucose containing 1 mM Phe) to an OD600 of 0.1, placed in microtubes, and incubated at 37°C for 1h. TCA concentration was determined with OD290 as shown in FIG. B.
3 Simultaneously determine the effect of pH on PAL enzyme activity, as shown in Figure C.
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{% block detail\_title\_a8 %}At week 8, the effect of the hypoxic promoter was tested to verify whether the hypoxic promoter could effectively activate PAL expression{% endblock %}
{% block detail\_content21 %}Experimental plan: To test the effect of the hypoxic promoter and PAL expression
Experimental procedure:
1. The tested engineering bacteria were statically cultured in a CO2 incubator (adjust the O2 concentration to 0%, 10% and 20%). After 16h of growth, the absorbance (OD600) and mRFP fluorescence intensity data were measured using a microplate reader.
2. The tested engineered bacteria were statically cultured in a CO2 incubator (O2 concentration was adjusted to 0%, 10% and 20%). The strains were resuspended in 1 mL Phe experiment buffer (M9 0.5% glucose containing 1 mM Phe) to an OD600 of 0.1, placed in microtubes, and incubated at 37°C for 1h. TCA concentration was determined using OD290. The Phe content was measured using an Elisa kit.
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{% block detail\_title\_a9 %}Trans-cinnamic acid (TCA) promoter test and determination of PAL activity at week 9{% endblock %}
{% block detail\_content23 %}Experimental plan: Promoter testing and determination of PAL activity
Experimental procedure:
1 The strain was resuspended in 1 mM Phe experiment buffer to an OD600 of 0.1 and placed in a microtube, and an additional 1 mM TCA was added to the test group. The cells were incubated at 37°C for 1h. Phe content was determined according to the phenylalanine ELISA kit, and TCA content was tested using OD290.
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{% block detail\_title\_a10 %}At the 10th week, the signal amplification switch of TP901 was coupled to test the fluorescent protein expression under different TCA content{% endblock %}
{% block detail\_content24 %}Experimental plan: The signal amplification switch of TP901 was coupled to test the fluorescent protein expression
Experimental procedure:
1. The same procedure as above was used to construct the plasmid, mRFP gene was coupled to the downstream of TCA promoter pSenCA, and the fluorescent protein expression was measured under different TCA content.
2. Fluorescence microscope verification experiment:
(1) Sample preparation: appropriate amount (about 30 μl) of wild type Rosetta and Rosetta-engineered bacteria droplets were dropped onto the slide and covered with a cover slip.
(2) Turn on the switch of the power control box of the high-pressure mercury lamp.
(3) Insert the light stopper to interrupt the light path.
(4) Preheat for 5-10 min.
(5) Place the slide with the sample on the stage.
(6) Select the objective lens (in order of first low power, then high power).
(7) Rotate the turntable of the spectroscope assembly to select the spectroscope assembly for observing mRFP (red fluorescence).
(8) Adjust the focal length through thick and thin screws.
(9) Open the computer connected to the microscope and click on the digital imaging system software to collect digital images.
3. Microplate reader was used to test fluorescent protein expression: 100 μL of wild-type Rosetta, recombinant strain solution was taken in an ultra-clean stage. The supernatant was added to a 96-well plate for 4-6 replicates. The detection conditions of the microplate reader were set (mRFP excitation λ: 584 nm/10 nm, emission λ: 607/10 nm), and the readings were taken. The data were recorded using Excel.
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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5013/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 and engineering, aiming to design and construct new biological systems and functions. It goes beyond studying the complexity of organisms in nature and seeks to optimize or reconstruct biological systems through artificial design, exploiting the programmability of life. This programmability of biological systems provides opportunities to address global challenges such as climate change, disease treatment, and food security.
In our project, we utilize a variety of components, including those previously commonly used or rarely employed, and test them multiple times to ensure their effectiveness. We have undergone numerous iterations to achieve microbial targeted therapy for diseases. The following provides detailed descriptions of the test results for each component, demonstrating the practical viability of our product.
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{% block detail\_title\_a2 %}Cycle 1 : Initial E. coli engineering aimed to absorb Phe faced a big challenge.{% endblock %}
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{% block detail\_title\_b1 %}1. Design{% endblock %}
{% block detail\_content4 %}We aim to reduce the absorption of phenylalanine in the human body by using E. coli to absorb the phenylalanine ingested by patients. We have generated a transport system by introducing a gene expressing the PheP transporter. Simultaneously, we express PAL, an important enzyme that catalyzes the deamination of phenylalanine (Phe) and removes the amino group that cannot be processed by PKU patients.
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Figure 1 Schematic representation of the overall principle of Phep and PAL.

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{% block detail\_title\_b2 %}2. Build{% endblock %}
{% block detail\_content6 %}In this section, we constructed engineered strains expressing both PheP and PAL to produce transgenic E. coli capable of absorbing phenylalanine and degrading Phe. We used a constitutive promoter to express the gene of PheP and PAL.
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{% block detail\_title\_b3 %}3. Test{% endblock %}
{% block detail\_content7 %}After expressing the engineered strains,measured Phe levels using a phenylalanine ELISA kit, while TCA concentration was determined by measuring OD290 with an microplate reader. The results, as shown in Figure 2A and 2B, demonstrate that after PAL expression, Phe levels decreased significantly, while TCA levels increased, indicating that PAL can effectively degrade Phe into TCA.
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Figure 2 Expression diagram of Phep and PAL.

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{% block detail\_title\_b4 %}4. Learn{% endblock %}
{% block detail\_content9 %}Through this test, it is known that there is an excessive amount of phenylalanine in E. coli cells, approaching a saturated state, which prevents the cells from further absorbing phenylalanine from the intestine. Therefore, it is considered to add a metabolic system led by phenylalanine deaminase.
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{% block detail\_title\_a3 %}Cycle 2 : Utilize PAL to metabolize Phe.{% endblock %}
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{% block detail\_title\_b5 %}1. Design{% endblock %}
{% block detail\_content11 %}Although our first version of the plan partially transferred phenylpyruvic acid into the bacterial cells, the capacity of bacteria is limited. When the production of Phe exceeds the tolerance of the bacteria, it still leads to intracellular Phe saturation and accumulation in the patient's body. Therefore, our second version of the plan replaced the promoter with the anaerobic promoter pPepT. When the engineered bacteria enter the patient's gut, the anaerobic environment activates the inducible anaerobic promoter, and the PAL gene starts to express. The engineered bacteria secrete PAL, which metabolizes Phe in the patient's body into trans-cinnamic acid (TCA). TCA is then transported through the intestinal capillaries into the liver via the bloodstream, where it further metabolizes into hippuric acid (HA), ultimately being excreted in the urine.
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Figure3 Hypoxia-induced promoter pPepT drives PAL expression effect.

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{% block detail\_title\_b6 %}2. Build{% endblock %}
{% block detail\_content13 %}In pSB1A3 vector, we placed PAL and Phep genes downstream of the hypoxia-induced promoter pPepT. After enzymatic cleavage, ligation, and transformation, they will be successfully transferred into Escherichia coli bacteria, allowing the target genes to be expressed and producing engineered bacteria capable of transporting phenylpyruvic acid and metabolizing phenylalanine into trans-cinnamic acid.
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{% block detail\_title\_b7 %}3. Test{% endblock %}
{% block detail\_content14 %}We tested the expression of the hypoxia-induced promoter, as shown in Figure 4A, and found that it was expressed normally under anaerobic conditions. Similarly, we measured the levels of Phe and TCA, as shown in Figures 4B and 4C. We observed higher TCA production under lower oxygen concentrations, indicating that PAL can play a significant role under anaerobic conditions. Furthermore, we validated the effect of environmental pH on PAL metabolic capacity, as shown in Figure 4D. PAL activity increased with increasing pH within the range of pH 5 to 8, with pH 8 being the optimal pH.
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Figure 4 Expression data graph of the anaerobic promoter.

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{% block detail\_title\_b8 %}4. Learn{% endblock %}
{% block detail\_content16 %}By comparison, we can conclude that the transformation ability of phe in bacteria containing Phep alone is weaker than that in bacteria containing both PheP and PAL. Therefore, it can be demonstrated that the inclusion of the metabolic system aligns with the expectations of the experiment.
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{% block detail\_title\_a4 %}Cycle 3 : Degradation of Phe in food in vitro.{% endblock %}
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{% block detail\_title\_b9 %}1. Design {% endblock %}
{% block detail\_content18 %}Considering that it is not feasible to directly administer Escherichia coli and that in vivo medication does not comply with local regulations on food and drug safety in China, we plan to design an ex vivo transformation system. This system includes the T7 promoter, RBS ribosome-binding site, PHEP transport protein, terminator, TCA promoter, TP901 integrase, and PAL phenylalanine ammonia-lyase. We retained the design of the transport protein gene and the overall structure of the PAL enzyme from the in vivo transformation system but made modifications to the promoter region of the PAL gene sequence. We modified the deoxy promoter to the T7 promoter and reversed its direction, adding the TP901 integrase sequence and TCA promoter before it. Prior to the entire translation process, a small amount of PAL enzyme had already been translated, converting some Phe to trans-cinnamic acid. Trans-cinnamic acid promotes the action of the TCA promoter, initiating the translation of the TP901 integrase. The TP901 integrase recognizes the attB/attP site，and enables the T7 promoter to reverse and face the PAL gene sequence, initiating the translation of PAL. The strong T7 promoter rapidly produces a large amount of PAL enzyme without inhibiting bacterial growth，the schematic diagram is shown in Figure 5.
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Figure 5 Schematic representation of the overall principle of PAL gene expression.

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{% block detail\_title\_b10 %}2. Build{% endblock %}
{% block detail\_content20 %}We designed a trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system to efficiently express PAL in an in vitro transformation system.
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{% block detail\_title\_b11 %}3. Test{% endblock %}
{% block detail\_content21 %} The schematic diagram of the trans-cinnamic acid (TCA) promoter is shown in Figure 6A, and the schematic diagram of the trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system is shown in Figure 6B. Firstly, we constructed mRFP downstream of the pTCA promoter and detected its expression, as shown in Figure 6C. After adding 1 mM TCA, there was a significant increase in mRFP fluorescence intensity. Secondly, we measured the content of Phe and TCA in the engineered strain using the same method, as shown in Figure 6D and E. After adding 1mM TCA, the Phe content decreased from around 1 mM to around 0 mM, while the TCA content increased from 0 mM to around 2 mM. Therefore, pTCA has a good activation ability for PAL expression.
To verify if the TP901 amplification signal switch system can promote gene expression downstream of pSenCA (TCA responsive promoter), we coupled mRFP downstream of pSenCA and added the TP901 amplification signal switch system. We tested the expression of fluorescent protein at different TCA concentrations. From Figure 6F, it can be observed that in an environment with 1mM TCA, the strain with the TP901 signal amplification switch system added downstream of pTCA had a fluorescence intensity of around 40 A.U, which was significantly higher compared to the control group. This indicates that TP901 has a significant effect in promoting downstream gene expression.
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Figure 6 Utilizing the trans-cinnamic acid (TCA) biosensor pSenCA and signal amplification switch for efficient gene expression.

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{% block detail\_title\_b12 %}4. Learn{% endblock %}
{% block detail\_content23 %}By analyzing the results, this design can basically meet the demand, but there is still the problem of slow degradation time. Therefore, we plan to use this technology in the factory production stage to produce edible products without phenylalanine.
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{% block detail\_title\_a5 %}Conclusion{% endblock %}
{% block detail\_content24 %}Through three iterations of microbes, we have obtained an engineered microorganism that can rapidly degrade Phe and complies with regulations. These iterations are based on our exploration and research on the market, patients, and regional regulations.
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{% block detail\_title\_a6 %}References{% endblock %}
{% block detail\_content25 %}1. Chien, Tiffany, et al. "Enhancing the tropism of bacteria via genetically programmed biosensors." Nature biomedical engineering 6.1 (2022): 94-104.
2. Courbet, Alexis, et al. "Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates." Science translational medicine 7.289 (2015): 289ra83-289ra83.
3. Flachbart, Lion Konstantin, Sascha Sokolowsky, and Jan Marienhagen. "Displaced by deceivers: prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns." ACS synthetic biology 8.8 (2019): 1847-1857.
4. Binder, Stephan, et al. "A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level." Genome biology 13.5 (2012): 1-12.
5. Pi, Jing, PETER J. Wookey, and A. J. Pittard. "Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli." Journal of bacteriology 173.12 (1991): 3622-3629.
6. Isabella, Vincent M., et al. "Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria." Nature biotechnology 36.9 (2018): 857-864.
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[{{self.detail\_title\_a5()}}](#a5)
[{{self.detail\_title\_a6()}}](#a6)

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```

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```

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### {{self.detail\_title\_b1()}}

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5013/wiki/cover/experiments.jpg{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5013/wiki/desc/desc-bg.jpg)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5013/wiki/cover/results.jpg{% 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 %}