iGEM 2021 — Team HiZJU-China

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# Team:HiZJU-China/Notebook

Team:HiZJU-China/Project-notebook

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* [Home](https://2021.igem.org/Team%3AHiZJU-China)

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* [Project](https://2021.igem.org/Team%3AHiZJU-China/Project)

  [Description](https://2021.igem.org/Team%3AHiZJU-China/Description)
  [Design](https://2021.igem.org/Team%3AHiZJU-China/Design)
  [Result](https://2021.igem.org/Team%3AHiZJU-China/Result)
  [Hardware](https://2021.igem.org/Team%3AHiZJU-China/Hardware)
  [Proposed Implementation](https://2021.igem.org/Team%3AHiZJU-China/Implementation)
  [Proof of Concept](https://2021.igem.org/Team%3AHiZJU-China/Proof_Of_Concept)
* [Team](https://2021.igem.org/Team%3AHiZJU-China/Team)

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  [Attributions](https://2021.igem.org/Team%3AHiZJU-China/Attributions)
* [Lab](https://2021.igem.org/Team%3AHiZJU-China/Project)

  [Experiments](https://2021.igem.org/Team%3AHiZJU-China/Experiments)
  [Notebook](https://2021.igem.org/Team%3AHiZJU-China/Notebook)
  [Protocols](https://2021.igem.org/Team%3AHiZJU-China/Protocols)
  [Model](https://2021.igem.org/Team%3AHiZJU-China/Model)
  [Safety](https://2021.igem.org/Team%3AHiZJU-China/Safety)
* [Parts](https://2021.igem.org/Team%3AHiZJU-China/Parts)

  [Parts](https://2021.igem.org/Team%3AHiZJU-China/Parts)
  [Engineering Success](https://2021.igem.org/Team%3AHiZJU-China/Engineering)
  [Contribution](https://2021.igem.org/Team%3AHiZJU-China/Contribution)
* [Human Practices](https://2021.igem.org/Team%3AHiZJU-China/Human_Practices)

  [Overview](https://2021.igem.org/Team%3AHiZJU-China/Overview)
  [Intergrated Human Practice](https://2021.igem.org/Team%3AHiZJU-China/Human_Practices)
  [Education and Public Engagement](https://2021.igem.org/Team%3AHiZJU-China/Education)
  [Collaborations](https://2021.igem.org/Team%3AHiZJU-China/Collaborations)
* [Awards](https://2021.igem.org/Team%3AHiZJU-China/Awards)

  [Judging](https://2021.igem.org/Team%3AHiZJU-China/Judging)
  [Sustainable Development Impact](https://2021.igem.org/Team%3AHiZJU-China/Sustainable)
  [Best Safety](https://2021.igem.org/Team%3AHiZJU-China/BestSafety)

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[### **Period Ⅰ-Engineered bacteria construction**](#card-element-1)

##### ***July 29th*** **OUTLINE**  prepare and sterilize LB medium  Prepare LB plates with ampicillin  Cultivate vector bacteria containing pET plasmid in LB liquid medium ***July 30th*** **OUTLINE**  Harvest the vector and extract pET plasmid  Amplify amoA gene by PCR. Gel imaging proved the success of PCR  Digest the pET plasmid with Nco I and HindⅢ **DETAILS AND ANALYSIS**  Extract pET plasmid After the first round of extraction, the concentration was very low, so we added another 2ml bacterium solution and centrifuged again. The final concentration of the pET plasmid is 10.4ng/ul.  Amplify amoA gene by PCR. Gel lmaging proved the success of PCR We acquired amoA gene from Nitrosomonas europaea ATCC 19718 The reaction system is listed below: ![](https://static.igem.org/mediawiki/2021/5/5b/T--HiZJU-China--notebook1.png) Gel imaging 10,000 DNA marker was used to identify the length of DNA fragment. The result shows the length of amoA fragment is approximately 800 bp, which is in accordance with theoretical data, 843bp. It proved we successfully acquired and amplify amoA fragment from AOB bacteria. Therefore, there is no need to extract the genome of AOB to acquire this gene fragment. ![](https://static.igem.org/mediawiki/2021/0/0e/T--HiZJU-China--notebook2.png) The concentration is 4.9ng/μl while the ratio is 1.99. ***July 31st*** **OUTLINE**  Digest amoA DNA fragment  Link amoA fragment and pET plasmid  Introduce the pET-amoA plasmid into E.coli DH 5α  Spread the bacteria onto the plate **DETAILS AND ANALYSIS**  Digest amoA DNA fragment The reaction system for enzyme ligation is 100μl. Theoretically, 2000-5000ng fragment is needed, but we only acquired 63μl 4.9ng/μl fragment. Therefore, all the fragment was used. ![](https://static.igem.org/mediawiki/2021/5/54/T--HiZJU-China--notebook3.png)  Link amoA fragment and pET plasmid The reaction system should have been 10μl，but due to the low concentration of pET and amoA fragment, the final volume was larger. The enzyme we used is T4 ligase. Details are shown in the table. ![](https://static.igem.org/mediawiki/2021/e/ec/T--HiZJU-China--notebook4.png)  Introduce the pET-amoA plasmid into E.coli DH 5α The steps are the same as in the protocols.  Spread the bacteria onto the plate The transformed E.coli DH 5α was spread onto the pre-made Amp plate to select ones carrying pET plasmid. The appearance of tons of bacterial colonies on the Amp plate the next day demonstrated the success of the introduction of pET plasmid. ![](https://static.igem.org/mediawiki/2021/0/06/T--HiZJU-China--notebook5.png) ***August 2nd*** **OUTLINE**  Verify if the colony on the Amp plate carries amoA gene  Pick and incubate the colony numbered 5 which carries amoA gene **DETAILS AND ANALYSIS**  Verify if the colony on the Amp plate carries amoA gene To confirm the colony survived carries whether pET or pET-amoA, we used PCR to amplify the amoA gene fragment with primer amoA YZ-1 and YZ-2 and then utilize Gel electrophoresis to check out its presence. We chose 8 colonies randomly and numbered them from 1 to 8. ![](https://static.igem.org/mediawiki/2021/8/8f/T--HiZJU-China--notebook6.png) Apparently, all of the 8 colonies we choose carry pET-amoA plasmid, although the copy number is different according to fluorescence intensity. ***August 3rd*** **OUTLINE**  Extract pET-amoA plasmid  Digest and purify pET-amoA plasmid  Acquire hao gene fragment by PCR  Digest hao fragment  Link hao and pET-amoA plasmid  Introduce the pET-amoA-hao plasmid into E.coli DH 5α  Spread the engineered bacteria onto the selective plate **DETAILS AND ANALYSIS**  Acquire hao gene fragment by PCR Gel electrophoresis is used to confirm the result of PCR. The gel image denotes the success. ![](https://static.igem.org/mediawiki/2021/2/2d/T--HiZJU-China--notebook7.png)  Spread the engineered bacteria onto the selective plate No colonies appeared on the plate, which meant the transformation in August 3rd failed. ![](https://static.igem.org/mediawiki/2021/a/ae/T--HiZJU-China--notebook8.png) ***August 4th*** **OUTLINE**  Prepare and sterilize LB medium  Prepare selective LB plates with ampicillin  Incubate colony 5 again and extract plasmid  Repeat the transformation in August 5th with another batch of competent E.coli DH5α  Spread the engineered bacteria onto the selective plate **DETAILS AND ANALYSIS** We speculate the failure yesterday derives from matters of plasmid extraction or competent E.coli DH5Α. We cultivate the bacteria containing plasmid of pET-amoA to extract the plasmid again. For the matter of competent bacteria, we transformed the pET-amo-hao plasmid extracted yesterday into another batch of competent E.coli DH5α. Some colonies but less than plate of pET-amoA appeared on the plate, so we infer the problem comes from the impurity of the plasmid. ![](https://static.igem.org/mediawiki/2021/0/05/T--HiZJU-China--notebook9.png) ***August 5th*** **OUTLINE**  Confirm the ligation of hao gene by PCR and gel electrophoresis  Pick and cultivate the colony numbered 6 overnight **DETAILS AND ANALYSIS** The gel image showed the success of inserting the hao gene into the plasmid. ![](https://static.igem.org/mediawiki/2021/0/0c/T--HiZJU-China--notebook10.png) ***August 6th*** **OUTLINE**  Harvest and preserve the bacteria cultured yesterday  Extract the plasmid of pET-amoA-hao for sequencing and transformation into E.coli BL21  Spread the engineered bacteria onto the selective plate and incubate overnight **DETAILS AND ANALYSIS** No colony appeared on the plate. ![](https://static.igem.org/mediawiki/2021/d/df/T--HiZJU-China--notebook11.png) ***August 7th*** **OUTLINE**  Repeat the transformation in August 6th  Spread the engineered bacteria onto the selective plate and incubate overnight **DETAILS AND ANALYSIS** No colony appeared on the plate. ![](https://static.igem.org/mediawiki/2021/7/78/T--HiZJU-China--notebook12.png) ***August 8th*** **OUTLINE**  Incubate the engineered strains from the preservation in August 6th ***August 9th*** **OUTLINE**  Harvest cells and extract the plasmid for transformation  Preserve the engineered E.coli BL21 **DETAILS AND ANALYSIS** No colony appeared on the plate. ![](https://static.igem.org/mediawiki/2021/0/0f/T--HiZJU-China--notebook13.png) ***August 10th*** **OUTLINE**  Prepare and sterilize LB medium  Transform the plasmid again and examine the vitality of competent bacteria **DETAILS AND ANALYSIS** Given the repeated failure, we decided to examine the vitality of competent bacteria with another plasmid. And once again, no bacteria can grow on the selective plate. Therefore, it was confirmed that the competent bacteria under poor condition caused the repeated failure, rather than the plasmid we extracted. ***August 11th*** **OUTLINE**  Prepare and sterilize 50mL of LB medium  Prepare 10% (vol/vol) glycerol  Cultivate E.coli DH5α which contains the recombinant plasmid for transformation ***August 12th*** **OUTLINE**  Cultivate E.coli BL21 at 37℃ for 3 hours  Harvest the culture yesterday and extract the plasmid  Transform the plasmid into the E.coli BL21 by electrotransformation **DETAILS AND ANALYSIS** Due to the low efficiency of chemical transformation, we changed our method and resort to electrotransformation. We succeeded for the first time and the efficiency is much higher. ![](https://static.igem.org/mediawiki/2021/c/ce/T--HiZJU-China--notebook14.png)

[### **Period Ⅱ-Protein induction and condition optimization**](#card-element-2)

##### ***August 13th*** **OUTLINE** Prepare and sterilize LB medium Pick and incubate a colony on yesterday plate ***August 14th*** **OUTLINE** Prepare and sterilize 1M IPTG solution Preserve the engineered E.coli BL21 (NB) ***August 15th*** **OUTLINE** Prepare Ammonium sulfate solution containing 300mg/l of NH4+-N Pick and incubate three colonies from plate (August 12th) ***August 16th*** **OUTLINE** Use ultrasound to break cells (BL21 and NB) **DETAILS AND ANALYSIS** The tubes were removed from the shaker and the cell culture fluids were transferred to centrifugal tubes. The cell culture tubes were centrifuged at 4000 rpm. for 10 minutes. The supernatant was discarded. The precipitates were cleaned twice with PBS solution. Then, the precipitates were suspended again with 1ml PBS. Use ultrasonic cell crusher to break the cell suspension solutions. Set the ultrasonic crushing parameters: power 30%, horn 3, ultrasonic on time 2s and off time 4s, and total working time 15 minutes. All operations take place on ice to ensure the temperatures of samples are under 4℃. But finally we failed. Analysis the results, we guess that’s because the setting of the power was a little small. We can increase the power or increase the ultrasonic time. ***August 17th*** **OUTLINE** Use ultrasound to break cells Prepare the AOB culture **DETAILS AND ANALYSIS** Set the ultrasonic crushing parameters: power 30%, horn 3, ultrasonic on time 3s and off time 7s, and total working time 15 minutes. All operations take place on ice to ensure the temperatures of samples are under 4℃. The results shows that the parameter settings are very appropriate. Attention: fill 2ml cell culture broth into 4ml tube for ultrasonic crushing. ***August 18th*** **OUTLINE** Confirm follow-up test method for degradation and purchase related reagents ***August 19th*** **OUTLINE** Prepare and sterilize the AOB culture The cells expansion in the tubes Add ITPG to induce expression **DETAILS AND ANALYSIS** We selected three colonies to amplify from the plate which transform the plasmid into the E.coli BL21 by electrotransformation on August 12th. Add 5μl BL21 into three LB culture media tubes for comparisons. Culture in an incubator for 12h and then add 1/1000 IPTG to induce the expression. Culture cells in a 30℃ shaker. ***August 20th*** **OUTLINE** Use ultrasound to break cells (BL21 and NB) The cells expansion in the tubes Culture ammonia-oxidizing bacteria (The same operations as before) ***August 21th*** **OUTLINE** Add ITPG to induce expression of the cells cultured yesterday ***August 22th*** **OUTLINE** Centrifuge the cell broth which has been induced expression for 22h Pretreat the culture broth for SDS-PAGE **DETAILS AND ANALYSIS** The cell culture broths were centrifuged at 4000 rpm. for 10 minutes. The supernatant was discarded. The precipitates were cleaned twice with PBS solution. Then, the precipitates were suspended again with 1ml PBS. Store the tubes in the -80℃ refrigerator. ***August 23th*** **OUTLINE** Use ultrasound to break cells pretreated yesterday Confirm the expression of the proteins amo and Hao by SDS-PAGE **DETAILS AND ANALYSIS** The SDS-PAGE experiment failed. The electrophoretic bands about amo and Hao are not apparent and legible. Forgetting to add the AMP antibiotics is a main reason which would cause the loss of plasmids. ![](https://static.igem.org/mediawiki/2021/e/ee/T--HiZJU-China--notebook15.png) ***August 24th*** **OUTLINE**  Prepare 100mM IPTG solution  Culture a new batch of BL21 and NB **DETAILS AND ANALYSIS** Culture BL21 and NB strains with LB culture media with conical flasks. When optical densities are 0.4-1.0, add four different concentrations IPTG (0, 0.2, 0.6, 1.0mM) to culture media. And then take the samples at 3h, 6h, 8h, 20h. ***August 25th*** **OUTLINE**  Pretreat the culture broth for SDS-PAGE ***August 26th*** **OUTLINE**  Confirm the expression of the proteins amo and Hao by SDS-PAGE **DETAILS AND ANALYSIS** Use the SDS-PAGE to confirm the expression of the proteins amo and Hao with the cell broth induced for 3h, 6h. But the electrophoretic bands about amo and Hao are not apparent and legible, which means the amounts of the protein amo and Hao were little. The amounts of the proteins may be more when genes are induced for 8h and 22h. ***August 27th*** **OUTLINE**  Confirm the expression of the proteins amo and Hao by SDS-PAGE(8h, 22h) **DETAILS AND ANALYSIS** The electrophoretic bands about amo and Hao are apparent and legible. But the lack of a control group made the results inconclusive. ***August 28th*** **OUTLINE**  Confirm the expression of the proteins amo and Hao by SDS-PAGE(8h, 22h) **DETAILS AND ANALYSIS** In order to confirm the electrophoretic bands, we did the experiment SDS-PAGE again with two control groups and four experimental groups (Induced at 0/0.2/0.6/1.0 mM ITPG 8h, respectively). The electrophoretic bands about amo and Hao are apparent and legible, which confirm the expression of the proteins amo and Hao. ![](https://static.igem.org/mediawiki/2021/8/84/T--HiZJU-China--notebook16.png)

[### **Period Ⅲ-Enzyme activity determination**](#card-element-3)

##### ***August 29th*** **OUTLINE**  Prepare 100ml NH4Cl solution with NH4+ concentration of 30 mg/ml  Inoculate three tubes of engineered bacteria and induced expression by IPTG **DETAILS AND ANALYSIS** When pH=7.5, the group produced purple-red color, indicating the existence of nitrite. The result confirm that the crude enzyme including amo and Hao is active. When pH=8.0, there is no discoloration, indicating that the enzyme may be inactive at pH=8.0. The experimental design needs to be further optimized. Orthogonal experiment can be used to determine the optimal reaction conditions. ***August 30th*** **OUTLINE**  Determine the influence of the reaction time and substrate concentration  Determine the influence of the induction time  Repeat the experiment with non-buffer reaction system  Test the parallel experiment setting above  Test the concentration of ammonium ion in reaction solution 2,3 and 4 using Nessler’s reagent  Prepare LB culture media and ammonia detection media  Use ultrasound to break cells **DETAILS AND ANALYSIS** 1、 Buffer solution with ammonium chloride and test reagent will change color while preparing for the control group. The buffer and ammonium chloride were respectively tested and it was found that the buffer at 7.5 could change color. So the experiment was abandoned. 2、 The discoloration could not be observed by naked eyes in the two groups, and the absorbance was similar to that of blank (distilled water + detection reagent). 3、 Absorbance significantly increased, and the group (IPTG 1.0mM) was greater than 0.2, but there was no parallel experiment. 4、 The reaction time was prolonged, but the absorbance decreased significantly. The absorbance of the group (IPTG 1.0mM) was slightly higher than the group (IPTG 0.2mM). The calculated concentration of nitrite is low. 5、 ① The reaction solution was not diluted, and the reagent was added with the reaction solution: Nessler’s reagent =100:3 (the ratio of documents in group files). Significant brown precipitates appeared in groups 2 and 3. ② Dilute 10/20/30mg/ mL ammonium chloride solution, with the same proportion of Nessler’s reagent, more brown precipitate appeared, after a period of shock standing precipitation disappeared. ③ The 4 reaction solution diluted 50 times, the same proportion of Tim Nessler’s reagent, still appeared insoluble precipitation, IPTG1.0 group was suspension, 0.2 group is closer to emulsion. ④ Dilute the reaction solution 500 times, basically no precipitation, but the absorbance value is small. Ammonium concentration cannot be calculated due to the lack of a standard curve. 6、 According to the non-dilution system experiment, the amount of enzyme liquid obtained by tube culture is insufficient, so use shaking flask culture liquid. ***August 31th*** **OUTLINE**  Make the standard curve of ammonium three times  Determination of the optimal experiment conditions by orthogonal experiment  Induction of the two shakers on the August 30th ![](https://static.igem.org/mediawiki/2021/c/cd/T--HiZJU-China--notebook17.png) ***September 1st*** **OUTLINE**  Make the standard curve of ammonium three times in Origin  Detection of the biomass, ammonium concentrations and nitrite concentrations  Prepare glucose standard solution  Pretreat the culture broth for ultrasonic cell diaruption **DETAILS AND ANALYSIS** The fitting degrees of the second and third groups are 0.986 and 0.975, respectively. So it is better to do the standard curve again with higher fitting degree. ![](https://static.igem.org/mediawiki/2021/7/78/T--HiZJU-China--notebook18.png) ***September 2nd*** **OUTLINE**  Detection of the biomass, ammonium concentrations and nitrite concentrations  Make the standard curve of EE2  Inoculate two shake flasks of engineered bacteria and induced expression by IPTG  Prepare test medium and sterilization  Make the standard curve of ammonium **DETAILS AND ANALYSIS** The fitting degree of the standard curve of ammonium is 0.998. But we failed to make the standard curve of EE2 because EE2 has very low solubility in water. ![](https://static.igem.org/mediawiki/2021/4/43/T--HiZJU-China--notebook19.png) ***September 3rd*** **OUTLINE**  Detection of the biomass, ammonium concentrations and nitrite concentrations  Use ultrasound to break cells (BL21 cultured 12h)  Inoculate a tube of engineered bacteria (NB) and a tube of BL21  Prepare test medium and sterilization ***September 4th*** **OUTLINE**  Detection of the biomass, ammonium concentrations and nitrite concentrations  Make the standard curve of ammonium with HPLC ![](https://static.igem.org/mediawiki/2021/7/7a/T--HiZJU-China--notebook20.png) ***September 7th*** **OUTLINE**  Test the biomass of medium 002.  Take a total of 6 samples from conical bottles that have been reacting for 3 days. Centrifuge the samples and take the supernatant for further detection.  Take the samples of AOB bacteria, numbered 1-3, and take the supernatant after centrifugation. Measure the ammonia nitrogen concentration of the supernatant after diluting the supernatant ten times.  Inoculate engineered bacteria and BL21 into the medium, with the purpose of facilitating the expansion of subsequent bacteria culture in conical flasks.  **DETAILS AND ANALYSIS**  Compared with the previous day, all ammonia nitrogen concentration of three groups of AOB bacteria were significantly reduced, indicating the survival of AOB bacteria. ***September 8th*** **OUTLINE**  Inoculate engineered bacteria and BL21 strain into the medium. This four conical flasks containing the bacteria are numbered 003. ***September 9th*** **OUTLINE**  Inoculate LB21 strain and engineered bacteria into the test medium. Join AMP into the medium at the same time  Test the pH of AOB’s medium, and adjust it to weak alkalinity.  Test the activity of crude enzymes under different temperature.  Inoculate engineered bacteria and BL21 into the medium, with the purpose of facilitating the expansion of subsequent bacteria culture in conical flasks. **DETAILS AND ANALYSIS**  The current pH of AOB’s medium is weakly acidic, which may be the reason of failure of the culture of AOB bacteria. ***September 10th*** **OUTLINE**  Sample from the test medium 003 for 29h’s detection of EE2 concentration and ammonia concentration.  Sample from AOB bacteria solution, and test the ammonia nitrogen content after centrifugation.  Transfer the bacteria in LB medium to the test medium, number this batch of strain 004.  Thaw, centrifuge, and crush the samples for 17h and 29h, take the supernatant and extracting it with ethyl acetate. The obtained solution will be used for HPLC analysis  Sample from the medium containing cultured BL21 and engineer bacteria induced by IPTG for 22h. Break the bacteria, and obtain the precipitates after centrifugation. Store the precipitates in a refrigerator at -80°C .The precipitates will be used in tomorrow’s SDS-PAGE electrophoresis.  Inoculate Y2H yeast into the culture medium. ***September 11st*** **OUTLINE**  Take the sample stored on September 10th and obtained the supernatant after centrifugation. Extract the EE2 with ethyl acetate from the supernatant. Ethyl acetate : supernatant=1:10, sample supernatant was 400μL.  Take the precipitation on September 10th . Dissolved it in water and conduct the SDS-PAGE electrophoresis.  Take Y2H yeast cultured on September 10th and spread the yeast onto the plate.  Test the biomass of test and ammonia content of medium 003.  Sample from test medium 004 for further detection of 24h’s EE2 and ammonia concentration. **DETAILS AND ANALYSIS**  After 60h, the biomass of BL21 decreased, but the biomass of engineering bacteria increased slightly, indicating 48 hours’ culture time of bacteria is sufficient. ***September 12rd*** **OUTLINE**  Extract the bait plasmid PGBKT7-ERα-LBD carries optimized human estrogen receptor ERα-421F .  Measure 47h’s the ammonia content of AOB bacteria group.  Prepare 500mL mobile phase for HPLC analysis, and carry out filtration and ultrasonic cleaning .  Inoculate 100μL engineered bacteria(glycerol tube at -80°C on September 5th ) along with 5μL AMP in 5mL LB medium.  Add 2.5μL IPTG into LB medium when the OD of the LB medium is 0.4-0.8.  Sample from test medium for detection of 48h’s ammonia content and EE2 content. Take the supernatant after centrifugation. Extract the EE2 with 400μL ethyl acetate from 600μL supernatant. ***September 14th*** **OUTLINE**  Prepare urea solution(6M/L).  Test the 47h’s ammonia content of AOB bacteria group.  Collect the supernatant after centrifugation. Extract crude enzymes from the samples. Collect the precipitate at the same time and dissolved the precipitate in urea solution. The obtained solution will be used to run the SDS-PAGE electrophoresis . Store the crude enzymes at -80°C. ***September 15th*** **OUTLINE**  Run SDS-PAGE electrophoresis of precipitation and crude enzyme.  Take the supernatant for HPLC analysis. ***September 16th*** **OUTLINE**  Add IPTG to induce engineered bacteria. ***September 20th*** **OUTLINE**  Extract crude enzyme and collect the precipitation preparing for SDS-PAGE electrophoresis. Dissolve the precipitation in urea solution.  Sample from 005 test medium for further detection of ammonia and EE2 content. ***September 21st*** **OUTLINE**  take the supernatant after centrifuged the urea solution. Run the SDS-PAGE electrophoresis with the supernatant.  Take the supernatant after centrifugation. Extract the EE2 with ethyl acetate from the supernatant. Ethyl acetate : supernatant=1:1. ***September 22nd*** **OUTLINE**  Prepare materials for the redetermination of EE2 standard curve when extracting EE2 with ethyl acetate.  Add EE2 and AMP to the test medium.  Take the supernatant after centrifugation. Extract the EE2 with ethyl acetate from the supernatant. Ethyl acetate : supernatant=1：4. ***September 23rd*** **OUTLINE**  Prepare LB medium.  Prepare the mother liquor of inorganic salt of test medium  Redetermination of EE2 standard curve when extracting EE2 with ethyl acetate  Inoculate LB21 bacteria and engineered bacteria into the culture medium. ***September 24th*** **OUTLINE**  Prepare LB medium.  Inoculate LB21 bacteria and engineered bacteria, with the purpose of facilitating the expansion of subsequent bacteria culture in conical flasks.  Prepare 4mL filtrate sterilized AMP solution ***September 25th*** **OUTLINE**  Test the activity of crude enzyme degrading the organic nitrogen.  Transfer the bacteria in LB medium to test medium named 006. **DETAILS AND ANALYSIS** ![](https://static.igem.org/mediawiki/2021/a/a8/T--HiZJU-China--note1.png) ![](https://static.igem.org/mediawiki/2021/1/1b/T--HiZJU-China--note2.png) Fig the detection of urea content  The urea content of engineered bacteria is evidently low than controlled group, indicating the degradation of ammonia. Our engineered bacteria has the ability to degrade ammonia. ***September 26th*** **OUTLINE**  Prepare the LB medium and test medium.  Sample from 006 test medium. Extract the crude enzyme after centrifugation. ***September 27th*** **OUTLINE**  Inoculate BL21 bacteria and engineered bacteria into LB medium, with the purpose of facilitating the expansion of subsequent bacteria culture in conical flasks.  Preserve the engineered strain.  Prepare SCD-TRP medium.  Expand the culture of yeast. ***September 28th*** **OUTLINE**  Inoculate the Y2H yeast into the culture medium.  Inoculate the CT yeast into the culture medium.  Prepare the urea medium, named 007.  Spread the Y2H yeast onto the plate, cultured at 30°C  Introduce the plasmid into the yeast, transform the yeast.  Prepare 15μg/ml estrogen acetone solution ***September 30th*** **OUTLINE**  HPLC anaylsis. **DETAILS AND ANALYSIS** ![](https://static.igem.org/mediawiki/2021/7/7b/T--HiZJU-China--note3.png) ![](https://static.igem.org/mediawiki/2021/3/36/T--HiZJU-China--note4.png)  Estrogen was found to have a larger absorbance at 281nm than 246nm.281 nm is more Suitable for estrogen detection.  The rest EE2 content of engineered bacteria is low than the control group, indicating the degradation of EE2. Our engineered bacteria is equipped with the ability to degrade EE2. ***October 1st*** **OUTLINE**  Conduct the HPLC analysis with Estrogen acetone solution, find the best method to detect EE2.  Sampled from 007 urea medium(48h)  Test the activity of crude enzymes to degrade the urea.  Inoculate the BL21 and engineered bacteria into culture medium. **DETAILS AND ANALYSIS**  Estrogen was found to have a larger absorbance at 281nm than 246nm.281 nm is more Suitable for estrogen detection. ***October 9th*** **OUTLINE**  Prepare 500mL mobile phase for HPLC analysis, and carry out filtration and ultrasonic cleaning .( Acetonitrile: water = 1:1\_  Take the supernatant after centrifugation. Extract the EE2 with ethyl acetate from the supernatant. Ethyl acetate : supernatant=2：15.  HPLC analysis.  Inoculate Y2H yeast into SCD-TRP medium, culturing at 30°C ***October 10th*** **OUTLINE**  Take the supernatant after centrifugation. Extract the EE2 with ethyl acetate from the supernatant overnight.  Prepare LB medium.  Preserve Y2H-ERα. Stored at -80°C ***October 11st*** **OUTLINE**  Add IPTG into medium. Induce the engineered bacteria at 28°C incubator.  Copy the data of HPLC analysis.

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# Team:HiZJU-China/Engineering

Team:HiZJU-China/Engineering Success

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[### **Overview**](#card-element-1)

##### This year HiZJU-China have tackled one or more of our project’s problems and use synthetic biology tools to generate desired results. Combined the teammates’ ideas with advice through Human Practice, we designed three systems for our project, namely the degradation, detection and kill switch system. we found two key enzyme amoA and hao enzyme have the ability to degrade EE2. With the purpose of pursuing better detection methods, we designed yeast two-hybrid technique to detect EE2. We have done a lot of amazing work to realize the goals we set. The proof on the success of our project is placed here. We hope our experience will serve as a helper for future iGEM teams.

[### **Engineer Success**](#card-element-2)

##### ***A. Degradation System*** **1. amoA gene and Hao gene from Nitrosomonas europaea ATCC 19718** The amoA gene encodes the AMO enzyme which produces electrons by ammoxidation reaction. The Hao gene encodes the Hao enzyme. Electrons produced by AMO enzyme are combined with oxygen on Hao enzyme to produce H2O2, which provides oxidant for EE2 degradation. **1-1. Agarose Gel Electrophoresis** Result: Two target gene, amoA gene and Hao gene are designed successfully as the gragh shown below. ![](https://static.igem.org/mediawiki/2021/9/98/T--HiZJU-China--ES1.png) ![](https://static.igem.org/mediawiki/2021/5/58/T--HiZJU-China--ES2.png) **1-2. SDS-PAGE** Result: amoA gene and Hao gene are expressed successfully, which means that the related gene worked well. We can found amoA and HAO protein both in supernatant and precipitate after cell breakage. ![](https://static.igem.org/mediawiki/2021/9/99/T--HiZJU-China--ES3.png) ![](https://static.igem.org/mediawiki/2021/e/ef/T--HiZJU-China--ES4.png) **1-3. Enzyme Activity** Method: amoA and Hao enzymes were used to react with ammonia. We use urea as a nitrogen source. Started testing the rest of the urea and the The products nitrate nitrogen that formed.(OD630 and OD420 respectively). Record the OD420 and OD630 value in real time. Result: We can see the OD630 of samples decrease very quickly, indicating the degradation of ammonia. ![](https://static.igem.org/mediawiki/2021/d/d8/T--HiZJU-China--ES5.png) Fig. 5 Enzyme activity of amoA and Hao enzyme(react with urea) **1-4 Ability of engineered bacteria to degrade EE2** Method: Our engineered bacteria were used to react with EE2 and nitrogen. We use a test medium to culture the bacteria. In the test medium, we add urea as the nitrogen source facilitating the cometabolic degradation of EE2. We used HPLC-hv to detect the concentration of EE2 after reaction. Result: We can found the concentration of EE2 decrease evidently after a certain time, indicating the success of the degradation of EE2. (organic amine will slowly transformed into ammonium.) ![](https://static.igem.org/mediawiki/2021/c/ce/T--HiZJU-China--ES61.png)Fig. 6 The standard curve of EE2 ![](https://static.igem.org/mediawiki/2021/6/6c/T--HiZJU-China--ES6.png) Fig. 7 The ability of engineered bacteria to degrade EE2 ***B. Detection System*** Our detection system based on yeast two-hybrid technique which required two expression plasmids. The bait plasmid PGBKT7-ERα-LBD carries optimized human estrogen receptor ERα-421F while the hunting plasmid PGADT7-GRIP1 carries GRIP1 gene. The GRIP1 gene can express glutamate receptor interacting protein and be widely used for yeast two-hybid assay. **2-1. Modification of chasis cells** Result: we successfully obtained two recombinant fluorescent yeasts-red Y2H-RFP and green fluorescent yeast Y2H-GFP regulated by T7 promoter. **2-2. ERα-421F gene and GRIP1 gene** Result: Two target gene, amoA gene and Hao gene are designed successfully as the gragh shown below. ![](https://static.igem.org/mediawiki/2021/2/26/T--HiZJU-China--ES8.png) Fig. 8 ERα-421F gene and GRIP1 gene **2-3. Protein Expression** Method: When the culture was grown to the logarithmic growth stage, add 5μL EE2 and DMSO with different gradients to 995μL yeasts solution to form exposure culture medium. Result: For yeast Y2H-RFP, the reporter gene RFP mCherry was activated and expressed red fluorescent protein, indicating the engineered can detect the EE2. ![](https://static.igem.org/mediawiki/2021/b/b7/T--HiZJU-China--ES9.png) Fig. 9 yeast Y2H-RFP cultured on different medium shows different color. a, positive control. b, negative control. [For the whole result, please visit the result.](https://2021.igem.org/Team%3AHiZJU-China/Result)

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# Team:HiZJU-China/Experiments

Team:HiZJU-China/Experiment

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  [Proposed Implementation](https://2021.igem.org/Team%3AHiZJU-China/Implementation)
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  [Overview](https://2021.igem.org/Team%3AHiZJU-China/Overview)
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* [Awards](https://2021.igem.org/Team%3AHiZJU-China/Awards)

  [Judging](https://2021.igem.org/Team%3AHiZJU-China/Judging)
  [Sustainable Development Impact](https://2021.igem.org/Team%3AHiZJU-China/Sustainable)
  [Best Safety](https://2021.igem.org/Team%3AHiZJU-China/BestSafety)

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[### **Plasmids**](#card-element-1)

##### petDuet: The pETDuet vector can combined both monoamine oxidase and hydroxylamine Oxidase coding gene. The petDuet vector use T7 promoter and lac promoter to express the protain also contains AmpR gene for selection. pGADT7: The pGADT7 vector expresses proteins fused to amino acids of the GAL4 activation domain (AD). In yeast, fusion proteins are expressed at high levels from the constitutive ADH1; transcription is terminated by the ADH1 transcription termination signals. The pGADT7 vector also contains T7 promoter, SV40 NLS and Kozak sequence. The vector carries the Amp+ for selection in yeast. Yeast strains containing pGADT7 exhibit a higher transformation efficiency than strains carrying other AD protein vectors. The pGADT7 vector is always used in yeast two-hybrid technology for detection. pGBKT7: It is a plasmid which can express a fusion protein of BD and ERa-421F. Coding sequence of target gene was inserted into an expression vectors with ERa-421F and Plasmid(BBa\_K289008) to obtain BBa\_K3978004. We transformed the constructed plasmid into yeast Y2H to construct yeast two-hybrid technology system.

[### **Fragment sequence**](#card-element-4)

##### amoA ATGGTGAGTATATTTAGAACGGAAGAAATCCTGAAAGCGGCCAAGATGCCGCCGGAAGCGGTTCATATGTCACGTCTGATTGATGCAGTTTATTTTCCAATTCTGATTATTTTGCTGGTGGGTACCTACCACATGCACTTTATGCTGCTGGCAGGTGACTGGGATTTCTGGATGGACTGGAAAGATCGTCAATGGTGGCCGGTTGTAACGCCAATCGTGGGGATCACCTACTGTTCGGCAATCATGTATTACTTGTGGGTCAACTACCGCCAACCGTTTGGTGCAACGTTGTGTGTGGTGTGTCTGCTGATTGGTGAGTGGCTGACACGTTACTGGGGATTCTACTGGTGGTCACACTACCCCATCAACTTCGTAACACCGGGCATTATGCTTCCGGGTGCGCTGATGCTGGACTTCACGCTGTATCTGACACGCAACTGGCTGGTGACGGCTCTGGTTGGAGGTGGATTCTTCGGTCTGCTGTTCTATCCGGGTAACTGGCCGATTTTTGGACCAACCCATTTGCCAATCGTTGTAGAAGGCACATTGCTGTCGATGGCTGATTACATGGGACATCTGTATGTTCGTACAGGTACACCCGAGTATGTTCGTCATATTGAGCAAGGTTCACTGCGTACCTTTGGTGGTCATACCACAGTTATTGCAGCATTCTTCTCTGCGTTCGTATCAATGTTGATGTTCACCGTATGGTGGTATCTTGGAAAAGTTTACTGTACAGCCTTTTTCTACGTTAAAGGTAAAAGAGGTCGTATCGTACATCGCAATGATGTTACCGCATTCGGTGAAGAAGGCTTTCCAGAGGGGATCAAATAA hao: ATGAGAATAGGGGAGTGGATGAGGGGTTTATTGCTCTGTGCGGGGTTGATGATGTGCGGGGTGGTTCATGCGGACATATCGACGGTACCGGACGAGACGTATGATGCGTTGAAGCTGGATCGAGGTAAGGCGACCCCGAAAGAGACGTATGAAGCGCTGGTCAAGCGTTACAAGGACCCGGCGCATGGAGCGGGGAAAGGGACGATGGGAGATTACTGGGAGCCGATAGCGATCAGTATATATATGGATCCCAATACTTTTTACAAACCACCGGTATCTCCCAAAGAAGTTGCGGAACGCAAAGACTGCGTGGAATGCCACTCGGACGAGACCCCGGTATGGGTAAGAGCCTGGAAGCGCAGCACCCACGCCAATCTTGACAAGATTCGCAACCTCAAGTCGGACGATCCCCTGTATTACAAGAAAGGGAAGCTGGAAGAAGTTGAAAACAACCTGCGCTCGATGGGCAAGCTTGGGGAAAAAGAAACCCTCAAGGAAGTAGGCTGTATCGACTGTCACGTTGACGTCAACAAAAAAGACAAAGCGGATCATACCAAAGACATCCGCATGCCGACGGCGGACACCTGCGGAACCTGTCACTTAAGAGAATTTGCCGAACGTGAATCGGAACGGGACACCATGGTCTGGCCGAACGGACAATGGCCTGCCGGACGCCCCTCCCACGCACTGGACTACACAGCCAACATCGAGACCACTGTTTGGGCAGCGATGCCGCAACGTGAAGTTGCCGAAGGTTGCACCATGTGTCACACCAACCAGAACAAATGTGACAACTGTCATACCCGTCATGAATTCTCGGCGGCGGAATCGCGCAAACCGGAAGCCTGTGCCACCTGCCACAGCGGGGTGGATCACAACAACTGGGAAGCGTACACCATGTCCAAGCATGGCAAACTTGCCGAAATGAACCGTGACAAATGGAACTGGGAAGTCCGTCTCAAGGACGCCTTCTCCAAAGGTGGACAGAATGCACCGACCTGTGCAGCCTGCCACATGGAATACGAAGGAGAATACACCCACAACATCACCCGCAAGACTCGCTGGGCGAACTACCCGTTCGTTCCGGGCATTGCAGAGAACATCACCAGCGACTGGTCGGAAGCCCGTCTGGACTCCTGGGTACTGACCTGCACCCAGTGTCACTCGGAACGGTTTGCCCGCTCCTACCTGGATCTGATGGACAAAGGCACCCTGGAAGGGCTGGCCAAATACCAGGAAGCGAACGCAATCGTACACAAAATGTATGAAGACGGCACCCTGACCGGACAGAAGACCAACCGTCCGAATCCGCCGGAACCGGAGAAACCTGGATTTGGTATCTTCACCCAGCTGTTCTGGTCGAAAGGCAACAACCCTGCCTCACTCGAACTGAAAGTTCTGGAAATGGCAGAAAACAACCTGGCCAAGATGCACGTTGGACTGGCTCACGTCAATCCGGGTGGCTGGACCTACACTGAAGGCTGGGGTCCGATGAACCGTGCCTATGTTGAAATCCAGGACGAATACACCAAAATGCAGGAACTGTCAGCCCTGCAGGCCCGTGTAAACAAACTGGAAGGCAAGCAGACCAGTCTGCTCGACCTCAAGGGAACCGGAGAGAAAATCTCCCTGGGAGGCTTGGGTGGTGGTATGTTGCTGGCAGGAGCCCTTGCTCTGATCGGCTGGCGCAAGCGTAAGCAGACCCGAGCTTGA T7 promoter AATACGACTCACTATAGG Lac operator GGAATTGTGAGCGGATAACAA AmpR promoter ACTCTTCCTTTTTCAATCATGATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA ERα-421F: CATATGTCTAAGAAGAACTCTTTGGCTTTGTCTTTGACTGCTGATCAAATGGTTTCTGCTTTGTTGGATGCTGAACCTCCTATTTTGTATTCTGAATATGATCCAACTAGACCATTTTCCGAAGCTTCAATGATGGGTTTGTTGACTAATTTGGCTGATAGAGAATTGGTTCACATGATTAATTGGGCTAAGAGAGTTCCTGGTTTTGTTGATTTGACTTTGCATGATCAAGTTCATTTGTTGGAATGTGCTTGGTTGGAAATTTTGATGATTGGTTTGGTTTGGAGATCTATGGAACATCCTGGTAAATTGTTGTTTGCTCCTAATTTGTTGTTGGATAGAAATCAAGGTAAGTGTGTTGAAGGTTTTGTTGAAATTTTCGATATGTTGTTGGCTACTTCTTCTAGATTTAGAATGATGAATTTGCAAGGTGAAGAATTTGTTTGTTTGAAATCTATTATTTTGTTGAACTCCGGTGTTTATACTTTTTTGTCTTCTACTTTGAAGTCCTTGGAAGAAAAAGATCATATTCATAGAGTTTTGGATAAGATTACCGATACTTTGATTCATTTGATGGCTAAGGCTGGTTTGACTTTGCAACAACAACATCAAAGATTGGCTCAATTGTTGTTGATTTTGTCTCATATTAGACACATGTCTAACAAAGGTATGGAACATTTGTACTCTATGAAATGTAAGAACGTTGTTCCATTGTACGATTTATTATTGGAAATGTTGGATGCTCATAGATTGCATGCTCCAACTTAAGGATCC GRIP1: AATTCATGATTGCTGTTTCTTTCAAGTGTAGATGTCAAATTTTGAGAAGATTGACTAAGGATGAATCCCCATACACAAAATCCGCTTCCCAAACCAAGCCACCAGATGGTGCTTTGGCTGTTAGAAGACAATCTATTCCAGAAGAATTTAAGGGTTCTACTGTTGTTGAATTGATGAAAAAGGAAGGTACTACTTTGGGTTTGACTGTTTCTGGTGGTATTGATAAGGATGGTAAACCAAGAGTTTCAAATTTGAGACAAGGTGGTATTGCTGCTAGATCTGATCAATTGGATGTTGGTGATTATATTAAGGCTGTTAATGGTATTAACTTGGCTAAATTTAGACATGATGAAATTATTTCCTTGTTGAAGAACGTTGGTGAAAGAGTTGTTTTGGAAGTTGAATATGAATTGCCACCAGTTTCTGTTCAAGGTTCTTCTGTTATTTTTAGAACTGTTGAAGTTACCTTGCATAAAGAAGGTAATACCTTCGGTTTTGTTATTAGAGGTGGTGCTCATGATGATAGAAATAAATCCAGACCAGTTGTTATTACTTGTGTTAGACCAGGTGGTCCAGCTGATAGAGAAGGTACAATTAAACCAGGTGATAGATTGTTATCTGTTGATGGTATTAGATTGTTGGGTACAACACATGCAGAAGCTATGTCAATTTTGAAACAATGTGGTCAAGAAGCTGCTTTGTTGATTGAATATGATGTTAGTGTTATGGATTCCGTTGCTACTGCTTCCGGTCCTTTGTTGGTTGAAGTTGCTAAGACTCCAGGTGCTTCTTTGGGTGTT GCTTTGACTACTTCTATGTGTTGTAATAAGCAAGTTATTGTTATTGATAAGATTAAGTCCGCTTCTATTGCTGATAGATGTGGTGCTTTGCATGTTGGTGATCATATTTTGTCTATTGATGGTACTTCTATGGAATATTGTACTTTGGCTGAAGCTACTCAATTTTTGGCTAACACTACTGATCAAGTTAAATTGGAAATTTTGCCTCATCATCAAACTAGATTGGCTTTGAAAGGTCCAGATCATGCTGCTTTGGTTTCTTCTTCTTTTTCTCCAACTTCCATGTCTGCATACTCCTTGTCTTCTTTGAATATGGGTACTTTGCCAAGATCCTTGTATTCTACTTCTCCTAGAGGTACCATGATGAGAAGAAGATTGAAAAAGAAAGATTTCAAGTCCTCTTTGTCTTTGGCTTCTTCTACTGTTGGTTTGGCTGGTCAAGTTGTTCATACTGAAACTACAGAAGTTGTTTTGACTGCTGATCCAGTTACTGGTTTTGGTATTCAATTGCAAGGTTCTGTTTTTGCTACTGAAACTTTGTCTTCTCCTCCATTGATTTCTTATATTGAAGCTGATTCTCCAGCTGAAAGATGTGGTGTTTTACAAATTGGTGATAGAGTTATGGCTATTAATGGTATTCCTACTGAAGATTCTACTTTTGAAGAAGCTTCTCAATTGTTGAGAGATTCTTCTATTACTTCTAAGGTTACTTTGGAAATTGAATTTGATGTTGCTGAATCTGTTATTCCTTCTTCTGGTACTTTTCATGTTAAATTGCCAAAAAAACATAATGTTGAATTGGGTATTACTATTTCTTCTCCATCTTCTAGAAAACCAGGTGATCCATTGGTTATTTCTGATATTAAAAAAGGTTCTGTTGCTCATAGAACTGGTACTTTGGAATTGGGTGATAAATTGTTGGCTATTGATAATATTAGATTGGATAATTGTTCTATGGAAGATGCTGTTCAAATTTTGCAACAATGTGAAGATTTGGTTAAATTGAAAATTAGAAAAGATGAAGATAATTCTGATGAACAAGAATCTTCTGGTGCTATTATTTATACTGTTGAATTGAAAAGATATGGTGGTCCATTGGGTATTACTATTTCTGGTACTGAAGAACCATTTGATCCAATTATTATTTCTTCTTTGACTAAAGGTGGTTTGGCTGAAAGAACTGGTGCTATTCATATTGGTGATAGAATTTTGGCTATTAATTCTTCTTCTTTGAAAGGTAAACCATTGTCTGAAGCTATTCATTTGTTGCAAATGGCTGGTGAAACTGTTACTTTGAAAATTAAAAAACAAACTGATGCTCAATCTGCTTCTTCTCCAAAAAAATTTCCAATTTCTTCTCATTTGTCTGATTTGGGTGATGTTGAAGAAGATTCTTCTCCAGCTCAAAAACCAGGTAAATTGTCTGATATGTATCCATCTACTGTTCCATCTGTTGATTCTGCTGTTGATTCTTGGGATGGTTCTGCTATTGATACTTCTTATGGTACTCAAGGTACTTCTTTTCAAGCTTCTGGTTATAATTTTAATACTTATGATTGGAGATCTCCAAAACAAAGAGGTTCTTTGTCTCCAGTTACTAAACCAAGATCTCAAACTTATCCAGATGTTGGTTTGTCTTATGAAGATTGGGATAGATCTACTGCTTCTGGTTTTGCTGGTGCTGCTGATTCTGCTGAAACTGAACAAGAAGAAAATTTTTGGTCTCAAGCTTTGGAAGATTTGGAAACTTGTGGTCAATCTGGTATTTTGAGAGAATTGGAAGCTACTATTATGTCTGGTTCTACTATGTCTTTGAATCATGAAGCTCCAACTCCAAGATCTCAATTGGGTAGACAAGCTTCTTTTCAAGAAAGATCTTCTTCTAGACCACATTATTCTCAAACTACTAGATCTAATACTTTGCCATCTGATGTTGGTAGAAAATCTGTTACTTTGAGAAAAATGAAACAAGAAATTAAAGAAATTATGTCTCCAACTCCAGTTGAATTGCATAAAGTTACTTTGTATAAAGATTCTGATATGGAAGATTTTGGTTTTTCTGTTGCTGATGGTTTGTTGGAAAAAGGTGTTTATGTTAAAAATATTAGACCAGCTGGTCCAGGTGATTTGGGTGGTTTGAAACCATATGATAGATTGTTGCAAGTTAATCATGTTAGAACTAGAGATTTTGATTGTTGTTTGGTTGTTCCATTGATTGCTGAATCTGGTAATAAATTGGATTTGGTTATTTCTAGAAATCCATTGGCTTCTCAAAAATCTATTGATCAACAATCTTTGCCAGGTGATTGGTCTGAACAAAATTCTGCTTTTTTTCAACAACCATCTCATGGTGGTAATTTGGAAACTAGAGAACCAACTAATACTTTGTAAG

[### **Microbial strains**](#card-element-5)

##### - Nitrosomonas europaea ATCC 19718 - Escherichia coli DH5α - Escherichia coli BL21 - Saccharomyces cerevisiae Y2H

[### **Media**](#card-element-9)

##### The pH was controlled in the range of 7.5~8.0 by manual addition of filter sterilized 0.1 M NaOH solution. Glucose, NH4Cl and 17α-Ethinyl Estradiol were sterilized by filtration and then added into sterilized medium. Solution B (NaFeEDTA Solution): 100mg NaFeEDTA( molecular formula C10H12FeN2NaO8·3H2O) dissolved in 100mL double distilled water. Solution C(microelement): 10mg Na2MoO4·H2O, 17.2mg MnSO4·H2O, 0.4mgCoCl2·6H2O, 12mg ZnSO4·7H2O, 50mg CuSO4·5H2O, dissolved in 100mL double distilled water. Media was sterilized in an autoclave. The pH was controlled in the range of 6.8~7.8 by manual addition of filter sterilized 2 M KHCO3 solution.

[### **Protocols**](#card-element-0)

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# Team:HiZJU-China/Results

# Results

You can describe the results of your project and your future plans here.

### 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.

### Inspiration

See how other teams presented their results.

* [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)