Team:HiZJU-China/Notebook
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:
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.
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.
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.
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.
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.
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.
Spread the engineered bacteria onto the selective plate
No colonies appeared on the plate, which meant the transformation in August 3rd failed.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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
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
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
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.
Fig the detection of urea content
