Difference between revisions of "Team:Gifu/Project"

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  • Abstract
  • Back ground
  • Purine metabolism
  • Experiments
  • Results
  • Conclusion
  • Modeling
  • Futurework
  • Reference
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Abstract

Back ground

Purine metabolism

Experiments

Results

Conclusion

Modeling

Futurework

Reference

　

PROJECT

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Abstract

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Urate oxidase or Uricase of Bacillus subtilis and Schizosaccharomyces pombe, allantoicase of Saccharomyces cerevisiae was cloned into Escherichia coli to degrade uric acid to urea. We extracted genome DNA from B. subtilis, S. pombe and S.cerevisiae and performed PCR amplification of these genes mentioned above. These gene fragments were inserted into pSB1C3. SDS-PAGE and His-link protein purification were carried out to determine the quantity of these enzymes in the cell lysates of the recombinant E. coli. In addition, we measured activity of uricase and allantoicase. We confirmed these enzymes synthesized in E. coli were successfully decomposed uric acid in this experiment.

Back Ground

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 In Gifu prefecture, Cormorant Fishing on the Nagara river (Ukai) is one of the most summer sights in Japan. Ukai is a traditional night fishing method in which an "usho"(Cormorant Fishing Master) and "u"(cormorant birds) work together to fish by the flames of Kagari-bi (fishing fire lanterns) reflecting on the dark surface of the river, with Mt Kinka and lofty Gifu Castle on its summit providing a dramatic backdrop to the scenery. Ukai has about 1,300 years of history. Now, it is popular as a tourism industry and a special world agricultural culture. On the other hand, damage caused by the cormorant droppings in the riverside is serious in Japan. The damage that we focus on is dieback of trees. In the river inhabited by cormorant, the leaves are stained with their droppings and interfered with photosynthesis. Birds’ excrement mainly consists of uric acid which is hardly soluble material. So trees cannot get adequate nutrition and die back. Cormorant is carnivorous animal, and the amount of uric acid in their droppings are larger than that of uric acid in other birds’ droppings. Also, cormorant is tufted and they tend to drop their excrement around their nests. In the city, deterioration of the landscape caused by birds’ droppings is also a serious problem. The droppings fell on the car and window glass cause corrosion if it left for a long time. Acidity of droppings leads to decaying of bridge, highway and signs which may cause serious accidents. So, we plan to resolve these problems by expression of birds’ "dropping cleanerase" and dissolving uric acid.

Purine metabolism

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Purine metabolism is a pathway of synthesizing and degrading purines. To breakdown uric acid into urea, five enzymes, Uricase, HIU hydrolase, OHCU decarboxylase, Allantoinase and Allantoicase are needed. This pathway is shown in Fig1. However, production of allantoin is preceded by two additional distinct unstable intermediate. HIU spontaneously decompose to OHCU and subsequently racemic allantoin at a low. So, we decided to reproduce the pathway shown in Fig2 in vitro. Enzymatic activity is required to degrade the intermediate products of purine metabolism. Ammonia, the final product of purine metabolism, is the most assimilated nitrogen source of most fungi and bacteria.

As template genes of Uricase, Allantoinase and Allantoicase, we used four microorganisms. Here, we mention why we used these microorganisms and characteristics.

B. subtilis has eight genes driving uric acid degradation. These genes are located in a cluster with the exception for the gene encoding allantoin permease. B. subtilis can degrade all of five intermediates of uric degradation described above.

In kegg pathway of purine metabolism in E. coli, it has HIU hydrolase and Allantoinase. It is high-growth with allantoin. Further, E. coli has a special anaerobic pathway to decompose uric acid to urea. This pathway doesn’t need Allantoicase. We grew E. coli in minimal media containing purines.

We performed growth test in minimal media containing purines of E.coli, B.subtilis, S.cerevisiae to confirm the existence of the target gene. The result is shown in fig1.

Experiments

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Growth test in minimal media

E.coli culture was grown in LB liquid at 37℃ overnight with shaking, B. subtilis culture was grown in LB liquid at 30℃, S.pombe culture and S.cerevisiae were grown in YPD liquid at 30℃ in advance. Minimal media containing 2mM urate or allantoin or (NH4)2SO4 as a sole nitrogen source and minimal media not containing nitrogen sources were also prepared. Each culture was washed with PBS twice and diluted 102 to 1010 times with each minimal media. Cell suspensions were incubated at the appropriate temperatures for one day and spotted onto LB broth or YPD broth. Results were imaged after one day incubation at appropriate temperature.

Plasmid construction

Assay

  Assay for Uricase
 We prepared 0.001% uric acid solution by dissolving in 50.0 mmol/L borate buffer containing 1.0mmol/L EDTA and detergent(pH8.5). 0.5mL of distilled water was added and the mixture was preheated at 25℃. E.coli culture was sonicated and diluted 1 to 100 times with the borate buffer. After that, the absorbance at a wavelength of 290.0nm and 25℃ were continuously measured.
  Assay for Allantoinase
 Enzyme solution was mixed with buffer for allantoinase(50.0mM Hepes, pH 7.75) containing 45.0mM allantoin and incubated at 37℃. We withdrew 0.5mL aliquots at regular timed intervals of 2 min. Each aliquot was quenched with 0.125ml of 0.20N HCl and heated in a boiling water bath for 4 min to degrade allantoic acid to glyoxylate and urea. After decomposition of allantoate, we degraded urea to ammonia by jack bean urease.(assay for allantoinase)Samples were neutralized with 0.5ml of 0.4N NaOH and buffered with 0.25ml of 150.0mM Hepes buffer, pH 7.75, containing 3.0mM EDTA. We added 0.125mg of urease in the reaction mixture. The released ammonia was assayed by Nessler’s reagent.
  Assay for Allantoicase
 We prepared 20.0mM potassium phosphate buffer containing 15.0mM allantoic acid. Enzyme solution was mixed with this buffer and incubated at 25℃. After that, we degraded urea and assayed the releasing ammonia by the method mentioned above.

Results

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  SDS-PAGE

Figure. SDS-PAGE

The marker show the point, 200kDa, 116.2kDa, 66.2kDa, 45.0kDa, 31kDa, 21kDa

 According to the molecular weight (56.5kDa) and comparison with negative control (lane 9 and 10), the strong band (red circle, lane 3 and 4) were predicted as uricase. The strongest band of lane 6 (less than 31kDa) was not thought as uricase because of the difference from correct molecular weight (38.7kDa).
In addition, we tried　the purification by His-tag, only to fail (no purification product was seen) because that uricase from B. subtilis hardly dissolve in PBS buffer we used (shown above Figure)

  Assay for Uricase from Candida sp. from Wako
 Positive control of Uricase assay was performed. This reagent was lyophilized powder. We diluted this with Borate buffer containing 1.0mM EDTA and detergent. The final concentration of the enzyme is 0.417 ng/μL. The result is shown in Table1,figure1.

Conclusion

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Modeling

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  Abstract
 Our goal of this project is to solve problems caused by birds’ droppings with genetically engineered E. coli. To acheive this goal, we manufactured E. coli that synthesizes uricase, which catalyzes uric acid to urea. Therefore, we are interested in "How fast can our E. coli process uric acid theoretically?" We analyzed data obtained from the experiment and calculated theoretically maximum rate of processing uric acid.

  Method
 We made a differential equation which represents time evolution of concentration of uric acid. To making the equation, we used the Michaelis-Menten equation, which is widely used to estimate rate of enzyme reaction. The conversion from uric acid to allantoin is catalyzed by uricase. For this reason, the conversion rate of uric acid is described with the Michaelis-Menten equation. The equation we made is as follows. d [uric acid] / dt = - Vmax * [uric acid] / (Km + [Uric acid])…(A) ([X] represents the concentration of a substance X.) Vmax is the conversion rate of uric acid under the condition that the ratio of the amount of uric acid to that of uricase is sufficiently high. This parameter is proportional to the concentration of uricase. Then we put the proportionality constant as Kcat. Here, we estimated the two kinetic parameters Vmax and Km in the equation by fitting these parameters to a measurement result of the concentration change of uric acid in the reaction mixture. Besides, we used an algorithm called "differential evolution" in this parameter fitting. A tool which estimates and outputs Vmax and Km by entering the measurement results of the concentration change was developed. We attached the source code of this fitting tool below.

<File:T--Gifu--Uricase tool.zip<>

 result
In the following data and description, the unit of concentration of any substances is ng/μL and the unit of time is minute. First, we measured the change of concentration of uric acid in the reaction mixture. Uricase in this mixture was standard reagent. The result of the measurement is summerized in this table. Then, we inputted these data into the parameter fitting tool mentioned above. Output of the parameter fitting was as follows. Vmax = 0.0083 Km = 0.045 Besides, this graph shows the result of parameter fitting. The blue curve means the fitted curve, and the red plots is the data obtained from the experiment.

The concentration of uricase as a standard reagent was [uricase] = 0.42ng/μL. Using the definition of Kcat, or Vmax = Kcat*[uricase], Kcat was calculated in the following way. Kcat = Vmax/[uricase] = 0.0083/0.42 = 0.020. Now two kinetic parameters which is independent of concentration of enzyme or substrate (Kcat and Km) were estimated. Second, we conducted His-Link purification of protein and measured the absorbance at 280.0nm to determine the concentration of uricase in the cell lysate. E. coli culture was sonicated to assay the enzyme. Uricase in this solution was synthesized by E. coli which we engineered. The result is below. [uricase] = 156.0ng/μL So Vmax of the producted uricase mixture was calculated in the following way. Vmax = Kcat*[uricase] = 0.020*156.0 = 3.1 We can conclude that the uricase mixture made with our engineered E. coli has an ability to process uric acid with theoretically maximum rate of 3.1ng/(μL*min).

 Discussion
Accoding to our modeling, a large amount of Uricase may be needed to solve the problem of birds’ dropping. Highly-improved Uricase should be developed by biosynthesis.

FUTURE WORKS

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This year, our purpose is to make it possible for E. coli to degrade urate and solve problems caused by birds’ dropping. So, we cloned genes of uricase, allantoinase and allantoicase into E. coli to assess production and activity of these three enzymes. We could extract genome from three kinds of bacteria, B.subtilis, S.cerevisiae and S. pombe and express three enzymes mentioned above. Meanwhile, this year’s result is not enough to solve the pollution of droppings. Now, we have three plans to improve our project. Firstly, we have to do more accurate assay using the allantoinase-loss-strain of E. coli. E. coli has a gene of allantoinase, and we can consider the possibility of affecting assay. Secondly, we think about the use of total synthesis. This year, we were going to use Candida utilis. However, Candida utilis has a high risk to human health. So, we used B. subtilis and S. pombe in place of that. They have the similar pathway of purine metabolism as Candida utilis . However, Candida utilis has the gene coded uricase which has high enzyme activity. If we use the gene coded uricase of Candida utilis by total synthesis, our project will improve more. Thirdly, each gene is cloned into B. subtilis instead of E. coli . B. subtilis have a function of secreting protein outside a cell. We can utilize expressed enzymes without sonication.

Referenc

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Determination of expressed Uricase activity

E. coli culuture were grown in the presence of IPTG. Cell suspensions were sonicated and centrifuged (150rpm, room temperature, 10min). The top layer was removed, and cells were buffered with 50mM borate buffer containing 1mM EDTA and detergent. Uricase from B.subtilis were measured respectively. These crude extractions were diluted 1 to 102 times with the borate buffer. The result is shown in Table1. We couldn’t confirm this enzyme catalyzes uric acid into allantoin.

Electrophoresis analysis of the cell lysates from SDS-PAGE

The growth test in minimal media

The growth test in minimal media containing purines of B.subtilis , S.cerevisiae ere performed. The result is shown in FigX. As expected, Bacillus subtilis grew well with uric acid, allantoin and ammonium sulfate. We confirmed that Bacillus subtilis utilized these intermediate as nitrogen sources. S.cerevisiae was positive growth with allantoin. However, it didn’t grow well with w/o ammonium, uric acid and ammonium sulfate.

The growth ability of the recombinant E. coli in minimal media containing uric acid

Growth test in minimal media of wild type E. coli and the recombinant E. coli were conducted to confirm Uricase activity which was expressed by the recombinant E. coli. The result of this assay is shown in FigX. No significant differences were observed in minimal media with 2mM uric acid and w/o ammonium. However, wild type is more positive growth in minimal media containing ammonia sulfate. We could’t understand why this incident happened.

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