Difference between revisions of "Team:Gifu/Project"

• 
  Project
  • Abstract
  • Back ground
  • Purine metabolism
  • Experiments
  • Results
  • Conclusion
  • Modeling
  • Futurework
  • Reference
• 
  Lab Note
  • Protocol
  • Calendar
  • InterLab
  • Proof
• 
  Parts
  • Basic Part
  • Composite Part
  • Part Collection
• Human Practices
  • Human Practices
• 
  About Us
  • Team
  • Collaborations
  • Safety
  • Attributions

PROJECT

Abstract

Urate oxidase or Uricase of Bacillus subtilis and S. pombe, allantoicase of S. cerevisiae was cloned into E. coli to degrade uric acid to urea. We extracted genome from Bacillus 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 Hislink 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 uricase activity and allantoicase activity. We confirmed these enzymes synthesized in E. coli successfully decomposed uric acid in this experiment.

Back Ground

 In Gifu prefecture, Cormorant Fishing on the Nagara river (Ukai) is one of the most summer sights. 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 1300 years of history. Now, it is popular as a tourism industry and became world agricultural culture. On the other hand, damage caused by the cormorant droppings in the riverside is one of problems of cormorant birds 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 consists mainly of uric acid and that is really insoluble material. So, trees cannot get adequate nutrition and die back. Cormorant is carnivorous, 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 indeed serious problem. The droppings fell on the car and window glass cause corrosion if it is left for a long time. Acidity of droppings leads to decaying of bridge, highway and signs. Serious accidents have sometimes happened. So, we plan to resolve the dieback by expression of birds’ dropping cleanerase and dissolving uric acid.

Purine metabolism

Experiments

Growth test in minimal media

E.coli culture was grown in LB liquid at 37℃ overnight with shaking, Bacillus 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 mmol/L borate buffer containing 1mmol/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 290nm and 25℃ were continuously measured.
  Assay for Allantoinase
 Enzyme solution was mixed with buffer for allantoinase(50mM Hepes, pH 7.75) containing 45mM 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 0.2N 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 150mM Hepes buffer, pH 7.75, containing 3mM 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 20mM potassium phosphate buffer containing 15mM allantoic acid. Enzyme solution was mixed with this buffer and incubated at 25℃. After that, we degraded urea and assay the released ammonia by the method mentioned above.

Results

Conclusion

Modeling

  Abstract
 Our goal is to catalyze uric acid into urea and lead our project to the solution of pollution by birds’ dropping. So, we decided to calculate Kcat and Km of expressed Uricase. To evaluate our system, We measured the enzyme activity of Uricase and analyzed the assay data. Therefore, we could determine the kinetic parameter of Uricase activity and calculate the maximum uric acid processing rate theoretically.

  Method
 Uric acid is degraded to allantoin by Uricase. The conversion rate follows the Michaelis-Menten equation. So, we used the following formula.

 d [Uric acid] / dt = - Vmax * [Uric acid] / (Km + [Uric acid])…(A)
 ([X] refers to the concentration of a substance X.)

Vmax is the degradation rate of uric acid under the condition of sufficiently high substrate. This parameter is proportional to the concentration of Uricase. So, we can design this as

 Vmax = Kcat * [Uricase].

Here, we determined the two kinetic parameters Vmax and Km in the formula by fitting a measurement result of the concentration change of uric acid in the reaction mixture. Besides, we used an algorithm, Differential evolution in this fitting.A tool which determines 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.

 Results
 In following tables and datas, a unit of Uricase concentration is ng/μL, a unit of time is min. The assay result of Uricase activity is shown in the table1.

 Table1

 The output result of this data is Vmax= , Km=

 Uricase of candida sp. from Wako was used in positive control of this assay. The concentration of Uricase is A and the assay result of Uricase activity is shown in the table2.

 Table2

 The output result of this data is Vmax=, Km=.

 Vmax is proportional to the concentration of the enzyme.(Vmax = Kcat * [Uricase])Therefore, the ratio of the Uricase concentration in suspension of disrupted cell to that of positive control experiment is equal to the ratio of Vmax in the suspension to that of positive control. So, we designed the following formula.

 (#Conc')*(#Vmax)/(#Vmax') = (#Conc)

 At this time, the speed parameter Km is, Kcat = (#Vmax) / (# Conc).

 On the other hand, Uricase concentration of the suspension (#Conc_SDS) is estimated by Hislink spin protein purification and SDS-PAGE. In consequence, we devised the following formula. Kcat = (#Vmax) / (# Conc_SDS)

 Discussion
In this study, we could obtain the theoretically feasible uric acid degradation rate by Uricase expressed in our project. When a substrate (uric acid) concentration against Uricase concentration keeps sufficiently high, uric acid decomposition rate can be maximized. In this case, we can conclude Vmax is or (#Vmax)*(#Volume). So, 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

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 Bacillus subtilis and Schizosaccharomyces pombe in place of that. They have the similar pathway of purine metabolism as Candida utilis . However, Candida utilis have 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, E. coli can’t be transported outside protein their body. So, we have to crush it to use the protein. Therefore, we plan to make this protein express in bacillus subtilis which have a function of secreting proteins outside a cell. In other word, there is a chance the protein is transported outside a cell, and we use it efficiently.

Reference