Team:Botchan Lab Tokyo/Integrated
Botchan Lab. Tokyo
Integrated Human Practices
Integrated Human Practices
Basic research make people’s lives more prosperous. It’s true that applied researches target to solve specific problems and its likely to generate beneficial economic effects in a short period of time. On the other hand, basic research tend to be ignored. However, basic research has unlimited potential. It may be difficult to have great effect on society right away, but by combining with new technology which develop in the future, it may change our daily lives.
This year, we decided to synthesize radioresistant E. coli for the first step to search for effective genetic combinations which can make bacteria resistant to radiation. As you know, in 2011, Japanese experienced the Great East Japan Earthquake and nuclear power plant accident, and have been facing various problems since now. That’s why we chose this project. By taking advantage of radioresistant in , we want to solve various problems. As we said before, it may take time to influence people’s lives, but we believe this technology may be useful inside or even outside the laboratory in the future.
In order to assess present problem, seek for application, and improve our project, we interviewed expert and company. Also, we were given advices from seniors at university. Thank you all for helping us and giving awesome advices.
The image below describe how our human practice influence and improve our project.
Visiting National Institute of Genetics
On December 28, we visited National Institute of Genetics and had a relationship with
biologists. We visited four different laboratories such as the Kurokawa lab, Arita lab, Oda lab, and Andachi lab, and talked with various researchers. We received explanations of the researches, and learned how researchers decide the contents of the research.
Mr. Andachi told us that cesium uptake was recent trend, and it was difficult to establish the method. Also, he told us there was no problem legally to conduct radiation experiment below the threshold, but we need to consider the affection of bacteria when it uptake radiation. If bacteria uptake cesium, cell death or mutation may occur, and cell death may be avoided below the threshold, so we need to consider the mutation.
This interview encouraged us to decide this year’s project. Many researchers have already been searching for cesium recovery method, so we decided to focused on radiation resistance. We thought searching for effective genetic combinations which make bacteria resistant to radiation may contribute to decrease the effect on bacteria when it uptake radioactive substances.
At the time, we were struggling with finding the theme, so this visit finally made a substantial contribution to our project. It was really precious opportunity to talk with researchers and we want to visit again to deepen exchanges. Thank you all for your cooperation!
Feedback from seniors and professors at university about project
On May 10, we participated in research presentation and we had an opportunity to present our project in front of seniors and professors at university. At this point, we just decided to synthesize radioresistant E. coli, and sought for application. Therefore, in order to get seniors to understand about iGEM, we introduced last year’s project, and then, we explained what would we do for this year. Most of seniors were interested in our project, and they gave us various piece of advice. Their questions made us consider again how to improve our project.
For example, someone asked us why we chose three genes, recA, ppr M, and pqqE, to make E. coli survive in environment under high radiation.We chose three genes based om other researches, but we recognized the importance of our speech need to be reasonable, and we tried to work hard on firming up our project.Also, someone advised us that if radioresistant E. coli can survive under high radiation, it can apply not only space but also in other harsh environments where can be used for research. We just imagined to use newly synthesized E. coli in space or bioremediation, so we noticed radioresistant E. coli may apply various situations which no one could imagine. This research presentation was precious opportunity to find problem and get outside opinions. Thank you all for your advice!
An interview with Mr. Kawahara from Earth Solution Inc.
Bioremediation is a process that uses mainly bacteria to remove contaminants in the soil and other environments. In Japan, bioremediation is used to detoxify contaminants such as Volatile Organic Compounds(VOC). If we can combine radioresistant E. coli with other technology such as uptake of cesium by E. coli which is the iGEM project of SYSU-China in 2011(https://2011.igem.org/Team:SYSU-China), we may establish more effiective way to decontaminate radioactive substances in environment with high radioactive concentration. Therefore, in order to search for application of radioresistant E. coli, we visited Earth Solution Inc. who developed bioremediation business and had an interview with Mr. Keiichiro Kawahara, representatives of the company.
The present situation of bioremediation
Currently, the mainstream of bioremediation is biostimulation that activates bacteria living in the soil and purifies contaminated soil. Biostimulation removes VOC such as tetrachloroethylene, and other substances. Nutrients are added to activate bacteria, but if this nutrients are added too much, groundwater will be eutrophicated, so it must be devised. Earth Solution Inc. pumps groundwater, adds a small amount of nutrients directly to it, returns it to the ground, and adds a small amount of nutrients to activate bacteria and purify the soil. As a result, eutrophication is less likely to occur.
On the other hand, it’s difficult to carry out bioaugmentation which is another method of bioremediation to add organisms to material in order to remove any undesirable chemicals. Since it’s difficult to evaluate the safety of the introduction of bacteria, and it costs more than the estimate, it’s hard to compete with other technologies.
According to Mr. Kawahara, when concentrating and recovering inorganic substances such as radioactive materials, they are stored in a more dangerous state, so there is a trade-off between the danger of concentration and the danger of spreading. It is necessary to perform bioremediation in consideration of trade-off.
We thought that it was important how the general public understand this purification method because bioremediation is not familiar to them. However, Mr. Kawahara said that these people would often accept bioremediation if they were superior to other methods due to cost or other reasons. Rather, experts who have a certain level of knowledge are reluctant to bioremediation.
Thank you Mr. Kawahara for your cooperation!
The Relation Between Our Project and bioremediation
Initially, we used bioaugmentation in a closed system using radiation-resistant bacteria to detoxify contaminants in the environments, for example, the large volumes of contaminated water and soil that are currently at issue. However, Mr. Kawahara advised that it is very expensive to culture large amounts of E. coli, so it is necessary to look for advantages over other technologies in order to develop as a business. In addition, we received advice that it may be good to search for substances such as harmful organic substances that are not yet a problem and make bacteria decompose it.
From the knowledge gained from this interview, we decided not to propose a technology that replaces the one currently used for processing radioactive substances, but use it for small-scale decontamination which is difficult to deal with by using current technology. We decided to propose our project as a method that can be used together with the current technology. This is because the Japanese government set a goal to conduct the final disposal of decontaminated soil outside Fukushima Prefecture in 2045, but it is difficult to assure a facility. Therefore, we would like to propose a decontamination approach taking advantage of our technology so that decontamination can be performed without constructing a large-scale facility. If we develop a method using biotechnology that can be used according to the situation as one approach to volume reduction, it may be possible to reduce the burden on Fukushima and treat decontaminated soil throughout Japan.
As for a future goal, not only discovering effective genetic combinations which provide radiation resistance to bacteria, but also we want to introduce genes that enable bacteria to collect radioactive substances such as cesium. Also we want to proceed with research with a view to recovering other harmful substances. We also want to create an environment where bacteria can be used more effectively by collaborating with researchers or other iGEM teams in various fields in order to secure data to convince experts to carry out bioremediation. To that end, we want to encourage more people to know iGEM and promote activities involving many branches of learning.
Other possibilities of radioresistant bacteria
The ultimate goal of this project is to establish efficient method of bioremediation, but as we proceeded with the project, we focused on the fact that radioresistant bacteria have increased DNA repair capacity, particularly considerable recombination activity. I thought that the characteristics could be used to increase the efficiency of cloning iVEC. Therefore we went to an interview with Prof. Motohashi who is doing research on cloning.
What Is iVEC(in Vivo E. coli Cloning)?1
Plasmid cloning is an essential part of any molecular biology project, and yet it is often a bottleneck in the experimental process. The majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, DNA assembly for plasmid construction can be achieved in vivo using iVEC method, in vivo E. coli cloning method. In this method, PCR products are engineered to contain terminal sequences identical to sequences at two ends of a linearized vector, and the method can directly transform a mixture of them into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Therefore, it can reduce the number of steps in DNA assembly, and also, it can minimize the time and cost. Furthermore, you don’t need to prepare purified enzymes.
Problems2
However, this simpler cloning method was not widely used despite of its introduction in the early 1990s. One of the main reason is low cloning efficiency. It’s said that it may be caused by low in vivo homologous recombination activity of E. coli. The other reason is low multiple insert cloning efficiency.
The Relation Between Our Project and iVEC
Then, we focused on the high DNA repair ability of the genes that we tried to introduce to E. coli. Especially, DrRecA directly promotes homologous recombination. The intrinsic bacterial recombination pathway assembles pieces of linear DNA through short homologous sequences at their termini, and likely functions as a bacterial DNA repair mechanism. Therefore, If the introduction of the genes that relate high DNA repair ability increased the in vivo homologous recombination activity of E. coli, we could improve the iVEC efficiency.
Discussion with an Expert and New Direction of Our Project
We have therefore consulted with an expert within academia. This time, we've got a chance to discuss the direction of our project with Professor Motohashi from Kyoto Sangyo University. Based on the advice from him, introduction of genes related to high DNA repair ability might probably increase iVEC efficiency, so we are planning to implement iVEC using transformants. We are also considering the introduction of genes related to homologous recombination activity other than recA, pprM and pqqE(See Project page). In addition, Professor Motohashi suggested to us that it might be effective to knock out/down genes related to the decrease in the activity of homologous recombination. We are now considering about not only adding genes, but also knocking out/down genes to achieve high iVEC efficiency.
Assay of iVEC Activity3
A DNA fragment containing an antibiotic resistance gene(e.g. a chloramphenicol resistance gene from pACYC184) and linearized pUC19(containing an ampicillin resistance gene) with 20-bp homologous overlapping ends is amplified by PCR and introduced into our transformants’ cells. In a standard assay of the iVEC activity, 0.15 pmol of the DNA fragment and 0.05 pmol of linearized pUC19 is used for the transformation of the transformants. Then, we will count the colonies resistant to both ampicillin and chloramphenicol after simultaneous introduction of the DNA fragment and linearized pUC19 into the transformants.
Summary
At first, our goal was to establish effective method of bioremediation, and it may be possible to realize by combining with other technologies. However, through our activities, we noticed the possibilities of radioresistant bacteria. iVEC are just examples, and radioresistant bacteria can be applied to other technology such as producing food in space by combining it with E. coli which can synthesize amino acids aimed at last year's our project. Radiation-resistant bacteria have the potential to make a significant impact by combining them with various technologies, and we hope that various iGEM teams will use this technology in the future.