Our project for iGEM 2019 is a component of "Demonstration of the system that E.coli die automatically after several times of divisions", which used to be our theme in the starting point. When we were brainstorming in last spring,we got 10+ ideas for our first year of iGEM. This theme was the most popular among members and mentors, so we decided to choose this.Initially we aimed to materialize the whole system of this idea inspired by UT-Tokyo team 2016, in this year, but it was highly demanding for the first year in iGEM.
Thus we decided to focus on building the main part of it; the part to kill E.coli without any stimulation from outside after once it settled down to the medium.
By the way this idea originally came from Japanese traditional cursing phrase, says "I curse you and all your descendants to the last generation". It's definitely scary but got members imagine the scene that E.coli suddenly die without any stimulation from outside after they increased. We thought it's an amazing system to tell people what Synthetic Biology can.
We have two goals.One is to experience all of iGEM 2019 activity and fully learn. Another one is to update "Genetic Literacy" in Japan.
In experiment our goal, in short, is to make “easily lyse-E.coli”.
BL21(DE3) pLysS, which is known as very famous strain of E.coli for protein expression, died in hypotonic water in our preliminary experiment. However it was not perfect, so we decided to try to enforce lysis capacity by transforming our part into BL21(DE3) pLysS.However the popular backbone plasmid pSB1C3 is Cam resistant as well as pLysS is. We tried to put it pSB1A3 which is Amp resistant. As a result we couldn't ligate our new composite part: BBa_K3050000,on pSB1A3, so instead we switched to simply make cell membrane weakened BL21(DE3) with magainin-2 that derives from BBa_K3050000 on pSB1C3 as the important step for our system.
We successfully confirmed to prove our hypothesis that BL21(DE3)pLysS tend to die in hypotonic solution by culturing BL21(DE-3)pLysS in hypotonic solution without and with 50μg/mL T7 RNA polymerase.
Wild E.coli don't die in hypotonic solution. However BL21(DE3)pLysS express T7 Lysozyme,and they lyse in hypotonic solution according to the result of our Preliminary Experiments (1).
T7 RNA polymerase is the inhibitor of T7 Lysozyme.It form a complex with T7 Lysozyme and inhibits its amidase activity. Therefore in hypotonic solution with T7 RNA polymerase, BL21(DE3)pLysS can survive and keep increasing until all T7 RNA polymerase combines to T7 Lysozyme as we proved in our Preliminary Experiments (2). After that, extra T7 Lysozyme start to kill BL21(DE3)pLysS. This means,it's supposed that we can control when BL21(DE3)pLysS dies dependently upon the quantity of T7 RNA polymerase to add.
This is also applicable for safety. Hypotonic solution exist everywhere, for example rain water outside. So even if they escape, it's hardly possible to survive.
Also we decided to characterize BBa_I746909 and even use it as a part of our new composite part to make our system visible for everyone without measuring OD600. This directly relates to Genetic Literacy. We don't ignore the fact that all researchers are supported by the society, and it's highly important to get our works understood by ordinary people. Though Genetic Literacy is not so common in Japan yet, we're sure we need to accept it as well as we did IT Literacy. Genomic test is used to know the type of cancer in hospital, or you can pay money for services just to know whether you're genetically easy to get fat or not. We all should know this kind of data can be accumulated and includes great amount of information, and probably be careful when give permission to test our genes.
See our Human Practice and Integrated Human Practice for more information.
We'd like to share our experience with people.
XIAODONG CHENG, XING ZHANG, JAMES W. PFLUGRATH, et al, The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase