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The cell death devices are required in many areas. In bioremediation, for example, this device can help to prevent introduced bacteria from disrupting the native ecosystem. It can also be applied to other ideas like E.coli capsules which is scattering drug or aroma by lysing responding to a certain signal. Therefore, the cell death devices based on diverse approaches have been developed, including previous iGEM projects. However, using them is still not easy and convenient, for strict restriction on promoters’ activity narrows the possible application, the function of the device is not enough to reduce the population, and so on. Therefore, we attempt to register on biobrick more useful cell death device to contribute to every future team.

The problems of present Cell-death devices

In order to design a universal and user-fridendly device, we need to tackle on mainly two issues: restriction on promoter-activity and the strength of the cell death function.

The switch of cell-death function should be dependent only on a specific signal which activates the promoter but should not be affected largely by leaks. As an example, a simple circuit consists of inducible promoter and killer gene can cause this kind of problems. We try to widen the range of applicable promoters by combining the anti-killergene with the killergene.

As for the ability of killing cell itself, we consider the lysis cassette of lambda phage is the most appropriate genes for this project among other candidates for killer gene we searched. It is because we found the data showing the lytic system is able to lead annihilation though some can only regulate cell proliferation at a certain population of the bacteria, and also the lysis cassette consists of anti-killergene and killer gene.

Lytic system derived from lambda phage

We take advantage of the lambda phage’s lysis cassette as killer gene, which consists of four genes: S, R, coding for holin, endolysin, respectively, and Rz/Rz1 gene. In this lytic system, endolysin accesses through the pores on cell membrane formed by holin, and degrade peptidoglycan, leading to E.coli to die. As anti-killer gene, we use SΔTMD1 cording for dominant-negative holin, which is deleted the first trans-membrane domein-TMD1 of wild type holin. This variant holin prevents holin from forming pores, and endolysin cannot access peptidoglycan.



Goal A Characterization of R0011, a strong lactose promoter

We used R0011, which is one of the most commonly used parts in iGEM, to characterize our genes because the strength of promoter activity is very high.However, in previous iGEM, no teams didn't characterize R0011 with RPU.

We tried to determine what is the ratio of activity of R0011,a lactose promoter when cell lysis occurs. Therefore, in oreder to characterize 'lysis box', we have to characterize R0011.

For the characterization of R0011, we moved R0011 onto pSB4K5, a low copy vector, and transformed it into E.coli KRX, the strain overexpressing lacI, so that R0011 is repressed fully at low IPTG concentraion. RPU (Relative Promoter Unit) is used to describe the activity of RPU, and GFP is used as a reporter. KRX transformed with BBa_I20260 is also used.

Goal B Characterization of λLysis cassette

E.coli transformed with the constructs below was grown in medium without IPTG and IPTG was added to the culture at proper time. The A550 of the culture was measured in order to find whether the killer gene works correctly.

Goal C Characterization of the anti-killer gene

E.coli transformed with the constructs below was grown in medium without IPTG and IPTG was added to the culture at proper time. The A550 of the culture was measured and the result was compared with that of the experiment of the killer gene in order to find whether the killer gene works correctly.


See below pages for details.




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