Team:Kyoto/Project

Project

Introduction

Background

The cell death device is required in many fields. In bioremediation, for example, this device can prevent introduced bacteria from disrupting the ecosystem. Also, It can be applied to other ideas such as E.coli capsules which scatter drug or aroma by cell-lysis responding to a certain signal. Therefore, the cell death devices based on diverse approaches, including previous iGEM projects have been developed. However, present cell-death devices are still not easy to use, because of the poor characterization or strict restriction on promoters’ activity and so on. Therefore, we attempt to register on biobrick better characterized and 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.

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Overview

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 killer and anti-killer, we have to characterize R0011.

Goal B: Characterization of λ Lysis cassette

To characterize λlysiscassette, we made below construct and observed E.coli transformed with the constructs in various concentration of IPTG.

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.

Protocols

See below pages for details.

• Protocols
• Materials

Notebook

• Notebook1: Construction for Lysisbox.
• Notebook2: Measurement of R0011.
• Team:Kyoto/LearnMore

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References

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3. PMID: 15973534 Davison J., Risk mitigation of genetically modified bacteria and plants designed for bioremediation., J Ind Microbiol Biotechnol. 2005 Dec;32(11-12):639-50. Epub 2005 Jun 23.
4. PMID: 19897658 White R, Tran TA, Dankenbring CA, Deaton J, Young R., The N-terminal transmembrane domain of lambda S is required for holin but not antiholin function., J Bacteriol. 2010 Feb;192(3):725-33. Epub 2009 Nov 6.
5. PMID: 7768829 Chang CY, Nam K, Young R., S gene expression and the timing of lysis by bacteriophage lambda., J Bacteriol. 1995 Jun;177(11):3283-94.
6. PMID: 18713319 Berry J, Summer EJ, Struck DK, Young R., The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes., Mol Microbiol. 2008 Oct;70(2):341-51. Epub 2008 Aug 18.
7. PMID: 11459934 Gründling A, Manson MD, Young R., Holins kill without warning., Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9348-52. Epub 2001 Jul 17.
8. PMID: 9573208 Smith DL, Struck DK, Scholtz JM, Young R., Purification and biochemical characterization of the lambda holin., J Bacteriol. 1998 May;180(9):2531-40.
9. PMID: 19298678 Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, Endy D., Measuring the activity of BioBrick promoters using an in vivo reference standard., J Biol Eng. 2009 Mar 20;3:4.
10. PMID: 2147680 Garrett J, Bruno C, Young R., Lysis protein S of phage lambda functions in Saccharomyces cerevisiae., J Bacteriol. 1990 Dec;172(12):7275-7.

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