Team:Kyoto/Project

Project

Abstract

The Fantastic Lysisbox: Genetically engineered cell death is essential for the application of biotechnology, such as in bioremediation area. In order to control the cell-death, we designed “Lysisbox”, which consists of a pair of modules: “Killer gene” and “Anti-killer gene.” As the Killer gene for Escherichia coli, we noted the lysis cassette [SRRz/Rz1 gene] of lambda phage coding for holin and endolysin. The holin form pores in the inner membrane and the endolysin access and degrade the peptidoglycan passing through the pores, leading the E.coli to death. As the Anti-killer gene, we chose S_ΔTMD1 coding for a dominant-negative holin that inhibits the formation of the fatal pores. The balance of these two genes' expression level has the key to the E.coli’s life or death. In addition, such controllable membrane pores must show critical functions for all living organisms with lipid membranes and a cell wall. “Lysisbox” must contribute a lot to future projects, thus you must say “FANTASTIC!!!"

Introduction

Background

The cell-death device is required in many fields ^{[1] [2]}. In bioremediation, for example, this device can prevent ecosystem disruption by introduced bacteria ^{[3] [4]}. 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 ^{[1] [3] [5] [6]}. However, present cell-death devices are not useful because they have two problems as follows:

1. They not quantitatively characterized^[7]
2. They are not applied to many promoters^[8].

Therefore, we attempt to solve these problems and design BioBrick better characterized and more applicable cell-death device to contribute to every future projects.

Problems of Present Cell-Death Devices

1.Need for quantitative characterization

In previous iGEM, many teams focused on λ lysis cassette, which caused cell lysis, and have characterized its lytic activity. However, in order to design a universal and applicable cell-death device using λ lysis cassette, we must characterize its lytic activity more quantitatively, for example,fosues when cell lysis occurs after induction of λ lysis cassette, how many cells lyse, whether all of cells lyse or not, and so on.

2. Solution to the Restriction on Promoter's Activity

In constructing a simple circuit consisting of an inducible promoter and λ lysis cassette , the combinations are limited by the promoter's leak, strength of killer-function and many other factors. We looked for the solution to this problem and came up with an idea of using an its antagonist, S_ΔTMD1, which is holin deleted trans membrem domain 1 and that loses the function of cell death. As well with two promoters (a constitutive one and an inducible one). With this circuit, we can apply more promoters to this device by using an appropriate constitutive promoter.

We considered that lysis cassette of λ phage and its antagonist, S_ΔTMD1 are the most appropriate to Killer-gene and Anti-killer gene ^[5]. This was because some iGEM teams in the past already dealt with lysis cassette and also because we found some articles that shows the effect of S_ΔTMD1 in the yeast ^[9]. [learn more]

Goals

Goal A:

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 characterized R0011 with RPU. We tried to determine how the activity of R0011^[10] changes.

Goal B:

We characterized λ lysis cassette quantitatively and linked between it and activity of the induction(RPU).

Goal C:Characterization of the anti-killer gene

To check function the anti-killer gene, we made upper construct and measured A550 of E.coli transformed with the device in medium with IPTG and without IPTG ^{[11] [7] [12 [13]}, and we compared this result with that of E.coli transformed with killer gene. Then, we find out the anti-killer gene can't inhibit this killer gene completely. see the detail of Goal C

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Goals

Goal A: Characterization of R0011, a strong lactose promoter

Goal B: Quantitative characterization of λ lysis cassette

Goal C: Characterization of the anti-killer gene, S_ΔTMD1

Conclusion

Achievements

1. We measured RPU of R0011 in eight kinds of concentration of IPTG, and made c
2. We found out a new fact that cell lysis become an apparent equilibrium state depending on the strength of induction(RPU). We established a standard method for lytic activity of λ lysis cassette and defined LAS（Lytic Activity Score）as a quantitative parameter of lytic activity, and we characterized the lytic activity of λ lysis cassette.
3. We
4. We checked the activity of S_ΔTMD1, which can't form pore of holin and cells don't die although endolyshin expresses.

Future work

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References

1. [http://www.ncbi.nlm.nih.gov/pubmed/20395970 PMID: 20395970] Khalil AS, Collins JJ., Synthetic biology: applications come of age., Nat Rev Genet. 2010 May;11(5):367-79.
2. [http://www.ncbi.nlm.nih.gov/pubmed/15832375 PMID: 15832375] Paul D, Pandey G, Jain RK., Suicidal genetically engineered microorganisms for bioremediation: need and perspectives., Bioessays. 2005 May;27(5):563-73.
3. [http://www.ncbi.nlm.nih.gov/pubmed/15973534 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. https://2009.igem.org/Team:Michigan
5. [http://www.ncbi.nlm.nih.gov/pubmed/19897658 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.
6. https://2009.igem.org/Team:UNICAMP-Brazil
7. [http://www.ncbi.nlm.nih.gov/pubmed/18713319 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.
8. https://2008.igem.org/Team:UC_Berkeley
9. [http://www.ncbi.nlm.nih.gov/pubmed/2147680 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.
10. [http://www.ncbi.nlm.nih.gov/pubmed/19298678 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.
11. [http://www.ncbi.nlm.nih.gov/pubmed/7768829 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.
12. [http://www.ncbi.nlm.nih.gov/pubmed/11459934 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.
13. [http://www.ncbi.nlm.nih.gov/pubmed/9573208 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.

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