Team:Kyoto/Project

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Contents

Project

Introduction

Background

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.

Overview

We, iGEM Kyoto2010, aim at designing a cell death device and characterizing closely to check the universal use. We named the new device, “Lysis box,” having circuit like this,

Lysis box consists of two parts: killer gene expressing constantly, and anti-killer gene expressing regulated by certain promoter. While inducible promoter is activated and the function of killer gene is inhibited by the products of anti-killer gene, E.coli are alive; Lysis box is off, When the inducible promoter is repressed, the bacteria die of lysing; Lysis box turns on.

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.

Goals

iGEM Kyoto 2010’s project will be carried out in 4steps below,

  1. We construct the circuit of “Lysis box”, regulated by lactose promoter and repressor, and check the functions of each modules, killer gene and anti-killer gene inducing by IPTG.
  2. As the first step of characterization, we’ll find out the effective range of inducible promoter’s activity for causing proper cell lysis, and define the range by adopting RPU, a standard unit for reporting promoter activity.
  3. In order to check the applicability of this device for other promoters, we measure their maximum and minimum activity levels. We will consider “Lysis box” is able to work properly when it is combined with the promoter whose activity levels are satisfied the criterition above.
  4. We will make some examples of application with this “Lysis box”

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Methods

See below pages for details.

Assay of measurement of the lactose promoter activity

To measure how the activity of a lactose promoter (BBa_R0011) changes at various IPTG concentrations, we use the GFP as the reporter. E.coli KRX transforemed with BBa_K358000 which express GFP at various levels depending on IPTG concentrations, E.coli KRX transformed with BBa_I20260 which express GFP at certain level constitutively and E.coli KRX transformed with pSB4K5 without insert are cultivated at 37℃. Cell growth was measured by using OD600 and GFP fluorescence expressed by each strain was measured.

OD600 is measured by BioPhotometer plus (Eppendorf).

GFP fluorescence is measured by Wallac 1420 Multilabel Counter.

To know detail procedures, see protocol.

Assay of measurements of cell lysis

Assay of lactose degradation

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Results

Charactarization of R0011, a lactose promoter

We tried to determine what is the ratio of activity of the constitutive promoter and that 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.

Construct the circuit of “Lysis box”, regulated by lactose promoter and its repressor

1. Check the function of the 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 in order to find whether the killer gene works correctly.

2. Check the function 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.

3. Make ‘’Lysis box’’, and combine it with the lactose promoter

E.coli strain which can degrade lactose was transformed with the constructs below and grown in the medium which contains lactose. The A550 of the culture and the concentration of lactose in the medium were measured every hour.

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Netbook

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References

  1. PMID: 20395970 Khalil AS, Collins JJ., Synthetic biology: applications come of age., Nat Rev Genet. 2010 May;11(5):367-79.
  2. 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. 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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