Bio-dosimeter iGEM Osaka

Toshiyuki Otake, Youfeng Lin, Shuhei Yasumoto, Takahiro Saka, Rie Takino, Shao Thing Teoh, Naoaki Ono

1.Introduction

2.Damage Tolerance 3.Detection of DNA Damage

4.Summary & Future Work

5.References

We constructed a DNA damage detection device by attaching the lycopene biosynthesis gene cluster (CrtEBI) downstream

D. radiodurans proteins related to DNA damage repair (PprI, PprM, PprA, RecA) were assayed for their ability to confer damage tolerance to host E. coli cells. Transformed cells were induced with IPTG, plated and exposed to varying doses of UV radiation or mitomycin C. Plates were further incubated and then CFUs counted. Viable fraction was used as an indicator of tolerance.

Parts & Characterization

PprM is a modulator of the PprI-dependent radiation response of D. radiodurans and regulates several proteins including PprA.

Deinococcus radiodurans is a bacterium with remarkable resistance to DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It has a complex DNA repair system comprising multiple unique proteins including the ones detailed below.

D. radiodurans RecA mutants are highly sensitive to UV and ionizing radiation, emphasizing the importance of this protein in DNA damage repair.

PprI, a protein unique to D. radiodurans, regulates multiple DNA repair and protection pathways, including PprA and RecA expression, as well as enhancing catalase activity.

PprA plays a critical role in the radiation-induced non-homologous end-joining repair (NHEJ) mechanism of D. radiodurans by preferentially binding to double-stranded DNA carrying strand breaks and catalyzing DNA end-joining reactions

1. Hua, Y. et al. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem. Biophys. Res. Commun. 306, 354-360 (2003). 2. Kota, S. & Misra, H.S. PprA: A protein implicated in radioresistance of Deinococcus radiodurans stimulates catalase activity in Escherichia coli. Appl. Microbiol. Biotechnol. 72, 790-796 (2006). 3. Schlesinger, D.J. Role of RecA in DNA damage repair in Deinococcus radiodurans. FEMS Microbiol. Lett. 274, 342-347 (2007). 4. Ohba, H., Satoh, K., Sghaier, H., Yanagisawa, T. & Narumi, I. Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans. Extremophiles 13, 471-479 (2009).

We also propose the following future work for our project: ・testing the response of the SOS promoter to other sources of DNA damage (ionizing radiation, hydrogen peroxide, mitomycin C etc) ・characterizing PprI, PprA, PprM, RecA and their combinations for their effects on tolerance towards other sources of DNA damage ・visually indicating the level of radiation exposure using multiple pigments/promoters ・incorporating DNA damage tolerance and detection into one device to build a complete Bio-dosimeter

Parts & Characterization

In this project, we ・BioBricked four proteins (PprI, PprM, PprA, RecA) implicated in the DNA damage response of D. radiodurans and assayed these parts and their combinations for the effects on DNA damage tolerance ・put together a DNA damage detector device by combining a lycopene biosynthesis part (Cambridge, 2009) with the SOS promoter (Bangalore, 2006) and assayed the promoter's response to varying levels of UV irradiation ・worked with the KIT-Kyoto team to conduct a widely-targeted survey of the public perception of radiation and synthetic biology in (West) Japan

We also wanted to gauge how well a biological dosimeter would be received by the public. To that end, we conducted a survey in conjunction with the KIT-Kyoto team, asking questions related to both synthetic biology and radioactivity. We discovered that most people are more concerned about the dangers of radiation than potential biohazards, and should support the development and use of our bio-dosimeters.

the building of a biological dosimeter. As shown below, there are two components to our "Bio-dosimeter": damage tolerance and detection. To confer tolerance to our E. coli chassis, we modularly introduced radiation resistance genes from the extremophilic bacterium Deinococcus radiodurans. For detection of DNA damage, we connected the native DNA damage response system of E. coli to color pigment production.

On March 11, 2011, the Great East Japan Earthquake struck off the coast of Eastern Japan and triggered a nationwide nuclear crisis centered on the Fukushima 1 Nuclear Power Plant. The need for low-cost, portable and easy-to-use dosimeters became apparent as measurements of radiation exposure could only be conducted at dedicated installations spaced far apart and the numbers reported only infrequently. Hence we have decided to tackle

PprA

RecA

blunt end

Binds / Catalyzes ligation

Recombination Repair

Damaged DNA

ssDNA

RecA

Activated RecA

Enhancement of the pprA promoter activation

PprI PprA

Unknown protein

Irradiation PprM

Binds

Regulates

Induces

Induces

Modulates Regulates

LexA2

End-joining Repair

Binds / Catalyzes ligation

LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated through binding to single-stranded DNA, and the activated RecA induces LexA auto-cleavage leading to derepression of LexA-regulated genes.

If DNA is significantly damaged (eg. by exposure to UV

radiation or chemicals), synthesis of several DNA

damage-related proteins occurs quickly. This reaction

to DNA damage is the SOS response. Central to this

response is RecA, which has multiple activities

essential for the repair and maintenance of DNA. In

the bacterial SOS response, it plays a co-protease role

in the autocatalytic cleavage of the LexA repressor.

We decided to employ lycopene biosynthesis as a reporter as it neither requires addition of substrate (eg. luciferase) or excitation at a specific wavelength

IPP

idi

Isopentenyl diphosphate Dimethylallyl diphosphate

FPP

GPP

GGPP

Phytoene

Lycopene

PPi

PPi

PPi

2PPi GGPP

IPP

CrtE

CrtB

CrtI

IspA

IspA Detection

SOS promoter crtB crtI crtE

Reporting

(eg. GFP). Lycopene biosynthesis is a stepwise process starting from farnesyl pyrophosphate (FPP) which is natively produced in E. coli. However, the conversion of colorless FPP to orange-red lycopene is catalyzed by a series of enzymes (CrtE, CrtB and CrtI) which are missing in E. coli.

of the SOS promoter. Activation of the SOS promoter by DNA damage-induced LexA derepression should trigger lycopene production. E. coli transformed with this part was irradiated with UV light, and then incubated for 2 hours to provide sufficient time for lycopene production. The lycopene was then extracted using acetone and quantified by absorbance at 474 nm. Promoter response was defined as the fraction increase in lycopene production upon UV induction. We observed promoter responses of up to 30% from the SOS promoter upon UV induction. The response was reduced beyond 600 J/m2, possibly as a consequence of intensive DNA damage interfering with lycopene biosynthesis. The high background of SOS promoter compared to LacI promoter may be a consequence of SOS genes being weakly expressed even in the absence of DNA damage.

Single-gene parts: Combination parts:

where CDS = PprI, PprA, PprM or RecA

Results indicated that induction by IPTG significantly increased cell survival; PprM and RecA increased tolerance to both UV and mitomycin C, especially in combination; and PprI had a synergistic effect with RecA in increasing UV tolerance.

Deinococcus radiodurans

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