Bio-dosimeter iGEM Osaka

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

1.Introduction

2.Damage Tolerance 3.Detection of DNA Damage

4.Summary & Future Work

5.Reference

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 and pre-cultured cells were plated and exposed to varying doses of UV radiation. Plates were then further incubated for 16h and colonies counted. Viability percentage relative to control was used as an indicator of tolerance. Results indicated that PprM, RecA and the combined part were all capable of increasing UV tolerance. Interestingly neither PprI or PprA were capable of increasing tolerance, suggesting their mutual requirement for DNA repair.

Parts & Characterization

PprM is a modulator of the PprI-dependent radiation response mechanism of D. radiodurans and regulates the induction of PprA as well as other, unidentified proteins.

The bacterium Deinococcus radiodurans shows 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.

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

PprI

PprI, a protein unique to D. radiodurans, is a central protein in the radiation response mechanism. Experimental data indicates that PprI regulates multiple DNA repair and protection pathways, including RecA and PprA expression, as well as enhancing catalase activity.

PprA preferentially binds to double-stranded DNA carrying strand breaks and stimulates the DNA end-joining reaction. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining repair mechanism in which PprA plays a critical role.

PprM

RecA

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).

・testing the response of the SOS promoter to other sources of DNA damage (eg. ionizing radiation, hydrogen peroxide, mitomycin C). ・assembling and characterizing other combinations of the DNA damage tolerance proteins PprI, PprA, PprM, and RecA. ・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.

PprA

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 for their 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. Results of two representative questions are as follows:

Hence we have decided to tackle 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.

Deinococcus radiodurans

PprA

RecA

blunt end

Binds / Catalyzes ligation

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.

Recombination Repair

Damaged DNA

ssDNA

RecA

Activated RecA

Enhancement of the pprA promoter activation

PprI PprA

Unknown protein

Irradiation PprM

Binds

Regulates

Induces

Induces

Induces Modulates

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

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UV Induction of Lycopene Biosynthesis

SOS promoter LacI promoter

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Promoter Response versus UV Energy Dosage

IPP

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Isopentenyl diphosphate Dimethylallyl diphosphate

FPP

GPP

GGPP

Phytoene

Lycopene

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2PPi GGPP

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IspA Detection

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PprI PprA PprM RecA Combined Vector only Wild type

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Viability After Various UV Energy Dosages

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DNA Damage Tolerance Conferred By Each Part

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LacI RBS pprI pprA pprM recA

We assembled test parts as follows;

SOS promoter crtB crtI crtE

We also propose the following future work for our project:

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.