Detector AmplificatorOutput

Aflatoxinbiosensor!

TeamITB_Indonesia

Designing a whole cell biosensor

for aflatoxin B1 detection in foods

aflatoxin

green fluorescentprotein

OurTeam! advisor

Ari Dwijayanti Indra Rudiansyah Nuke Ayu F. Lili Melani Dimas Dwi A. Riandy Rahman N. Dr. MaelitaR. Moeis

Dr. SonySuhandono

Dr. Adi Pancoro

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BANDUNGINSTITUTE OF TECHNOLOGY

SCHOOL OF ELECTRICAL ENGINEERING ANDINFORMATICS

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FACT about aflatoxinAflatoxins are naturally occuring mycotoxins that are mutagenic, carcinogenic., and heat stable produced by Aspergillus (fungi). Aflatoxin contamination of foods that are found in many developing countries such as Indonesia. It may cause a serious problem for human health. The presence of aflatoxins especially aflatoxin B1 on food such as peanuts and corn (leguminose) can increase the risk of liver cancer. One fourth Indonesian who have liver cancer caused by aflatoxin exposure.

aflatoxin activationmodul i

üWe have constructed cyp ( ) which is one member of the cytochrome P450 superfamily of proteins derived from human. CYP3A4 has a high efficiency to convert AFB1 into its active form (Exo-AFB1-8,9-epoxide, known as aflatoxin B1-oxide).

üWe have removed the illegal restriction site (EcoRI, XbaI, PstI, and SpeI) on native CYP3A4 sequence.

üWe cut the signal peptide to localize CYP3A4 in the cytoplasm. Cytosolic CYP3A4 is intended to only activate AFB1 that has entered the cell.

üWe have constructed CYP expression modul ( ) that consisted of cyp (Bba_K1064004) under the control of a constitutive promoter ( ) and strong RBS ( ). So CYP (~50 kDa) can be expressed constitutively and be localized in the cytoplasm. (Fig 1)

Bba_K1064004

Bba_J223119 Bba_B0030

Bba_K1064000

The biosensor uses Escherichia coli as the chassis to build a genetic circuit using CYP to oxidize the Aflatoxin B1 to become aflatoxin B1 oxide (more reactive form) and SOS response system to detect DNA damage caused by the aflatoxin B1-oxide attack. For the ease of usage, we will design a syringe shaped device with our whole cell biosensor in it. This device would allow aflatoxin B1 to enter the device, but would not permit the cells to leave the device.

mechanism of detection

medium aflatoxin

membrane

positive responseof aflatoxin

grinded sample

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CYP gene

sos gfp

Our device will be designed to be easily used by user. So, we make a syringe-shaped device with a little amount of our dried cell inside of it. User then insert medium to prepare the cell for analyzing, and insert some amount of prepared sample (grinded sample dilluted with alcohol). After several minutes, user can see the change in fluorescent concentration as an effect of aflatoxin presence.Diffusion of aflatoxin

into our cell

Aflatoxin

Cell producingcytochrome P450

Aflatoxin is enzymaticallyactivated to very reactive form :aflatoxin B1 oxide (AFB1 oxide)

AFB1 oxide attack DNA andactivating SOS response

Cell will produce GFPas reporter

aflatoxin whole cell biosensor

modelling

We have simulated our complete biological system model using Simbiology (Mathwork). We estimate the time needed for analysis after we modelled diffusion of aflatoxin, CYP synthesis, aflatoxin oxidation reaction, DNA damage kinetics, reporter kinetics, and integrated the models we have. We want our device to report aflatoxin's presence as low as Indonesia's regulation on food safety (BPOM RI No. Hk.00.05.1.4057 stated that aflatoxin safety threshold in Indonesia is 20 ppb). We simulate the first 3600 seconds of our device and we can see that fluorescent concentration for different aflatoxin concentrations can be distinguished clearly after 2500 second (approximately 40 minute). See Fig 4.

GFP

(m

ole

)

time (s)

Fig 1. SDS PAGE analysis of CYP expression

Fig 2. Cell with SOS response systemcaused by UV irradiation (10 s)observed in fluorescence microscope

Fig 4. Simulated GFP output with various aflatoxin concetration

Fig 3. Comparationpromotor activitiestowards DNA damage

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how much do people knowabout aflatoxin?

future development

Because the importance of aflatoxin knowledgement for Indonesian, We conducted a survey to surrounding community (96 correspondent) about aflatoxin and it’s risk.

To make Aflatoxin whole cell biosensor much better we have planned to improve our system by adding another more visible chromophore as reporter, constructing the reactive sequence, amplifying the DNA damage signal, and adding buoyancy system.

sos response systemmodul ii

üAflatoxin B1-oxide attacks DNA randomly causing DNA adduct and ssDNA is produced. ssDNA will activate the RecA protein. Activated RecA protein will cleave LexA protein that is bound to SOS response promoter. The LexA cleavage will induce the expression of reporter gene downstream of this promoter.

üThe SOS response promoter is followed by a reporter gene coding a chromophore, therefore the concentration of aflatoxin B1 in food samples could be easily detected by the color change of the bacteria. We have constructed two devices to detect SOS response caused by DNA damage. These devices consist of the following BioBricks parts :

, , , and .

Bba_K1064002 Bba_K1064003 Bba_K1064005Bba_K1064006

üWe have characterized both promoter activities towards the DNA damage for and

using UV irradiation (10 sec expossure) and observed the results as glowing object by fluorescence microscopy. (Fig 2)

üWe have characterize both of promoter activities towards the DNA damage response and found that

has 1.2 fold activity of . (Fig 3)

Bba_K1064002Bba_K1064003

Bba_K1064003 Bba_K1064002

http://2013.igem.org/Team:ITB_Indonesia

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According to the data we gathered :of the respondents feel that developing a cheap and reliable aflatoxin-sensing device is an urgent need. 32%

reference:¬Farombi, EO. 2006. Aflatoxin contamination of foods in developing countries: Implications for hepatocellular carcinoma and chemopreventive strategies. African Journal of Biotechnology Vol. 5 (1), pp. 001-014, 2 January 2006¬Gallagher, E. P., Kunze, K. L., Stapleton, P. L., Eaton, D. L. (1996) The Kinetics of Aflatoxin B1 Oxidation by Human cDNA-Expressed and Human Liver Microsomal Cytochromes P450 1A2 and 3A4. Toxicology and Applied Pharmacology 141, 595–606. Article No. 0326¬Guengerich FP (January 2008). "Cytochrome p450 and chemical toxicology".Chem. Res. Toxicol. 21 (1): 70–83¬Ming Ni, Si-Yuan Wang, Ji-Kun Li, and Qi Ouyang. Simulating the Temporal Modulation of Inducible DNA Damage Response in Escherichia coli¬Rawal, Summit., S. M. Yip, Shirley., Roger A. Coulombe, Jr.2010. Cloning, Expression and Functional Characterization of Cytochrome P450 3A37 from Turkey Liver with High Aflatoxin B1 Epoxidation Activity. Chem. Res. Toxicol. 2010, 23, 1322–1329¬Shimoni Y, Altuvia S, Margalit H, Biham O (2009) Stochastic Analysis of the SOS Response in Escherichia coli. PLoS ONE 4(5): e5363. doi:10.1371/journal.pone.0005363