Project

Mosquito as a natural syringe

In our project,we designed a system to resolve the problem caused by a common yet horrible toxin-"aflatoxin". Our project, named"Aflatoxout", consists of three parts-Firstly the antidote to alleviate harm caused by aflatoxin,secondly the test paper to detect aflatoxin, thirdly the app & device as a platform for food safety information.This is a creative system that not only prevents the public from consuming aflatoxin but also creates safe detoxifying products and platform to assist with public health.

GFP System

To create a reporter system, we constructed a GFP expression vector. We amplified a constitutive promoter from Drosophila actin 5c gene and an eukaryotic poly A signal by PCR from pAc5.1 vector. The resulting DNA fragments were assembled with a BioBrick existing part of GFP to generate the reporter vector of Ac5-GFP-polyA / pSB1C3 (K2543004).

There are at least 17 different types of aflatoxins found in nature, and B1, B2, G1, G2 are the commonest type. The “B” and “G” indicate the blue and green fluorescent colors produced by aflatoxins under UV light on thin layer chromatography plates, while the subscript numbers 1 and 2 indicate major and minor compounds, respectively_[1]. In dairy products, aflatoxin M1 (AFM1) is easily detected when contaminated_[2]. In the public’s conceptions, cooking or heating can remove harmful substances. However, aflatoxins are a group of stable compounds that would not be degraded unless heated to 280℃. Chances are we may all be exposed to these mycotoxins.

The harm of Aflatoxin B1

Aflatoxin B1 (AFB1) is the most toxic and carcinogenic in mammals. Animals that consume AFB1-contaminated food can develop acute and chronic health problems. For acute aflatoxicosis in animals, AFB1 causes liver necrosis in rats_[3] and hepatitis X in dogs_[4]. It also causes hemorrhagic necrosis of the liver, bile duct proliferation, edema, and lethargy in human_[5].

In addition, it is usually reported that more often children rather than adults die from acute aflatoxicosis because adults have higher tolerance for aflatoxin. Despite a certain extent of tolerance in adults, aflatoxins are yet to be feared since they are well-known mycotoxins for their chronic carcinogenesis. AFB1 is the most potent hepatocarcinogen in mammals and it is included in category 1A_[6]. When aflatoxins are taken into the body, they will first undergo phaseⅠmetabolism in liver. There are a group of heme-binding enzymes called cytochrome P450 (CYP450) involving in the metabolism of endogenous substrates and biotransformation of xenobiotics like aflatoxins. When AFB1 is metabolized into AFB1-exo-8,9-epoxide (AFBO), it can bind to DNA and form DNA adducts_[1]. If this damage cannot be repaired, it will lead to mutation and probably result in cancer.

Mosquito Signaling

To create a reporter system, we constructed a GFP expression vector. We amplified a constitutive promoter from Drosophila actin 5c gene and an eukaryotic poly A signal by PCR from pAc5.1 vector. The resulting DNA fragments were assembled with a BioBrick existing part of GFP to generate the reporter vector of Ac5-GFP-polyA / pSB1C3 (K2543004).

The gene of aflatoxin-degrading enzyme

There are various enzymes found in many microorganisms which have the ability to degrade aflatoxins_[7]. F420-dependent reductases (FDR) are in an enzyme family produced in some species, like Actinomycetales, Nocardia corynebacterioides, Mycobacterium smegmatis and have almost 100% degradation ability_[8]. Because MSMEG5998 from Mycobacterium smegmatis has the best enzyme ability_[9] and has the suitable reaction pH and temperature for human body, we put the gene of this protein into our vector to express it.

Enzyme cofactor

In addition, this enzyme needs a special cofactor, F420, which is only produced in the bacteria when detoxifying aflatoxins. Its structure and function are like the important cofactor in human bodies, FAD. When it becomes the active form, F420H2, it can supply two electrons to be a oxidant. However, F420H2 is unstable and easily oxidized into F420, thus we needed another enzyme, F420-dependent glucose-6-phosphate dehydrogenase (FGD) that is also produced in Mycobacterium smegmatis. This enzyme can maintain the reduced form of F420.

Linking thioredoxin

In order to understand whether our two enzymes were soluble in E. coli and whether they would become inclusion bodies when E. coli expressed them on a large scale, we modified them. In previous studies, E. coli thioredoxin (trxA) were used to form a fusion gene expression system to increase the solubility of target proteins_[10]. Therefore, in our project, we linked thioredoxin with our two proteins through some linkers, which were designed for some restriction sites.

Comparing original and modified proteins

Moreover, we produced two original enzymes by two plasmids obtained from Taylor, M.C. in CSIRO from Australia and compared whether the two modified version proteins and two original proteins have different solubility and different enzyme activities. We found that both the original and modified (synthetic) MSMEG5998 were able to degrade aflatoxin B1 while only the original FGD could help the reaction.

Finally, we chose the better MSMEG5998 and better FGD to test for effects in HepG2, human hepatoma cell line. We discovered that MSMEG5998 could alleviate DNA damage induced by aflatoxin B1 and therefore decrease activation of p53 pathway.

What we do

For our antidote, we synthesize enzymes that degrade aflatoxin to a large extent, as well as alleviate some of the DNA damage induced, to reduce the harm of aflatoxins. In addition, we also use yeast to express our recombinant proteins because it is a common and safe vector which is generally recognized as safe (GRAS) approved from FDA.

AMP System

To solve the problem suffered from aflatoxin B1,the best way is to prevent having contaminated food. The traditional way to detect aflatoxin B1 is using HPLC or ELISA. Although the results are more accurate than the others, it is time wasting and expensive. So, we want to development a new model to make public more intuitive and easier to use. For this, we spend many time to search better way to detect aflatoxin. Fortunately, we found the immune strip is very suitable with our topic. This way is common, low cost and most important that it is very easy to do.

Secret of “Y/N”

Immune reagents work mainly through the antibody and antigen between the specific characteristics of the operation. Then, we can produce antibody specific to aflatoxin. The immunochromatography strips are based on this feature, and the structure of strips are composed by three part. The major part combined with Anti-aflatoxin antibody and gold nano particle is release pad. Second part is coated with antigen and antibody on nitrocellulose membrane. The last part is composed by sample pad and absorbent pad. The absorbent pad provides force for sample to move.

To make antibody be recognized by eyes, the antibody will be combined with gold nanoparticles_[11]. The process of immunochromatography need to extract aflatoxin in the sample first. Then, according to the capillary phenomenon, the sample will be attracted to the Anti-aflatoxin antibody with gold nanoparticl.If the sample has aflatoxin, the active site of first antibody would be occupied and couldn’t binding with antigen on nitrocellulose membrane until arrive second antibody. Then complex of first antibody would move toward absorbent pad. Because the complex has the antigen of sample, it couldn’t bind with antigen on nitrocellulose membrane until arrive at second antibody. The result can be recognized through red line on the strip.

The New Generation of Antibody!!!

The traditional antibody on the release pad of test strip is produced with hybridoma. In our perspective, if we can have bacteria such as E. coli that are able to express antibody for us, we can access to higher efficiency in producing antibody. However after we search for the papers online, we found that the process of glycosylation and formation of disulfide bond in post-translational modification of prokaryotes is different with those in eukaryotes. Thus it will not be able to produce the full-length antibody.

Then we go further to search whether there is someone who has the similar idea with us. We found that there are methods like phage display that can produce scFv (single chain variable fragment)_[12]. In our project, besides finding out the scFv combine to aflatoxin from a research published in 2012_[13], we will further more improve its function. In the traditional process, we have to bind gold nanoparticles to the antibody in order to observe the result on test strip. To replace this step, we put the sequence of Red Fluorescent Protein into DNA sequence when designing the fusion protein. What’s more the structure of scFv only contain the variable region of antibody, so the control line of the strip which have secondary antibody that bind to the constant region of primary antibody can’t work anymore.

To deal with this problem and facilitate the process of purification, we add a His-tag at the end of the fusion protein. After putting the three-dimensional structure of protein into consideration, we add a rigid linker between the RFP and scFv to avoid interference between the two proteins when folding.

What we do

To improve the way of making the test strip, we constructed the DNA sequence which is composed of scFv and Red Fluorescent Protein.There are two main advantages about this fusion protein. One is that it can reduce the cost to produce aflatoxin antibody compared with using hybridoma because our fusion protein can be expressed abundantly by E.coli. The other is that we don’t need to use gold nanoparticles to show the result. It could simplify the process of making the antibody tested on strip.

Reference

• 1. Bbosa, G.S., et al., Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. Health, 2013. 5(10): p. 14.
• 2. Galvano, F., V. Galofaro, and G. Galvano, Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. Journal of Food protection, 1996. 59(10): p. 1079-1090.
• 3. Butler, W., Acute toxicity of aflatoxin B1 in rats. British journal of cancer, 1964. 18(4): p. 756.
• 4. Newberne, P.M., R. Russo, and G.N. Wogan, Acute toxicity of aflatoxin B1 in the dog. Pathologia veterinaria, 1966. 3(4): p. 331-340.
• 5. Williams, J.H., et al., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. The American journal of clinical nutrition, 2004. 80(5): p. 1106-1122.
• 6. Creppy, E.E., Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology letters, 2002. 127(1): p. 19-28.
• 7. Adebo, O., et al., Review on microbial degradation of aflatoxins. Critical reviews in food science and nutrition, 2017. 57(15): p. 3208-3217.
• 8. Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.
• 9. Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.
• 10. Lavallie, E.R., et al., A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Nature biotechnology, 1993. 11(2): p. 187-193.
• 11. Chandler, J., T. Gurmin, and N. Robinson, The place of gold in rapid tests. Vol. 6. 2000. 37-49.
• 12. Hammers, C.M. and J.R. Stanley, Antibody phage display: technique and applications. The Journal of investigative dermatology, 2014. 134(2): p. e17.
• 13. Li, X., et al., Molecular characterization of monoclonal antibodies against aflatoxins: a possible explanation for the highest sensitivity. Analytical chemistry, 2012. 84(12): p. 5229-5235.