Preliminary Project Description
Aflatoxins, specifically, aflatoxin B1 have been troubling food and agriculture industry all over the world. As secondary metabolites produced by certain species of Aspergillus, aﬂatoxins have been one of the most toxic and problematic . Though many past traditional biological methods have been put in this fight against these toxins, the purposes were mostly realized by mixing whole strains and degrading toxins passively.
Our goal this year is to create a more sensitive cellular device to simultaneously detect and degrade AFTB1 in the environment. We have two subsystems, the Detection Subsystem and the Degradation Subsystem. We will take and modify some past parts designed by Tsinghua 2017 as our biosensor to detect AFTB1. And we will utilize this part to induce downstream genes to produce enzymes that will degrade aflatoxin outside cells. We have some candidates now that show the ability to degrade AFTB1 and they will be tested by us then.Detection Subsystem
Inspired by Tsinghua 2017, we will reconstruct the sensor based on the yeast two-hybrid technique. There are two domains in this part, the transcription-activation domain (AD) and the DNA-binding domain (BD). They correspondingly use two different single-chain antibodies of AFT ScFv1 and ScFv2 to link together when AFTB1 exists and activate Gal4 promoter then. When the promoter is activated, it can open downstream genes, the Degradation Subsystem.Degradation Subsystem
The degradation subsystem is designed to degrade aflatoxin which will be induced by the Detection Subsystem.bacC
bacC is a bacilysin biosynthesis oxidoreductase gene from Bacillus subtilis subsp. subtilis str. 168. As a new report says, bacC may contribute aflatoxin degradation in some degree. The BLAST results show that bacC has some similarities and identities to reductase with nine reductase enzymes of Mycobacteria smegmatis, which have been proven to clean aflatoxin. Some experiments were also completed by the author to support that. As a good candidate to us, we will design experiments to express bacC and test its functions. Finally, it may be in use as part of our Degradation Subsystem.ADTZ
ADTZ, which is also called aﬂatoxin oxidase (AFO) , is the ﬁrst enzyme identiﬁed as able to degrade AFB1. It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project, we will induce it by our Detection Subsystem and regulate its expression.
Concerning the effectiveness of our project, we will preliminarily take Saccharomyces cerevisiae as our chassis. As a completed cellular device, we hope our modified strain can automatic feel aflatoxins from environment and begin to express relative degradation enzyme to keep there non-toxic, ensuring a healthy condition to human beings.