- Human Practices
Great importance is attached to safety in our project design. Firstly, we use E.coli as our chassis organisms, which pose no harm to individuals or environment, and do not use any other organism. E.coli DH5α, MG1655 and BL21 (DE3) are applied as the chassis when we implement the experiments of molecular cloning and expression. This year our team fight for engineering the E. coli that can rapidly detect glyphosate (most common pesticide) in tea and effectively degrade it in soil, which means there are two aspects in our program: the detecting part, the degrading part and the kill switches.
1. The detecting part.
GOX, from Ochrobactrum antrophi strain LBAA, makes glyphosate converted into one AMPA and one glyoxalic acid and AMPA, then the glyoxalic acid was reduced and consumed NADPH by the enzyme named GRHPR.
The quantity of NADPH could be converted into fluorescence signal by iNAP, a kind of fusion protein of cpYFP and the NADP(H)-binding domain (which was designed pointed mutations of the NAD(H) from Thermus aquaticus according to the article). So the quota of NADPH could be determined inversely by the fluorescence intensity, and ultimately the amount of glyphosate is quantified.
All the enzymes were displayed on the surface of engineered bacteria through several kinds of anchor proteins.
2. The degrading part.
E. coli is chosen as the chassis bacteria for its broad space and advanced background to be operated in genetic engineering. However, the transportation and degradation system of organophosphate is inefficient in E. coli. To make the all the sets functioning well, the following parts should be involved:
a) High efficiency phosphonate acid transmembrane transportation system: phnCDE from Sinorhizobium meliloti 1021 transport organophosphorus across membrane.
b) The enzyme for C-P cleavage of glyphosate: phnGHIJK (core gene: phnJ) from Enterobacter cloacae K7 degrade organophosphorus to small molecule.
c) The enzyme for derivation and degradation of AMPA: phnO from Salmonella enterica derivatize AMPA into glyphosate analogue.
All the proteins are totally harmless to human body. All the plasmids are harmless for human beings and none of our parts would raise any safety issue according to the current expertise.
The building of laboratory includes the location of emergency exits, emergency ventilation system, fire-fighting equipment, first aid kit and so on. Every year after recruiting new members, we spend several weeks training them in basic laboratory operations and handling safety accidents. Laboratory coat, gloves and glasses are necessary when entering the laboratory. All culture fluid and culture medium with bacteria were processed properly to prevent the potential biological pollution. We handled all the biological materials, wastes and equipment strictly following the BSL-1 requirements, our lab is also equipped with professional operators of dangerous goods and waste biological products, they will disposal all the waste properly. We turn off the water and electricity, close window and door before we leave the lab.
At the same time, we invited laboratory safety engineer, Wenyao Shao，to join our team as an advisor and to conduct safety education for all team members when they officially entered the experimental phase consistently.