Overall Construction

During the process of research and construction, our team attach importance on engineering ideas and principles: The engineering of a biological system is the central guidance of our iGEM project. Initially, considering about SDZ residue in the environment could screening bacteria’s mutation with high drug resistance ability, which could lead to severe damage to human body and medical system, our team proceed with designing and constructing Engineering strain with high degradation of SDZ.

First Round:Design

Because long-term misuse of SDZ antibiotics leads to accumulative antibiotic residues in livestock and feces, which divulge into the environment through various ecological cycle, water and other ecological environments will eventually induce a variety of drug resistant bacterial strains and pose a serious threat to public health. Therefore, our team betake to research on effective biocatalyst in degradation of SDZ residue. During the experiment, our team first used genetic engineering technology for the construction of eligible biocatalyst. Based on the advantages of Ecoli for a variety of available genetic operation tools, mutant strains and high transformation efficiency, we introduced Ecoli Nissle 1917 cell (EcN) as the chassis. Considering laccase has the functions of substrate universality, pollutant concentration reduction, detoxification and decolorization, we will utilize Ecoli containing lacc6 target gene to degrade sulfonamides antibiotics with the intracellular expression of functional genes.

First Round:Build

Separately constructed required gene fragments, utilizing PCR technique for the amplification of Lacc6 gene fragments, modifying the plasmid pSB1A3-mRFP from iGEM headquarter before Enzymatic linkage process. With regard to gene fragments and products of Enzymatic linkage, The recombinant plasmid was constructed with the product of T4 ligase enzymatic digestion, using Calcium ion conversion method conversing recombinant plasmid into engineering strain and culturing the strain with medium under appropriate condition. In order to screen the successful constructing strain, we first used ampicillin to screen the cells containing and successfully express target gene, EcN Lacc6 would present with green fluorescence under fluorescence microscope.

First Round:Test

First Round:Learning

According to the test results, we found that although EcN-Lacc6 has the ability to degrade sulfonamide antibiotics, the degradation rate couldn’t reach the expected value, and could only reach the range of 15% - 30% of expectancy. Therefore, we seek for other technologies to improve the degradation rate of SDZ. Considering that the enzyme cannot function in the cell after the death of the host cell, so as to improve the expression efficiency of the enzyme, we assume that the efficiency would be better if the laccase is degraded on the surface of the cell using Cell Surface Display Technology. Therefore, we started the second round of experiment to authenticate the hypothesis.

Second Round:Design

In this experiment, we still use EcN as a recipient cell, using Lacc6 from Pleurotus ostreatus, a type of fungus in the nature environment as degradation enzyme. The difference is that because the N-terminal of INP contains hydrophobic amino acids and can be connected to the outer membrane through glycosyl phosphatidylinositol, coordinate with highly efficient expression rate, which does not affect the viability of Ecoli. Utilizing the Gram negative cell surface display technology and INP anchoring Lacc6 on the EcN cell membrane by INP, which is overall called EcN-IL. This enables Lacc6 to secrete in EcN and recognize specific cell surface structures on the membrane, So it can be directly expressed on the surface of host cells for practical application. In terms of testing, we still use HPLC to measure the degradation rate of EcN Lacc6 on sulfonamide antibiotics, and verify its effectiveness.

Second Round:Build

Similar with the first round experiment, constructions were separately been in progress, which is conducive to making other modifications more convenient and accurate in the future. First, the target gene Lacc6 was obtained, and then the plasmid pSB1A3-mRFP provide by iGEM Headquarters was similarly modified. Secondly, the gene fragment and modified plasmid were still digested separately, and the product of T4 enzyme ligase digestion was used to construct the recombinant plasmid and PCR amplification was carried out for further amplification. Then, the recombinant plasmid was anchored to the cell membrane of EcN by INP-N using cell surface display technology. In order to screen the correct construction, we first used ampicillin to screen the cells containing and successfully expressing the target gene Lacc6, and detected the fluorescent protein to screen EcN IL under fluorescence microscope.

Second Round:Test

After successfully constructing EcN-IL with Lacc6 Laccase presented on the plasma membrane, we were still wondering the degradation rate of SDZ after using cell surface display technology on EcN. We undoubtedly repeated the experiments with three groups of different SDZ concentration of 30 mg/L, 50 mg/L and 100mg/L respectively, and measured the initial SDZ content with HPLC instrument for obtaining the degradation rate of SDZ under the presence of EcN-IL. The following diagram shows the results of the experiment. It could be observed from the figure that EcN-IL could indeed degrade SDZ residue better than EcN Lacc6 under every different SDZ concentrations in comparison with two bar-diagram.

Second Round:Learning

We could choose better substrate in order to improve efficiency and reproductive rate, DE3 has higher activity and faster proliferation rate compared with E-Coli Nissle 1917 cell we used during experiment. Using real animal manure instead of reagents containing sulfadiazine, because microorganisms and environmental factors in feces may affect the final effect of EcN-IL, which could provide more accurate results under realistic environmental simulation.