Here, we tried to improve the former antimicrobial peptide(mccb17) expressing system of 2019 Fudan BBa_K3245010 .By dividing into the peptide expressing part and the immunity part, we firstly tested whether the polycistron could work properly and separately, then tried to manipulate each of their expression levels with more efficiency an better function.

First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids(with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed successfully them into E.coli BL21, then purified the protein products with his-tag via immobilized metal affinity chromatography to check if the basic protein-coding could all be realized correctly as the basis of our design for the system. We will make further confirmation that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.

As our design page describes, mcbABCD should show a certain degree of restrain to both the growth and condition of its cocultured cell. While with mcbEFG, engineered E.coli are supposed to have better immunity themselves against the mccb17 coded by mcbABCD, so as to gain advantage, affect the surrounding WT’s metabolism(the expression of GFP), and better survive in the environment.

We performed 2 different kinds of experiments, rendering verification for both antimicrobial expressing part mcbABCD and immunity part mcbEFG, by testing the effect mccb17 may exert on the metabolism and survival state of microbes they cocultured with.

Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM, where MEFL stands for the absolute units of Fluorescent Intensity for Green Fluorescent proteins and particle stands for the absolute units of cell count, so that our y axis represents fluorescent intensity per cell under iGEM standard.

We could see an overall decrease of GFP expression in nearly all groups cocultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide(MccB17) encoded by mcbABCD does have a negative effect on microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the toxic environment, proving that our part mcbABCD has worked successfully.

While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of mcbEFG is working successfully, as the E.coli expressing mcbEFG can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage.

Among the three graphs, it is also obvious the system works better when the population occupies a larger percentage. When the ratio of WT:Px meets 50：7, nealy all of the groups shows very weak competitiveness or even no survival advantage at all(as in the case of P1), indicating the need of a certain amount of population to get the system working efficiently. This result also affirms it is vital to have the antimicrobial system in our design to gain enough survival advantage for our engineered E.coli to colony in human intestine and further express the desired product.

Due to the difference in strength of these promoters, we have also screened out the most suitable promoter P2 for our system via this experiment.

Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM. In this chart, Calculated Bacteria Counts stands for the relative increase of OD, which represents the total number of cells, and MERL, which represents the total number of WT cells. As we have screened out P2 in front, here we used the mixture ratio of P2(P2AD):WT=1:1/1:4 to find out more detailed effect that antimicrobial peptide expressing system can exert.

We could see the population increase are not significant in total over time, yet WT grows rather fast at first in the P2AD group, indicating its lack of immunity part mcbEFG can lead to the death of engineered E.coli. more, thus shows less restrain on the WT cells. Yet, as mccb17 accumulates, a clear decrease can be observed when time extends in both P2 and P2AD, proving our antimicrobial peptide expressing system are working to help the engineered E.coli. gain better survival advantage.