Verification and improvement of the antimicrobial peptide expressing system
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.
Problems of this system in the past
The concept of quorum sensing system described above was proposed by Fudan 2019. Since MccB17 plays a vital role in the entire quorum sensing system, the better the MccB17 is expressed, the faster the engineered bacteria will achieve the balance of quorum sensing. However, we found that the expression level of MccB17 tested by Team Fudan 2019 last year was so low that the engineered bacteria cannot be competitive enough in the intestine.
Verification of expression
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 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 confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.
Verification of function
Figure1. flowchart of this verification experiment
As our design page describes, with mcbEFG, E.coli are supposed to have better immunity themselves against the antimicrobial peptide 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 culture WT(E.coli expressing GFP driven by plac), PxAD(E.coli expressing mcbABCD driven by P1\2\9\11\12\13)and Px(E.coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time. When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1, 10:1, then induce them with IPTG for 2h. Here, the control group would be merely WT with IPTG and WT without IPTG. After mixture, we immediately put the engineered E.coli on ice to cease the proliferation and measure their OD later for a precise mixture ratio. Thus the ratio you see in the graph on the left has been adjusted to be more accurate in the ratio of cell number in the mixture. Finally our data is measured by plate reader.
Figure2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7)
Figure3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7)
Figure4. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7)
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. Firstly, we could see a overall decrease in nearly all groups that are cocultured than the control group which only contains WT. We can clearly see a better inhibition effect in E.coli with Px-mcbABCD-mcbEFG-ptetR(Px) than those merely with Px-mcbABCD(PxAD), as the MEFL/particle is much lower in the former group than the latter one, indicating WT's function in creating GFP is strongly restricted by the more toxic environment. While comparing to the control group, we can also see a decrease in the GFP expression result indicating that the antimicrobial peptide(mccb17) encoded by mcbABCD does have a strong restraining effect on other microbial.
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