Verification and improvement of the antimicrobial peptide expressing system
Here, we tried to improve the former antimicrobial peptide(mccb17) expressing system of 2019 Fudan < a href="http://parts.igem.org/Part:BBa_K3245010 "> LINK .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.
Improving the system
To solve the problem, we have proposed some improvement plans. We though about using the high-copy plasmids to express MccB17, but they are more difficult to regulate and response sensitively, and will greatly increase the expression burden of engineered bacteria.
By researching on the literature and talking with Teacher Lin(iHP), who is engaged in prokaryotic expression work at the Fudan University Academy of Sciences, we decided to use low-copy plasmids to reduce the expression burden of engineered bacteria. We tried to keep the main part of MccB17 (McbABCD) a relatively low expression and high express the immune part (McbEFG) to achieve high secretion of MccB17.
The highly expressed channel proteins and immune proteins will export more MccB17 and reduce the accumulation of antimicrobial peptides in the engineered bacteria cells, thereby inducing them to produce more antimicrobial peptides.
Lin also recommended a series of promoters from a kit which contains combinations of specific constitutive bacterial promoters that vary in strength to us in order to have the expression of mcbABCD in better performance.
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 will make further confirmation that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.
Verification of function
Methods
Figure1. flowchart of this verification experiment
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 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 cultured 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 in the graph down below has been adjusted to be more accurate in the ratio of cell number in the mixture, different from the ratio on the left. Finally our data is measured by plate reader.
Result
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.
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 over 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.
Discussion
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.
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