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.
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 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.
Verification of function
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.
influences on metabolism
Methods
Figure 1. flowchart of this verification experiment
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
Figure 2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7)
Figure 3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7)
Figure 4. 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 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.
influnces on survival states
Methods
Figure 5. flowchart of this verification experiment
We cultured WT(E.coli expressing rFP driven by constitutive promoter), 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.
Meanwhile, we culture plates with different antibiotics(since WT and Px/PxAD possess different resistance) to measure the precise ratio of mixture.
Finally our data is measured by plate reader.
Result
Figure 6. Calculated Bacteria Counts when the ratio of mixure(Px/PxAD:WT=1:1)
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 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.
Improvement in reduced leakage of ptetR
We have measured the expression level of PtetO2, PtetO3 along with the original PtetR(BBa_R0040) via plate reader by combining them with GFP, when they were induced by different concentration of hydrotetracycline or not induced.
figure1.When aTc=0, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure3.When aTc=200, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure2.When aTc=100, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure4.When aTc=400, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
Under the induction of various concentrations of aTc, the expression level of ptetO2 is very low, but the expression level of ptetO3 is moderate. Although the expression level of ptetO3 is lower than ptetR, it is very suitable for our project design. Since ptetO3 is used to drive McbEFG, if McbEFG translates too much channel protein, then when McbEFG is turned off, the expressed channel protein will be difficult to recover completely, reducing the sensitivity of quorum sensing.
Figure 7.When aTc=100,fold of increase of promoters
Figure 8.When aTc=200,fold of increase of promoters
Figure 9.When aTc=400,fold of increase of promoters
The results show that among the three promoters, ptetO3 has the highest fold of increase. This means that it is the most sensitive to the induction of aTc, which can improve the sensitivity of quorum sensing.
CaAP(Calcium Absorption Peptide)
We hope that our project can solve the problem of calcium deficiency in the elderly. According to our investigation, due to the diet structure, the calcium absorption of Chinese people is not high, so we found CaAP from the perspective of improving the calcium absorption rate of the elderly.
We have successfully cloned our designed CaAP(Calcium Absorption Peptide) into pFAB plasmid and transformed the construct into E.coli. BL21 strain to demonstrate its expression.
We chose to use a set of promoters to drive the expression of the specially designed CaAP(Calcium Absorption Peptide). Thus, the expression intensity of the gene of interest can be quantified and regulated.
P1-P14 is a collection of precisely quantitated constitutive bacterial promoters that vary in strength and effectiveness. These promoters can be used to initiate the expression of the target gene in E. coli in a controlled, reliable way.
To test the calcium-chelating ability of the designed CaAP, we purified it from the bacterial lysate of our successfully cloned strains(E.coli. BL21 transformed with pFAB-CaAP) using Ni-chelating affinity chromatography(CaAP contains an 6x-His tag at the C terminal). In this way, the effect of CaAP can be demonstrated through the differences of calcium ion concentration in the divalent calcium ion solution with or without CaAP.
Secretion Peptides guided CaAP
To promote calcium bioavailability, CaAP needs to be expressed in our probiotics and subsequently secreted into the extracellular space. Therefore, we linked 5 signal peptides which belong to the Type II secretion system in E.coli., to the N terminal of CaAP, respectively. The 5 secretion peptides are listed below:
| NSP4 | BBa_K3606042 |
|---|---|
| OmpA | BBa_K3606043 |
| DsbA | BBa_K3606030 |
| PelB | BBa_K208004 |
| PhoA | BBa_K808028 |
To test the secretion efficiency of our 5 secretion peptides and compare their secretion capabilities, we also fused them to MBP(Maltose Binding Protein) that normally expressed in the cytoplasm of E.coli. cells. Driven by an iPTG inducible promoter Ptac, we can achieve quantified characterization of these secretion peptides through differences of the amount of signal-peptide-MBP fusion protein in bacterial lysate and culture medium.
We have successfully cloned OmpA guided MBP(Maltose Binding Protein) and expressed it in E.coli.BL21. As shown in Figure.1 ,the amount of MBP in the lysate shows no difference with or without iPTG induction, suggesting that OmpA has the strong ability to secrete MBP into the extracellular space.As for the relatively low amount of MBP in the culture medium concentrated by TCA than in the cell lysate(shown in Figure.2), it may be caused by other interference factors during the preparation of protein samples.
To test the secretion efficiency of our 5 secretion peptides fused CaAP(Calcium Absorption Peptide) guided by 5 secretion peptides, we cloned the Secretion peptide-CaAP segment into the pGEX vector respectively and transformed into E.coli. BL21 strain. Thus, quantified characterization of these secretion peptides fused with CaAP is expected to be achieved through the differences of calcium ion concentration in bacterial lysate and culture medium.
Figure 1.NSP4/PhoA/OmpA-CaAP expression with and without iPTG induction in the bacterial lysate.
Figure 2.NSP4/PhoA/OmpA-CaAP expression expression with and without iPTG induction in the culture medium concentrated by TCA.
Reference
[1] Huanna Qiu, et al. Construction of promoters with tight regulation on chromosome of Escherichia coli. Microbiology China,2018,45(08):1693-1704. (Chinese)