Improvement of the antimicrobial peptide expressing system
Here, we tried to improve the former antimicrobial peptide (Mccb17) expression, based on 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 for better results.
Verification of the expression
First, to affirm the system could work properly, we started from the very beginning by testing the expression of each mcb gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform IPTG induced expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed successfully them into E. coli BL21. We have made further confirmation that every part can be induced successfully due to the correct bands on the SDS-PAGE gels, only shown in IPTG induced samples. If needed, we could purify these protein products via immobilized Ni affinity chromatography (GST was replaced with His-tag in our constructs).
Verification of the function
As our design page describes, McbABCD should show a certain degree of restrain to both the growth and metabolism of its co-cultured cells. 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 (expressing of GFP in our experiments), and better survive in time.
We performed 2 different kinds of experiments, verifying 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 co-cultured with.
Influences on metabolism
Figure 1. Flowchart of the IPTG induction experiments
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 with similar OD.
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 2 hours. Here, the control group would be merely WT with IPTG and WT without IPTG.
After mixture, we immediately put the starting cultures on ice to cease the proliferation and measure their OD 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 numbers in the mixture (different from the ratio on the flowchart). Finally, all samples were measured by a plate reader.
Results from IPTG induction experiments
Figure 2. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=5:7
Figure 3. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=20:7
Figure 4. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=50:7
Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee, 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 co-cultured 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 unsuitable environment, proving that our part mcbABCD works as designed.
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 McbABCD 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 McbABCD 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 (P1 to P14) driven the expression of mcbABCD, our initial test suggested the most suitable promoter P2 for the system.
Influnces on survival
Figure 5. Flowchart of the survial (time-course) experiments
We cultured WT (E. coli expressing RFP driven by a 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. Later, we collect samples from the mixture at different time points, to be measured by a plate reader.
Meanwhile, we plate the mixture onto plates with different antibiotics (since WT and Px/PxAD possess different antibiotic resistance) to measure the precise ratio of mixture.
Results from survial experiments
Figure 6. Calculated Bacteria Counts with 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 Measurement Committee. 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. 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, suggesting our antimicrobial peptide expressing system is working to help the engineered E. coli gain better survival advantage.
Reduced leakage of PtetR (improved part)
We have measured the expression level of PtetO2 (BBa_K3606006), PtetO3 (BBa_K3606007) along with the original PtetR (BBa_R0040) via plate reader by combining them with GFP, when they were induced by different concentration of anhydrotetracycline (aTc) or not induced.
Figure 7. When aTc=0, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
Figure 9. When aTc=200, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
Figure 8. When aTc=100, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
Figure 10. 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 take a long time to degrade completely, reducing the sensitivity of quorum sensing.
Figure 11. When aTc=100, fold of increase of promoters
Figure 12. When aTc=200, fold of increase of promoters
Figure 13. 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.
Demonstration of CaAP and signal-peptide guided CaAP secretion system
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 enough, so we design 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.
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 14,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 15), it may be caused by other interference factors during the preparation of protein samples.
Figure 14. OmpA-MBP expression with and without IPTG induction in the bacterial lysate.
Figure 15. OmpA-MBP expression with and without IPTG induction in the culture medium concentrated by TCA.
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. Further, 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 16. NSP4/PhoA/OmpA-CaAP expression with and without IPTG induction in the bacterial lysate.
Figure 17. NSP4/PhoA/OmpA-CaAP expression expression with and without IPTG induction in the culture medium concentrated by trichloroacetic acid (TCA) precipitation.
Please note that our modeling results are at /Team:Fudan/Model, and our online survey results are at /Team:Fudan/Human_Practices.