1. Measurement of signal peptide (kp-sp) efficiency and β-galactosidase (lacZ) enzyme activity
Kp-sp is a signal peptide of Actinomyces origin that promotes protein secretion to the extracellular [1]. We constructed a plasmid with kp-sp added at the N-terminal end of lacZ (BBa_I732005), BBa_K4183003 (Figure 1), and transformed it into the EcN.
Figure1. BBa_K4183002 and BBa_K4183003 schematic
We coated the engineered EcN bacteria containing BBa_K4183003 on LB agar plates containing IPTG as well as X-Gal and incubated them overnight at 37°C. The results showed that the breakdown of X-Gal could be clearly observed around the monoclonal colonies (Figure 2), which proved that lacZ was secreted extracellularly.
Figure2. Micrographs of X-Gal disassembled on LB agar plates containing X-Gal and IPTG
(A) When the N-terminal end of lacZ did not contain kp-sp, no disassembly of X-Gal was clearly observed around the monoclonal colonies.
(B) When the N-terminal end of lacZ contains kp-sp, X-Gal is clearly observed to be disassembled around the monoclonal colony, showing a blue color.
To further measure the efficiency of kp-sp transport, we measured the efficiency of lacZ in the bacteriophage and supernatant using the β-galactosidase (β-GAL) activity assay kit (Solarbio, BC2580). The results showed (Figure 3) that the enzyme activity of lacZ in the supernatant accounted for 21.93% of the overall enzyme activity, which also proved that the efficiency of kp-sp transport was about 21.93%.
Figure3. Determination of enzymatic activity of lacZ in bacteriophage and supernatant
(A) Using the standards in the kit, the standard curve was measured with R2 = 0.9997.
(B) The enzyme activity of lacZ in the bacterium was 31048.32061 and 8723.369848 in the supernatant, as measured using the kit and visible light spectrophotometer.
2. Validation of CAP expression and acid resistance testing
CAP is an EcN-derived non-toxic bacterial capsular polysaccharide that has been used in bacterial therapy for tumors in vivo. [2] It protects the bacterium from external environmental disturbances, such as extreme pH, enzymes, and other microorganisms. We hope to overexpress CAP on the surface of EcN to protect our engineered bacteria from being harmed by gastric acid. We designed it to survive in the complex flora of the intestine without affecting the original microenvironment of the intestine.
The synthesis of CAP requires the participation of various enzymes, such as kfiABCD, kpsCFSU and etc. Among them, kfiC is one of the key enzymes [2], and if the kfiC gene is overexpressed in the bacterium, CAP can be synthesized in large quantities. Therefore, we first constructed a kfiC expression system controlled by the lactose promoter, BBa_K4183004 (Figure 4).
Figure4. BBa_K4183004 schematic
We transferred the BBa_K4183004 plasmid into EcN and cultured it overnight in LB medium supplemented with IPTG. The CAP was subsequently purified by chloroform-phenol extraction, electrophoresed in SDS-PAGE (4%-20%), and stained using alcian solution. The results showed that an increased amount of CAP could be easily observed in the engineered bacteria overexpressing kfiC compared to the negative control (Figure 5).
Figure5. SDS-PAGE verified the expression of CAP.
Since CAP improves bacterial tolerance to extreme PH environments in the stomach, we tested the engineered bacteria in different acidic environments.
The results showed (Figure 6) that the engineered bacteria overexpressing CAP could grow slowly in PH = 4 compared to the negative control. We also found that it could survive in the environment with PH ≤ 3 for about 30 min. After a person eats, the food will stay in the stomach for 1-1.5 h. This indicates that the engineered bacteria overexpressing CAP have some acid tolerance, but they cannot fully survive in the stomach acid environment.
Figure6. The growth of bacteria in different PH environments, (A) is the negative control group and (B) is the experimental group.
3. Engineering bacteria for lactose degradation
3.1 The first generation of engineered bacteria-lacMAN1.0
From the above experimental data, we obtained the engineered bacteria that can secrete lacZ into the extracellular and have some acid resistance. Therefore, we planned to combine the two (Figure 7), and thus developed and tested the first generation of engineered bacterium-lacMAN1.0 for lactos degradation.
Figure7. BBa_K4183006 schematic，lacMAN1.0
The results showed that the lacZ secretion capacity and acid tolerance of lacMan1.0 were reduced to different degrees (Figure 8). Only 13.33% of lacZ was secreted into the extracellular compartment, lower than the previous 21.93%; its growth remained slow at pH=4 but it barely survived at pH≤3. We think that this is caused by BBa_R0010 exhibiting insufficient promoter strength for an overly long expression frame.
In addition, the induction of IPTG is extremely difficult in the intestine. Therefore, we planned to replace BBa_R0010 with the current promoter BBa_J23119, which is more strongly expressed, and deleted the original lacI element on the pET28a plasmid to reduce the replication pressure of bacteria.
Figure8. Measurement of enzyme activity and acid resistance of LacMAN 1.0
(A) The enzymatic activity of lacZ was determined in lacMAN1.0 in the bacteriophage as well as in the supernatant using the kit.
(B) To determine the growth of lacMAN1.0 in acidic environment with different pH.
3.2 Second generation engineered bacteria-lacMAN2.0
To improve the transcription rate of the expression frame and to reduce the use of IPTG in humans, we replaced BBa_R0010 with BBa_J23119 (Figure 9) and deleted the self-contained lacI element on pET28a, constructed a new complex element, BBa_K4183009, and transferred it into EcN, thus developing the second generation engineered bacterium lacMAN2.0.
Figure9. BBa_K4183009 schematic, lacMAN1.0
We then performed the same experiment on it. The results showed that the lacZ secretion efficiency of lacMAN2.0 was somewhat improved to 14.49% relative to lacMAN1.0, but still lower than the initial 21.93%. In terms of acid tolerance, lacMAN2.0 could grow normally in the first 30 min in an acidic environment with pH ≤ 4, consistent with that in a normal environment.
Figure10. Measurement of enzyme activity and acid resistance of LacMAN 2.0
(A) Determination of enzymatic activity of lacZ in lacMAN2.0 in the bacteriophage as well as in the supernatant using the kit.
(B) Determination of the growth of lacMAN2.0 in acidic environments with different pH.
In summary, during these months, we have developed an engineered bacterium, lacMAN2.0, that can overexpress CAP and secrete lacZ extracellularly, which is acid-tolerant and can degrade lactose extracellularly, making it possible to degrade lactose in the intestine without worrying about lactose uptake and gas production by E. coli, in line with the normal lactose degradation process in humans. However, we still have some problems in the experiment.
1) the secretion efficiency of kp-sp is still low, which requires us to find more efficient signal peptides to improve the secretion efficiency of lacZ.
2) the acid resistance of CAP is still insufficient for gastric acid, which needs further improvement and testing
3) Other properties of CAP, such as the nature of colonization in the intestine and protection from other microorganisms, were not performed due to time and experimental conditions, and we only have relevant literature to prove it.
1 Cui, Y. et al. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide. Protein Expression and Purification 129, 69-74, doi:https://doi.org/10.1016/j.pep.2016.09.011 (2017).
2 Harimoto, T. et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nature Biotechnology 40, 1259-1269, doi:10.1038/s41587-022-01244-y (2022).