In vitro Validation of Effector Genes
To confirm the functionality of the synthetic effector genes within engineered bacteria, we conducted in vitro functional assays on individual effector gene-engineered strains.
IsmA Verification
To provide a more intuitive assessment of IsmA's efficacy, we first activated the IsmA single-gene strain in LB medium. Subsequently, we transferred the activated strain to a cholesterol-enriched egg yolk medium for short-term cultivation. We then quantified the cholesterol content in the culture medium, as illustrated in Figure 1. In comparison to the control group, the IsmA single-gene engineered bacteria displayed a remarkably significant cholesterol-reducing capability. This robustly demonstrates IsmA's cholesterol degradation potential."
We further utilized a gradient egg yolk medium to emphasize the exceptional cholesterol degradation capabilities of our IsmA engineered bacteria, as depicted in Figure 2. The CK group represented the 2% egg yolk medium without bacterial inoculation. Due to the remarkable cholesterol degradation capabilities of the IsmA gene, a significant difference in cholesterol levels from the CK group only became evident in the 8% egg yolk medium group (excluding the 2% group). This robustly underscores the favorable expression effect of the IsmA gene.
BSH Verification
For the BSH gene-engineered bacteria, we employed the bile salt MRS qualitative solid medium to assess the activity of the BSH gene. As shown in Figure 3, after 24 hours of cultivation, the BSH gene strain exhibited a pronounced precipitate ring, indicating the evident expression and bile salt hydrolysis activity of the BSH gene.
BCoAT Verification
For the BCoAT gene-engineered bacteria, we employed GC analysis to measure the production of short-chain fatty acids, as illustrated in Figure 4. The acetic, propionic, and butyric acid contents in the BCoAT gene samples were significantly higher than those in the two control groups. Butyric acid showed significant differences compared to both control groups, propionic acid displayed significance compared to the empty vector group, and acetic acid exhibited significance compared to the EcN group.
Through in vitro efficacy validation of single-gene strains, we have demonstrated the proper functionality of the selected genes in an extracellular environment.
Functional Verification of Engineered Strains
By concatenating effector genes (IsmA, BSH, BCoAT) in different configurations, we have generated engineered strains capable of performing a diverse range of functions. The triad gene combination (IsmA-BSH-BCoAT), achieved through iterative engineering, serves as a well-rounded engineered strain capable of simultaneously carrying out three distinct functions. However, the concurrent expression of these three functions may potentially weaken a specific function compared to other concatenated gene combinations. To validate the functionality of the engineered strains, we conducted IsmA, BSH, and BCoAT verifications and compared the strain's capabilities.
In terms of cholesterol degradation testing, we cultured the engineered strains in a basal cholesterol medium and employed the OPA method to measure the remaining cholesterol content in the culture medium, as shown in Figure 5. The results indicate that the triad gene combination exhibited the highest cholesterol degradation capability, with a degradation rate of approximately 30%, showing remarkable significance compared to the control group.。
In the BSH gene assessment, as depicted in Figure 6, the triad gene combination exhibited a significant precipitate ring similar to that of the single BSH gene single strain.
Considering that the host, E. coli, originates from an anaerobic intestinal environment, we also conducted anaerobic cultivation in the BSH verification, as illustrated in Figure 7. It is evident that the triad combination displayed a more pronounced precipitate ring under these conditions.
Furthermore, to confirm the protein activity of the engineered strains, we extracted BSH crude enzyme solutions and measured enzyme activity using the 3-ketosteroid assay, determining the levels of bound bile salt hydrolysis amino acids, as depicted in Figure 8. Although the enzyme activity did not exhibit statistical significance, it is notable that the triad combination displayed slightly higher enzyme activity compared to other gene concatenations. This finding reaffirms the expression of BSH within the triad combination.
On this basis, we measured the protein content and calculated unit enzyme activity based on the protein content, as shown in Figure 9. It is noteworthy that the triad combination exhibited a slightly lower unit enzyme activity, but overall, the difference is not substantial.
In the BCoAT gene assessment, as depicted in Figure 10, the triad gene combination exhibited notably lower capability in producing short-chain fatty acids compared to the BCoAT single-gene strain. This suggests that in further research, we must delve deeper into the influence of short-chain fatty acids on the ultimate outcome. Alternatively, the pursuit of improved results may involve experimenting with a mixed culture of BCoAT single strains and the triad combination
SDS-Page Electrophoresis
To further validate the expression of BSH and IsmA proteins in the engineered strains, we conducted SDS-Page electrophoresis experiments to separate and identify the proteins. As shown in Figure 11, both BSH and IsmA proteins are secreted in the concatenated gene-engineered strains.
UHPLC Analysis of Cholesterol and Its Degradation Products
To further validate the role played by the IsmA gene, we supplemented the crude enzyme extract from the IsmA gene-engineered bacteria into a cholesterol culture medium for reaction. After 24 hours of reaction, we employed UHPLC technology to analyze the cholesterol culture medium for cholesterol and its downstream products. As depicted in Figure 12, the UHPLC results indeed detected downstream products, confirming the functional role of the IsmA gene.
Oleic Acid Inducer Efficacy Verification
To confirm the efficacy of the oleic acid inducer, we fused the mRFP gene downstream of the oleic acid inducer gene to construct a reporter gene. We cultured the engineered strains in different oleic acid induction conditions under both anaerobic and aerobic environments. The inducer's expression effect was assessed by normalizing the fluorescence signal intensity. As illustrated in Figure 13, the oleic acid inducer exhibited a favorable response to gradient oleic acid, particularly evident under anaerobic conditions, with a notable difference starting at the 10% oleic acid gradient compared to the previous gradient.
In comparison between anaerobic and aerobic conditions, as depicted in Figure 14, there is a slight difference in the expression level of the oleic acid inducer gene, but it does not exhibit statistical significance.
Oxygen Suicide Switch Verification
To ensure the safety of the engineered strains, we collaborated with Beijing University of Chemical Technology on an oxygen suicide switch project. For more details about their project, please refer to their wiki.
We employed the Gibson assembly method to assemble five genes and successfully obtained the constructed plasmid. Gel verification, as illustrated in the following figure, indicates a length of approximately 4.7kb, consistent with the expected plasmid length.
In the case of Nissle 1917 with the successfully introduced plasmid, cultivation took place inside a glove box. After extensive experimentation to optimize reaction conditions, it was observed that Nissle 1917 exhibited significantly slow growth in an oxygen-depleted environment, necessitating a growth period of 48-72 hours to discern distinct colonies. However, when cultivated in aerobic conditions, there were no discernible signs of engineered bacterial growth, making it feasible to confirm the functionality of the suicide system.
