Strain construction Gene deletion in Cronobacter malonaticus 3267 was done using the lambda red recombinase system protocol with minor modifications. The ΔfimE mutant overexpressed type 1 fimbriae leading to the formation of microcolonies. Target cells within these microcolonies were protected from contact-dependent killing mediated by the type 6 secretion system (T6SS), leading to their survival. This defense mechanism could represent a nonspecific resistance mechanism, unlike immunity proteins, which could potentially lead to resistance against a broad range of assailants.
So, we wanted to make EcN have this function as well, and we knocked out the FimE fragment in EcN. We chose the ADP1 strain, which has a more aggressive type VI secretion system, and demonstrated this function of FimE on EcN by bacterial antagonism experiments with ΔfliC EcN and ADP1.
· knockout using homologous recombination method
(Nissle1917) using ultra fidelity DNA polymerase, and the Kn resistance gene was amplified from the pKD4 plasmid. After fusion PCR ligation, the complete targeting fragment Δ fimE:: Kn was obtained and cloned into the suicide plasmid pCVD442 to obtain the targeting plasmid; Then, the target plasmid was transformed into E. coli β 2155 using electroporation to obtain a donor bacterium. After conjugation with the recipient bacterium, clones of the genome integrated target plasmid were screened on kanamycin plates; Finally, these cloned bacterial solutions were spread on LB agar plates containing 10% sucrose and cultured until they became monoclonal. Then, PCR technology was used to screen for clones in which the fimE gene was replaced by the Kn resistance gene.
Electropherogram: M: DNA molecular weight standard. From top to bottom the molecular weights are: 1200, 900, 700, 500, 300, 100bp, with 700bp brightened. 1-14: Internal primer amplification results for clones 1- 14; 15: Internal primer amplification result of the original strain, product length: 268bp; 16: Negative control amplification results.
·Bacterial antagonism experiment verifies the defense ability of knockout strains
We used the T6SS system to attack the highly effective and safe bacterium Acinetobacter baumannii (ADP1) as our target bacterium, and the FimE knockout strain EcN (BBa_K5245248021 FimE Knockout) as the prey bacterium. The two strains were mixed and cultured, and it was finally verified that the strain with fimE knockout had a higher survival rate. The Δ fimE mutant could lead to the formation of microcolonies, and the target cells in the microcolonies were protected from contact dependent killing mediated by T6SS, resulting in the survival of the engineered bacteria.
