Part:BBa_K2387066
Colicin E7 lytic mechanism controlled by inducible araC/pBAD promoter
Colicin E7 gene encodes for a lysis protein. Colicin E7 is a 4872Da protein that has two main functions,the detachment of ColE7 from the Immunity protein, therefore resulting in the switch on of the endonuclease activity of ColE7 and the induction of the lysis of host cells, leading to the exposure of the interior cell contents and allowing then the free diffusion of ColE7 proteins. Colicin E7 gene is expressed under the pBAD promoter which is an E.coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [1]. However, when arabinose is addedd to the medium, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [2]. pBAD promoter allows then for the control of the lysis process to take place.
An SOS response operon regulates the expression of ColE7. This SOS response operon is formed by three genes (ceaE7, ceiE7 and celE7) whose products are ColE7, ImE7 and LysE7, respectively [1]. ColE7 can be inhibited by complexing with the immunity protein ImE7. The inducible COLE7 system (BBa_K2387066) codes for an L-arabinose inducing lysis gene (BBa_K117000) encodes for the lysis protein of 4872 Da in molecular weight in those colicin-producing strains of bacteria. Once this gene becomes activated, two main functions of Colicin E7 system are covered by the lysis protein. The activation of the gene, leads to the partial lysis of the host cell membrane. This first function leads then to the lysis of host cell, uncovering the intracellular environment of the cell and thus allowing for ColE7 proteins free diffusion. Moreover, the lysis gene also removes the immunity protein from colicin protein, resulting in the activation of the endonuclease activity of colicin protein. In this case, the detachment of ColE7 from Immunity protein is triggered, activating this the ColE7 endonuclease activity.
The BBa_K11700 codes for the lysis gene but not for the ColE7 or ImE7 proteins suggesting this that a specific E. coli strain (able to synthesize colicin) might be required. However, it was hypothesized that the only expression of E7 lysis gene (BBa_K117000) by E.coli DH5α , will cause partially lysis. The results indicated that E7 lysis protein might also have function in noncolicin-secreted strain of E.coli as the lysis was fully achieved after inducing the system (Figure 1) with L-arabinose (BBa_K2387066). Future approaches could study the differences in the lytic effect by comparing the lysis achieved when inserting the pK317 plasmid which contains the entire cole7 system and the inducible COLE7 system (BBa_K2387066).
Four different lytic mechanisms (Colicin E7 system, T4 Endolysin, T4 Holin and T4 Endolysin + T4 Holin cassette) were assessed in two backbones (pSB1C3 and pSB4K5). All the mechanisms are expressed under the pBAD promoter (Figure 2) and therefore all the mechanisms were induced at different arabinose concentrations. The figure (Figure 1) depicts the OD after 20 hours of arabinose induction. Based on the results, it was concluded that already low inductions of L-arabinose are able to induce a complete lysis of the bacterial cells (Figure 1).
References
[1] K.-F. Chak, W.-S. Kuo, f. –. Lu, and R. James, “Cloning and characterization of the ColE7 plasmid,” J. Gen. Microbiol., vol. 137, no. 1, pp. 91–100, Jan. 1991.
[2]Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.
[3]Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.
Sequence and Features| None |