Difference between revisions of "Part:BBa K1497008"

Latest revision as of 21:36, 17 October 2014

hokD (relF)

The hokD (formerly known as relF) gene of E. coli transcribes for a small polypeptide that causes cell death by elimination of vital cell wall functions. The hokD gene is located in the E. coli chromosome as part of the relB operon. It’s gene product shows 40% homology to the polypeptide encoded by the hok (host killing) gene and induces the same characteristic cell deaths when over expressed (Gerdes, Bech et al. 1986). It has been shown that the relF gene can be used to design efficient suicide functions to contain bacterial growth (Knudsen and Karlstrom 1991, Knudsen, Saadbye et al. 1995).

Functional Parameters

In order to investigate the killing ability of hokD, we put the gene under control of an arabinose-inducable promoter (BBa_K808000), which can be controlled tightly if glucose is added to the medium.

Culture grown in media containing glucose showed a simmilar amount of colony forming units (CFU) as the control culture without the hokD gene but the culture cultivated with arabinose showed a 1000-fold reduction in CFU per ml culture (Fig. 1).

Addtionally to the spread-plate assay a 10⁵-fold dilution of each culture was plated on LB-CMP-agar plates containing either 2 g/l glucose or 0.3 g/l arabinose and incubated for 24 hours at 37°C in order to investigate the ongoing proliferation of the bacteria with glucose and arabinose.The amount of colonies formed by the control cultures were identical for both types of plates (136 CFU with glucose, 132 CFU with arabinose). The culture containing the hokD-gene cultivated in LB-media with glucose showed simmilar amounts of colonies on the plate containing glucose as the control culture (134 CFU) but a 2-fold reduction in CFUs on the plates containing arabinose (63 CFUs). The culture containing the hokD-gene which already had reduced cell-growth because it was cultivated in LB-media containing arabinose only formed 3 colonies on the plate containing glucose and no colonies on the plate with arabinose (Fig. 2).

Additional experiments with different sets of promoters should be performed to further characterize the toxicity of this BioBrick.

Figure 1 Calculated CFU per ml culture for three cultures. Control (empty plasmid), pSB1C3-araC-pBAD-hokD with 2 g/l glucose and pSB1C3-araC-pBAD-hokD with 0.3 g/l arabinose.

Figure 2 Total counted number of colonies fore three different cultures on plates containing glucose or arabinose. Cultures are Control (empty plasmid), pSB1C3-araC-pBAD-hokD with 2 g/l glucose and pSB1C3-araC-pBAD-hokD with 0.3 g/l arabinose.

References

Gerdes, K., F. W. Bech, S. T. Jorgensen, A. Lobner-Olesen, P. B. Rasmussen, T. Atlung, L. Boe, O. Karlstrom, S. Molin and K. von Meyenburg (1986). "Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon." Embo j 5(8): 2023-2029.

Knudsen, S. M. and O. H. Karlstrom (1991). "Development of efficient suicide mechanisms for biological containment of bacteria." Appl Environ Microbiol 57(1): 85-92.

Knudsen, S., P. Saadbye, L. H. Hansen, A. Collier, B. L. Jacobsen, J. Schlundt and O. H. Karlstrom (1995). "Development and testing of improved suicide functions for biological containment of bacteria." Appl Environ Microbiol 61(3): 985-991.

Sequence and Features