BBa_K4028000 is a coding sequence of tke2 from Pseudomonas putida KT2440. Tke2,
known as a toxic Rhs-type
effector, is an effect factor in type VI secretion system (T6SS).
A wide range of Gram-negative bacteria have been shown to have antibacterial T6SSs, including opportunistic
pathogens such as Pseudomonas aeruginosa,[4] obligate commensal species that inhabit the human gut
(Bacteroides spp.),[5] and plant-associated bacteria such as Agrobacterium tumefaciens.[6] Under natural
conditions, bacterial cells encoding T6SS transport effect factors with cytotoxic or antibacterial effects
(amidase, glycoside hydrolyase, lipase, etc.) to recipient cells through physical contact, thus inhibiting
the growth of recipient cells. The bacteria encoding T6SS can translate and produce corresponding immune
proteins to counteract the damage caused by toxic effector factors.[1,2,3]
Team:Shanghai Metro/Contribution
Parts Contribution
==Contribution==
Figure1. Principle diagram of T6SS.
==Reference==
1. Bingle, l.E.H. et al. (2008). Type VI secretion: a beginner’s guide. Current
opinion in microbiology. 11:3-8.
2. Silverman, J. M. et al. (2012). Structure and regulation of the type VI
secretion system. 66:453-472.
3. Hernandez, R. E. et al. (2020). Type VI secretion system effector protein:
Effective weapons for bacterial competitiveness. Cellular microbiology. 22:e13241.
4. Hood RD, . et al (January 2010). "A type VI secretion system ofPseudomonas
aeruginosa targets a toxin to bacteria". Cell Host & Microbe. 7 (1): 25–37. doi:10.1016/j.chom.2009.12.007.
PMC 2831478. PMID 20114026.
5. Russell AB, . et al (August 2014). "A type VI secretion-related pathway in
Bacteroidetes mediates interbacterial antagonism". Cell Host & Microbe. 16 (2): 227–236.
doi:10.1016/j.chom.2014.07.007. PMC 4136423. PMID 25070807.
6. Ma LS, . et al (July 2014). "Agrobacterium tumefaciens deploys a superfamily of
type VI secretion DNase effectors as weapons for interbacterial competition in planta". Cell Host & Microbe.
16 (1): 94–104. doi:10.1016/j.chom.2014.06.002. PMC 4096383. PMID 24981331.
This is the naturally occurring version of the TetR class B promoter. It can be
induced by tetracycline to express downstream proteins.
==Contribution==
We set different tetracycline concentrations to test the protein expression induced
by TetR(B) promoter. In our design, the immune protein (ike4) is regulated by TetR(B) promoter. The
expression of the immune protein (ike4) directly affects and is positively correlated with strain growth.
Therefore, the strain growth can indicate the regulation of the TetR(B) promoter.
The results of the test are below.
Table 1. OD600 value of the strain D: Pus232-ike4-tke4 after 6 hours
Figure 1. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline
The results show that concentrations at the range of 300-500µg/L are more
effective. The results also proved that adding tetracycline to strain will boost the growth of strain. It
determines that the optimal concentration of tetracycline is 300µg/L to 500µg/L.
==Contribution==
With this idea coming from the T6SS, we designed to express the effector components
of T6SS in industrial strains by inserting the gene segment tke2-ike2 to induce the controlled expression of
the corresponding immune proteins. The normal growth of industrial microorganisms can be ensured only by
adding certain components (tetracycline) in the fermentation broth, so as to achieve the purpose of the
experiment.
Figure1. The gene design of TetR promoter-ike2-lac promoter-tke2.