Part:BBa_K4028001

ike2

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: ike2

Base Pairs: 447bp

Origin: Pseudomonas putida KT2440, genome

Properties: Immunity effector in type VI secretion system

Usage and Biology

BBa_K4028001 is a coding sequence of ike2, an immunity protein in Pseudomonas putida KT2440. Ike2 is used for protecting bacteria from 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. Meanwhile, the bacteria encoding T6SS can translate and produce corresponding immune protein (ike2) to counteract the damage caused by toxic effector factors.[1,2,3]

Figure1. Principle diagram of T6SS.

Experimental approach

1. ike2/4 Electrophoresis

Figure 5. Gel electrophoresis of ike2 and ike4 PCR products.

Channel 1: ike2 electrophoresis failed maybe because bacteria added is too much.

Channel 2: ike2 succeeded

Channel 3: ike2 electrophoresis failed maybe because bacteria added is too much.

Channel 4: ike4 succeeded

Channel 5: ike4 succeeded

Channel 6: ike4 succeeded

Channel 7: ike4 succeeded

This step is used to check if the ike2 and ike4 extracted from Pseudomonas putidas are successful and could be used to do double enzyme digestion later in the process.

PCR clean-up ike2 and ike4 DNA fragments to do double enzyme digestion of BamHI and XbaI overnight.

Channel 1 and 3 failed the test. One possible explanation could be that the bacteria added into the PCR solution is too much.

2. Pus232-ike2 Transformation Plates

Control group E.coli DH5α/Pus232 and E.coli DH5α/Pus232-ike2 show blue strains, which are desired results because without the tke presence, lacZ protein in Pus232 won’t be replaced, and X-Gal we added under the catalysis of lacZ protein, blue products will be produced, thus the strain appearing blue(Fig. 10 left and middle). However, the white single strain may be microbial contamination (Fig.10 left).

Figure 9. Transformation plates of recombination reaction.

Experimental group Pus232-ike4-tke4 had a white single strain when the picture was taken (Fig. 10 right). The result could be satisfactory because tke4 replaced lacZ protein in the plasmid, stopping it from expressing thus the strain won’t demonstrate any blue color.

References

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.