Part:BBa_K4028006
tet pro-ike2
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name: tet pro-ike2
Base Pairs: 533 bp
Origin: Escherichia coli, Pseudomonas putida KT2440, synthetic
Properties: Gene technology for protecting patented bacterial strains
Usage and Biology
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 to counteract the damage caused by toxic effector factors.[1,2,3]
Construct design
The immunity effector ike2 is under tet promoter. The composite part is inserted in the pUS232 vector (Figure 3).
The profiles of every basic part are as follows:
BBa_K4028001
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).
BBa_K4028005
Name: tet promoter
Base Pairs: 56bp
Origin: Escherichia coli, synthetic
Properties: Inducible promoter regulated by tetracycline
Usage and Biology
This is the naturally-occuring version of the TetR class B promoter. Note that this will promote bidirectionally. (pR1 and pR2 will promote in the forward direction; pA will promote in the reverse direction.) The three transcription start sites are not labeled, but can be found from the reference.
Experimental approach
1. Pus232 Electrophoresis
Channel 1~14: Purified plasmid Pus232 from 1~14 E. coli DH5α cultures.
This step is used to test if the plasmids Pus232 extracted from the E. coli DH5α are successful and could be used to do double enzyme digestion later in the process.
Based on the three types of structures plasmid may have, we collect the supercoiled bands. Channel 2, 5, 6, and 7 are the best bands and are selected for making double enzyme digestion. Channel 3, which has a high concentration of plasmid, was once selected but failed.Therefore, channel 2, 5, 6, and 7 plasmids are selected to do double enzyme digestion of BamHI and XbaI for 2 hours.
Channel 3 might have undergone too long of a P2 cracking phase, which leads to the fracture within the DNA. The DNA secreted contains groups of open circular DNA , showing as the bright white color in channel 3, but less circular plasmid DNA that we are targeting to collect.
2. ike2/4 Electrophoresis
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.
3. Pus232 Double Enzyme Digestion
Channel 1&2: Pus232 control group
Channel 3~6: Products of Pus232-BamHI+XbaI Double Enzyme Digestion
Channel 7~10: Products of Pus232-BamHI+XbaI Double Enzyme Digestion
Channel 3~10 have shorter bands after the double enzyme digestion, and all of them are successful. Gel clean-up the product of Pus232-BamHI+XbaI double enzyme digestion to obtain Pus232-backbone. Clean-up the product of ike2 and ike4-BamHI+XbaI overnight double enzyme digestion to obtain ike2-fragment and ike4-fragment.
T4 DNA ligase is used to connect Pus232-backbone with ike2-fragment and ike4-fragment overnight separately.
4. Pus232-ike2 sequencing analysis
The sequencing results show that Pus232-ike2 is constructed successfully.
5. Electrophoresis of Pus232-ike2/4 Enzyme Digestion
Channel 1~4: Products of Pus232-ike2-SacII single enzyme digestion, succeed, cut the target bands for gel clean-up.
Channel 5~8: Products of Pus232-ike4-SacII single enzyme digestion, succeed, cut the target bands for gel clean-up.
Channel 9~12: Products of tke4 PCR, contains some unneeded bands, cut the 4k+ bands for gel clean-up.
6. 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).
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.
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