Difference between revisions of "Part:BBa K4028007"

 
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<partinfo>BBa_K4028007 short</partinfo>
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<partinfo>BBa_K4028005 short</partinfo>
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<partinfo>BBa_K4028005 SequenceAndFeatures</partinfo>
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== Profile ==
 +
 
 +
Name: tet pro-ike4
 +
 
 +
Base Pairs: 560 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]
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_1.png|700px|thumb|center|Figure1. Principle diagram of T6SS.]]
 +
 
 +
== Construct design ==
 +
 
 +
The immunity effector ike4 is under tet promoter(Figure 2). This composite part is inserted in the pUS232 vector (Figure 3).
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_2.png|700px|thumb|center|Figure 2. Immune protein expression box.]]
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_3.png|700px|thumb|center|Figure 3. Schematic map of immune expression plasmids.]]
 +
 
 +
The profiles of every basic part are as follows:
 +
 
 +
=== <partinfo>BBa_K4028003</partinfo> ===
 +
 
 +
Name: ike4
 +
 
 +
Base Pairs: 504bp
 +
 
 +
Origin: Pseudomonas putida KT2440, genome
 +
 
 +
Properties: Immunity effector in type VI secretion system
 +
 
 +
==== Usage and Biology ====
 +
 
 +
BBa_K4028003 is a coding sequence of ike4, an immunity protein in Pseudomonas putida KT2440. Ike4 is used for protecting bacteria from type VI secretion system (T6SS).
 +
 
 +
=== <partinfo>BBa_K4028005</partinfo> ===
 +
 
 +
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 ===
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_4.jpg|700px|thumb|center|Figure 4. Gel electrophoresis of plasmids Pus232.]]
 +
 
 +
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 ===
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_14.jpg|700px|thumb|center|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.
 +
 
 +
=== 3. Pus232 Double Enzyme Digestion ===
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_5.jpg|700px|thumb|center|Figure 6. Gel electrophoresis of Pus232 double enzyme digestion products.]]
 +
 
 +
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-ike4 sequencing analysis ===
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_6.jpg|700px|thumb|center|Figure 7. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of Pus232-ike4.]]
 +
 
 +
The sequencing results show that Pus232-ike4 is constructed successfully.
 +
 
 +
=== 5. Electrophoresis of Pus232-ike2/4 Enzyme Digestion ===
 +
 
 +
[[File:T--Shanghai_Metro--BBa_K4028007-figure_7.png|700px|thumb|center|Figure 8. Gel electrophoresis of Pus232-ike2, Pus232-ike4 enzyme digestion products.]]
 +
 
 +
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.
 +
 
 +
== References ==
 +
 
 +
1. Bingle, l.E.H. et al. (2008). Type VI secretion: a beginner’s guide. Current opinion in microbiology. 11:3-8.
  
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tet pro-ike4
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2. Silverman, J. M. et al. (2012). Structure and regulation of the type VI secretion system. 66:453-472.
  
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<!-- Add more about the biology of this part here
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3. Hernandez, R. E. et al. (2020). Type VI secretion system effector protein: Effective weapons for bacterial competitiveness. Cellular microbiology. 22:e13241.
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===Usage and Biology===
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<!-- -->
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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.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4028007 SequenceAndFeatures</partinfo>
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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.
  
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<!-- Uncomment this to enable Functional Parameter display
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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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===Functional Parameters===
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<partinfo>BBa_K4028007 parameters</partinfo>
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<!-- -->
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Revision as of 00:41, 19 October 2021

tet ptomoter


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: tet pro-ike4

Base Pairs: 560 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]

Figure1. Principle diagram of T6SS.

Construct design

The immunity effector ike4 is under tet promoter(Figure 2). This composite part is inserted in the pUS232 vector (Figure 3).

Figure 2. Immune protein expression box.
Figure 3. Schematic map of immune expression plasmids.

The profiles of every basic part are as follows:

BBa_K4028003

Name: ike4

Base Pairs: 504bp

Origin: Pseudomonas putida KT2440, genome

Properties: Immunity effector in type VI secretion system

Usage and Biology

BBa_K4028003 is a coding sequence of ike4, an immunity protein in Pseudomonas putida KT2440. Ike4 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

Figure 4. Gel electrophoresis of plasmids Pus232.

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

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.

3. Pus232 Double Enzyme Digestion

Figure 6. Gel electrophoresis of Pus232 double enzyme digestion products.

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-ike4 sequencing analysis

File:T--Shanghai Metro--BBa K4028007-figure 6.jpg
Figure 7. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of Pus232-ike4.

The sequencing results show that Pus232-ike4 is constructed successfully.

5. Electrophoresis of Pus232-ike2/4 Enzyme Digestion

File:T--Shanghai Metro--BBa K4028007-figure 7.png
Figure 8. Gel electrophoresis of Pus232-ike2, Pus232-ike4 enzyme digestion products.

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.

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.