Team:Shanghai Metro/Contribution

Shanghai_Metro

Parts Contribution

BBa_K4028000

== Profile ==

Name: tke2
Base Pairs: 4158 bp
Origin: Pseudomonas putida KT2440, genome
Properties: Anti-bacterial effector in type VI secretion system

== Usage and Biology ==

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]

Figure1. Principle diagram of T6SS.

BBa_K4028001

== 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]

BBa_K4028005

== Profile ==

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.
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.

BBa_K4028006

== 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).

Figure 2. Immune protein expression box.

Figure 3. Schematic map of immune expression plasmids.

BBa_K4028007

== 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]

== 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.

BBa_K4028008

== Profile ==

Name: lac pro-tke2-tet pro-ike2
Base Pairs: 4746 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 ==

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.

Figure2. The gene design of TetR promoter-ike2-lac promoter-tke2.

Figure 3. Schematic map of T6SS effector and immune expression plasmids.

BBa_K4028009

== Profile ==

Name: lac pro-tke4-tet pro-ike4
Base Pairs: 5208 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 ==

Tke4 is a key functional factor that inhibits bacterial growth which is controlled by a lac promoter (Figure 2). The immunity effector ike4 is under tet promoter. The anti-bacterial effector tke4 and the immunity effector ike4 are inserted in the pUS232 vector (Figure 3).

Figure 2. T6SS effector and immune protein expression box.

Figure 3. Schematic map of T6SS effector and immune expression plasmids.