Team:Washington/Gram Negative/Design


Designing the Transfer of the Type Six Secretion System into E. coli

Using a Fosmid to Transfer the T6SS Genes into E. coli

The T6SS is comprised of 23 genes across several operons. Capturing and moving these genes via standard restriction digest cloning was determined to be impractical. We discovered that the sequence of the P. aeruginosa strain (PAO1) we were using was solved using fosmids (essentially large plasmids). We were able to locate these fosmids and were very excited to find that one of the fosmids contained all of the necessary T6SS genes organized nicely in two divergent operons. We successfully transferred the fosmid into E. coli. It was not clear, however, whether or not the genes would be expressed in E. coli, since the promoters that controlled the expression of the T6SS genes on the fosmid were from P. aeruginosa. In order to verify expression of the T6SS genes we performed a Western blot using antibodies against Fha1, a critical protein of the secretion system and a reporter for T6SS activity.

Obtained Fosmid Contains T6SS Genes Organized into Two Divergent Operons

Testing the Native Pseudomonas aeruginosa Promoter-regulated Type VI Secretion System in E. coli

To determine whether the T6SS genes from the fosmid were being expressed from the native P. aeruginosa promoter, the fosmid was tranferred into E. coli, and a Western blot was performed on cell extracts for Fha1, one of the proteins critical for T6SS activity. As expected, the Western blot showed a band for Fha1 in the P. aeruginosa cell extract sample, but no bands were seen in the E. coli cell extract samples (shown below), indicating that E. coli was probably not transcribing the T6SS genes from the native P. aeruginosa promoter.

Expression of T6SS Components from the Fosmid Occurs in P. aeruginosa but Not in E. coli

Strategy to Improve Type VI Secretion System Expression in E. coli

To allow expression of these genes in E. coli, we designed a promoter system that would allow the same E. coli - recognized promoter to drive expression of both divergent operons. This promoter system would replace the native P. aeruginosa promoter system. We chose a bidirectional T7 promoter, which would promote transcription in both the 5' and 3' direction. T7 promoters are well characterized, and known to be highly robust for expression in E. coli.

Designing Regulation of the Toxin/ Antitoxin System

Separating the Regulation of the Toxin/Antitoxin from the Type VI Secretion System

We chose to separate the regulation of the toxin antitoxin system (Tse2.Tsi2) from the regulation of the T6SS, since the T6SS has too many proteins to be easily induced. The purpose of generating novel Tse2/Tsi2 regulatory circuits is to activate the killing activity of our E. coli antibiotic only when necessary. If our probiotic system were constitutively producing Tse2, this would adversely affect natural gut flora. In addition, natural gut flora might develop resistance to Tse2 and pass this resistance to potential Gram-negative pathogens. As a BioBrick, the Tse2.Tsi2 circuit is modular, and therefore promoters and other regulatory components can be swapped out. For example, one could ingest the probiotic, have it exist in the gut, but only induce its killing behavior by ingesting some user-defined signal, such as arabinose, which would activate expression of the toxin, which would then travel through the already-present T6SS into Gram-negative targets. In addition, one could envision a scenario in which it would be advantageous to separately regulate Tsi2, to kill the probiotic when it is no longer useful by preventing Tsi2 expression, causing cell suicide due to the production of Tse2. For the purposes of this project, we decided to make a probiotic that produced the Tse2 toxin only when the probiotic detected the presence of a pathogen, and to express Tsi2 on the same operon.

Separating the toxin and the antitoxin allow us to make us to make a probiotic that can respond to a pathogen but that can also be killed off through repression of the antitoxin

Inducing Toxin Expression Upon Detection of Pathogen: Proof of Concept

In order to activate Tse2 production in response to a pathogen, we needed a promoter that is inducible by some molecular stimulus unique to a specific pathogen. In addition, the expression of Tsi2 would need to be either constitutive or induced by the same stimulus that induces Tse2 expression. As a proof-of-concept, this project uses the LuxR-pLux transcription factor - promoter system from Vibrio fischeri to regulate expression of the Tse2-Tsi2 locus. V. fischeri excretes 3OC6HSL, a small membrane permeable molecule (hereafter referred to as HSL). HSL binds to the transcription factor LuxR, thereby inducing its DNA transcriptional activity. Thus, expression from the pLux promoter is linked to cell density. This is referred to as quorum sensing. Quorum sensing is found in many bacterial species including pathogenic species, making the use of the pLux-LuxR system a good proof-of concept for induction of toxin production when the probiotic detects the presence of a pathogen. When our probiotic detects a Gram-negative pathogen-specific molecule (modeled by HSL), transcription is induced by an inducible promoter (modeled by pLux). This leads to expression of Tse2 (a toxic protein) and Tsi2 (its antitoxin). The T6SS then attacks the pathogen, puncturing the cell wall. Tse2 is then secreted into the Gram-negative pathogen, killing the pathogen. This system could easily be modified to target a wide range of Gram-negative pathogens by just changing the regulation of the Tse2 and Tsi2 locus.

In Response to Pathogen Detection, Probiotic Produces and Secretes Toxin into Pathogen Cytoplasm

Diagram of the Toxin and Antitoxin HSL Inducible Circuit

The Tse2 Tsi2 toxin antitoxin system has a relatively simple circuit design. Tse2 and Tsi2 are present in one operon (as in Pseudomonas aeruginosa) and are regulated by the pLux promoter. For the pLux promoter, we used the well-characterized BioBrick F2620. The LuxR transcription factor is constituitively expressed because no tetR is present to repress the production of LuxR. When HSL is present it binds to LuxR resulting in the induction of Tse2 and Tsi2 production. This BioBrick is part K314203.

Washington_Diagram_F2620_Tse2_Tsi2.png B0010 B0012 B0034 BOO34 luxR pLux pLux1 pTet tse2 tsi2

Overview of the Gram(-) Therapeutic       Building the Gram(-) Therapeutic