Background

The human intestinal microflora is considered an essential “organ” which plays an important role in human health. This complex ecosystem is composed of approximately 500 anaerobic and aerobic bacteria species, most of them localized in large intestine.
Many studies on animals bred under germ-free conditions have shown that microflora has specific functions:

• Metabolic: fermentation of non-digestible dietary residue and endogenous mucus, salvage of energy as short-chain fatty acids, production of vitamin K, absorption of ions;
• Trophic: control of epithelial cell proliferation and differentiation; development and homoeostasis of the immune system;
• Protective: protection against pathogens.

A disruption or an alteration of human gut microflora equilibrium leads to severe auto-immune diseases, types of colon cancer and non-allergic food hypersensitivities; therefore, it is very important to keep its integrity. In fact already a century ago, the first probiotics have been commercialized. Probiotics are bacteria species (normally present in human gut) that are administered as food components or supplements. Nowadays, with the emerging research field of synthetic biology the potential applications of probiotics could further increase. In addition, the introduction of novel functions to this “organ” via probiotics could lead the way to new therapeutics in order to cure or prevent many pathological conditions.

Project overview

We engineered an indigenous strain from the gut microflora in order to create a safe, controllable and versatile molecular platform, which can be used for production of a wide range of molecules. For this purpose we used the Escherichia coli strain Nissle 1917 (commercialized as Mutaflor) as the host organism for our platform. This particular strain has been used as a probiotic for decades and its beneficial effects on human health are well documented. We introduced into the strain a gene guard system based on an inducible cumate gene guard switch and we trasformed it with a particularly designed plasmid for protein expression.
The gene guard system is divided in two parts:

• the killing mechanism
• the regulating pattern

The killing mechanism is placed on the same plasmid used for protein expression. It consists of two different proteins, T4 Holin and Cathelicidin LL-37, both toxic for prokaryotic cells. These antimicrobial proteins are both regulated by a cumate responsive promoter (T5 promoter Cumate Operator) placed upstream the structural genes. Holin is the biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein comes from bacteriophage T4; it is a small membrane protein which depolarizes and permeabilizes the membrane allowing the secretion of Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial peptide produced by human macrophages. It has an amphypathic structure that allows it to associate with bacterial membrane and form pores which lead to cell lysis; its presence in the periplasm is pivotal for its killing effect. As an alternative killing mechanism the complex T4 Holin + Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE 2 arrests the bacterial growth.
The cumate-responsive regulator CymR, which regulates the expression of the T4 Holin and Cathelicidin LL-37, will be introduced into the bacterial chromosome by recombination using the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2 which codes for the Tn7 transposase. The regulator cassette consists of a constitutive promoter (J23100) and the cymR gene, put in tandem in order to increase the quantity of the repressor to tightly control toxin expression.

No Cumate Expression Mode: CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37. Bacteria live!

Cumate-Induction Kill Mode: Cumate enters by diffusion into bacteria, binds and inactivates the repressor. The cumate responsive promoter (T5 Cumate Operator) is derepressed and the expression of T4 Holin and Cathelicidin LL-37 is turned on. T4 Holin interacts with the cytoplasmatic membrane, permeabilizes it allowing the secretion of Cathelicidin LL-37 into the periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms pores, which leads to cell lysis. Bacteria die!

Plasmid Transfer:

1. The plasmid transfers from the E. coli Nissle into other bacteria via horizontal transfer. The receiving bacteria do not produce the CymR repressor, so the cumate responsiv promoter (T5 Cumate Operator) is derepressed and the T4 Holin and Cathelicidin LL-37 expression is activated.
   Receiving bacteria die!
2. E. coli Nissle looses the plasmid. It continues to produce the CymR repressor, which does not appear to be toxic for prokaryotic cells.
   Bacteria live and is harmless!