Introduction

There has been a wide array of peptides, either innate or synthetic, used as drugs or vaccine in combat of different diseases. For instance, insulin was synthesized and mass produced in bacteria; long, synthetic peptide vaccine has also brought to bedside to help treat patient with invasive cancers. (Nat Rev Cancer. 2008 May;8(5):351-60.) However, the delivery of the peptide into human body is a big issue, since it cannot be administered orally, and has a poor distribution and absorption as compared to small molecules drugs in human body. With the advent of biotechnology and the growing forum of synthetic biology, we try to engineer and finely design different circuits in bacteria, which can deliver peptides through epidermal, buccal, rectal, or enteral. As for a simple exemplification of our ultimate goal of microbial peptide delivery system, we choose GLP-1, the endogenous hormone as our delivery model. Our design features efficiency, quick response to the environment changes, and sustainable release of GLP-1. This is the first model of our peptide delivery system, and opens a new room for the synthetic biology in medicine applications.

GLP-1, a human innate neuro-peptide for energy balance, is chosen to combat for obesity and metabolic syndrome. We engineer the non-pathogenic E. coli which senses fatty acids in intestines and secretes synthetic GLP-1. Appropriate signal peptides and penetratin are used to facilitate peptide secretion and intestinal uptake. Furthermore, we design a circuit with quorum sensing and double repressors, which aims to generate quick but sustainable responses and serves as an anti-noise filter. Plasmid stabilization modules including partition system and multimer resolution system are also incorporated to circumvent the undesirable loss or segregational instability of our artificial device. With this general concept of delivery of short peptide into human body, we can also target other human diseases with alternative circuit designs.

Circuit

Fat Extinguisher

The main circuit is designed to detect the presence of fatty acid in the intestinal environment and produce the peptide drug (GLP-1 in this case) and cell penetrating peptide (CPP) as response. There are two core systems: double repressors and quorum sensing, which aims to corroborate a quick responsive to fat ingestion and stable functioning circuit. We start the circuit with fatty acid sensor (fadR), which is followed by double repressor (tetR and LacI). The fadR is a novel promoter which can detect the presence of fatty acid; the double repressor tetR and LacI combine to make the circuit more stable and form a prominent threshold.

Thermal Avenger

We designed a thermal sensitive promoter in our circuit to sense the change of temperature of ambient. The temperature sensitive repressor CI would dimerize in room temperature and suppress the expression of downward gene sequence; however, when the temperature rises, the dimer would break and lose the function of repression. As our experiment proves, the circuit works differently under room temperature from inside human body (37⁰C). As for application of the thermal sensitive device, first, we incorporate toxin and anti-toxin in our thermal promoter, which works as a self destructor. Second, we can utilize the thermal sensitive characteristic of the circuit to make a baseline secretion level of the GLP-1.

Stability and Safety

Every genetically modified (GM) system that functions outside of the laboratories will face two major problems: system stability and safety! Without selective pressure, we have to deal with plasmid segregation instability, which may lead to a incomplete system due to loss of circuit pieces; to make our lab E. coli colonized bowel we have to use recA+ strains which may cause plasmid multimer. Our GM lab E. coli will also be in contact with many kinds of wild bacteria and is under the risk of horizontal gene transfer.