With two specific bacterial species defined along with their respective signal peptides, the team then focused on experimental protocols necessary to attach the signal peptides to each of the desired gut peptides. This was accomplished via a Ligation Cycle Reaction (LCR) in which each of the signal peptides – PhoA, PelB, Lp3050, and M6 – were combined with each of the gut peptides – GLP-1, PYY, and Ghrelin – for a total of twelve reactions. Upon the completion of these ligations, each combination underwent a PCR with primers that had flanking regions which included a stop codon and his-tag for the gut peptides.
The technique employed for testing the synthesis and secretion of the gut peptides the team used is known as a Western Blot. Bacterial colonies were grown up and upon screening were placed in growth media dependent on the bacterial species – LB for E. coli and MRS for L. reuteri. After a period of time, a sample of the media supernatant was saved for the Western Blot detection of gut peptide secretion. The cells were then spun down and lysed to serve as a sample for the Western Blot detection of the gut peptide initial synthesis. Supernatant and lysis samples for the same constructs were blotted side-by-side for a comparison of peptide concentration inside and outside of the bacterial cells.
With the synthesis and secretion of the signal-gut peptide combinations characterized through the Western Blot procedures, the team’s next focus was the construction of the inter-species quorum sensing communication system necessary for a true interactive consortium of bacteria.
Research into known quorum sensing structures within our team’s desired bacterial structures led to the design of a bacterial communication system that also was able to incorporate a lysis cassette. The LuxR/I family of quorum sensing structures is found naturally within Vibrio fischeri and facilitates a bio-density detection mechanism; our team’s system implemented a 3-oxoheanoyl-homoserine lactone (AHL) variant. AHL is unique and indeed integral to quorum sensing for its ability to flow within and out of the cellular membrane. The AHL is meant to travel from cell to cell, complexing with protein LuxR (a product we can constitutively express within our system) to then engage with pLux promoter regions.Credit: Boston U 2013 iGEM
For the purposes of our experiments, we utilized two known pLux regions: BBa_R0061 and BBa_R0062. In the presence of AHL and LuxR they are repressed and induced (respectively) from otherwise contrary constitutive states (that is, constitutively on or off). These served as the basis of our quorum sensing system.
Below is an outline of the entire system in the presence of AHL.
Breaking down each component, let us further analyze the system.
The AHL Factories
Consists of LuxI production; experimentally under anhydrotetracycline inducible control
Within clinically functional system this might represent the gut colonizing species where production is controlled via pH or fatty acid sensing
The Target Molecule Production Lines
Consists of constitutively expressed LuxR and inducible pLux (active transcription in the presence of AHL). Design allows any target molecule and not just our targeted hormone (modularity is key in any factory).
Protein Dependent Timed Cellular Lysis
The Suicide Cassette is our take on the popular BBa_K112808 enterobacteria phage T4 lysis cassette. This system consists of endolysin and holin under pCI control as well as a constitutivly active T4 antiholin to suppress basal expression levels
The timer relies upon the activity Enterobacteria phage lamda repressor protein cI under control of repressible (constitutively promoting transcription) variant of pLux to generate a time delayed and protein dependent lysis. In the presence of AHL. cI production ceases and lysis ensues.