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From 2010.igem.org

Description

Abstract

We have designed a universal platform for polypeptide release within the small intestine of the human gut. Our model system release beta-galactosidase, a functional homologue of human lactase, once it reaches the duodenum to help a lactose intolerant patient metabolize lactose. The chassis for this system is the common probiotic in yoghurt, Lactobacillus acidophilus. Once the Lactobacillus acidophilus has reached the duodenum, they will lyse by either by a timed inducible/repressible system, a bile-inducible system, or an encryption system.

Background

Small Intestine

Stomach

Probiotics

Modules

Enzyme Production

Capsule Basics

Our goals is to have each cell be surrounded by a protective 'capsule' to allow them to safely travel through the harsh acidic environment of the stomach to arrive in the small intestine for their main purpose. A past iGEM team used Transcription Factor RcsB to stimulate a the production of Colonic Acid from the capsule synthesis pathway of E.coli. Colonic Acid is a polymer of {=====] and has been shown to increase cell survivability in acidic conditions. We investigated the pathway for capsular polysaccharides synthesis and found more transcription factors that could give a similar or even better results!

RcsA and RcsB are transcription factors that are know to be positive regulators of capsular polysaccharides synthesis. We placed RcsA, RcsB, and a combination of the two under a IPTG inducible promoter to test both quantity of colonic acid produced and cell survivability. These two transcription factors form a heterodimer that is know to activate around 19 genes related to colonic acid synthesis. RcsB is also know to form a homodimmer and positively regulate cell division. RcsA and RcsB belong to the multicomponent RcsF/RcsC/RcsD/RcsA-RcsB phosphorelay system.

More information on these transcription factors and their usage in Biology can be found here: [Ecocyc-RcsA] & [Ecocyc-RcsB]

We wanted to test what IPTG induction levels were appropriate to protect our cells from an acidic environment of pH=2 before placing our devices under constitute control with appropriate ribosome binding sites.

[IMAGE OF 3 DEVICES]

Based on our experiments quantifying the amount of colonic acid produces and cell survivability after acid shock, we have chosen [----------] to use in our final device which will be under constitutive promotion

(IMAGE OF FINAL DEVICE]

One caution we had to consider in up-regulating this pathway is biofilm formation. Cholonic Acid synthesis is one of the first steps of the biofilm formation pathway. To ensure a biofilm is not produced we have taken advantage of an inhibitory transcription factor YgiV that blocks the activation of further steps in this pathway.

pH Sensitive Expression

Inducible-Repressible Expression

Lysis

Animation