Difference between revisions of "Part:BBa K1598005"

Revision as of 22:58, 21 September 2015

pYear-RBS-TPH1-6xHis-Terminator

This is a composite part consisting of a nitric oxide sensitive promoter pYeaR, an RBS, the TPH1 expressing gene and a double terminator. The subparts in the biobricks <a href="http://2015.igem.org/Team:UCL/Sensors">BBa_K381001</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1598002">BBa_K1598002</a> have been tested by iGEM UCL 2015.

Usage and Biology

Clinical depression is likely caused by a chronic low grade-response to inflammation [1]. Although the pathway from inflammation to depression is complex and not fully understood it has been shown that the immune response is often accompanied by symptoms such as oxidative and nitrosative stress in the gut. [2]. Therefore, we have used the PyeaR promoter, which is sensitive to nitric oxide in the cell, upstream of human TPH1. The rate-limiting step of synthesis of serotonin is catalyzed by tryptophan hydroxylase, TPH, which converts tryptophan, an essential amino acid, into 5-hydroxytryptophan (5-HTP) [8]. It was shown that microbial colonization of the gut is essential for maintaining normal levels of tryptophan hydroxylase and serotonin in the blood [9]. We have created a synthetic device that produces functional human tryptophan hydroxylase to restore healthy serotonin levels in affected patients. Thus, we have created a composite system, which senses and responds to mood.

Sequence and Features

Testing

Introduction

To demonstrate a functional prototype of our project, we decided to show our system working under real-world conditions simulated in the lab using a Gut-on-a-Chip design similar to the one made at Harvard University[1].

The idea is to model the rate at which our genetically engineered bacterial culture (E. Coli Nissle) grows and colonizes the gut, and to characterize its expression of 5-HTP, a serotonin precursor that acts as an anti-depressant. With the assistance of Dr. Chiang, from UCL’s Microfluidics Lab, we designed using SolidWorks a 3D version of the chip model described in the attachment.

We improved the original Gut-on-a-Chip designed at Harvard University by making it a more realistic mimic of reality and more financially feasible.The new design doesn't require a porous membrane, and is inspired by a bulging bioreactor. In addition to replicating the peristaltic motion of the longitudinal muscles in the intestines like Harvard's design, this model will also replicate the motions created by circular muscles.

Mammalian Cell Culture

Results

Column: Cell Count (Cells per ul)

50000	25000	12500	6250	3125	1563
781	391	195	98	49	Negative Control

Protocol for Determining Adherence Time:

1. Repeat steps 1 to 12 as described in the protocol above.
2. Seed 6000 cells into 3 wells respectively of 4 96 well plates.
3. Repeat steps 14 to 24 for one plate at intervals of 1 hour.

Adherence Results

1 hour

2 hours

3 hours

4 hours