Composite

Part:BBa_K1598005

Designed by: Marta Napiorkowska   Group: iGEM15_UCL   (2015-09-17)
Revision as of 23:20, 21 September 2015 by Superjack15 (Talk | contribs)

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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.


SolidWorks1 SolidWorks2

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.



GoC Design1 GoC Design2

Mammalian Cell Culture


mammalian cells cells1 mammalian cells cells2 mammalian cells cells2 mammalian cells cells2


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




We cultured bacterial cells on mammalian cells in the chip to simulate the gut environment and measured the bacterial cell density

[edit]
Categories
//collections/immune_regulation/devices
Parameters
None