Difference between revisions of "Part:BBa K1598005"

Revision as of 14:09, 21 September 2015

pYear-RBS-TPH1-6xHis-Terminator

Human tryptophan hydroxylase 1 gene under the control of Nitrate-sensitive pYear promoter

Usage and Biology

Sequence and Features

[1]

[2]

Functional Parameters

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 described in:
<a href="http://pubs.rsc.org/en/Content/ArticleLanding/2012/LC/c2lc40074j#!divAbstract" target="_blank">http://pubs.rsc.org/en/Content/ArticleLanding/2012/LC/c2lc40074j#!divAbstract</a>

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.

<img src="" alt="SolidWorks1" style="height:200px;"> <img src="" alt="SolidWorks2"style="height:200px;">

Dr. Paul Sharp, who works with human intestinal epithelial cells at Kings College London, kindly agreed to collaborate with us on this project. He gave us the Caco-2 cells we needed, and advised us on the best ways to culture them. After attending a lecture by Dr. Marques (UCL Biochemical Engineering), we decided to improve 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 Dr. Marco's bulging bioreactor. It has been designed under the guidance of Dr. Paul Sharp. 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.

<img src="" alt="GoC Design1" style="height:320px;"> <img src="" alt="GoC Design2" style="height:320px;">

Experiments were carried to determine the optimum seeding cell density of the cells, and the time they need to adhere.
</div>

mammalian Cell Culture

<img src="" alt="mammalian cells cells1"style="height:200px;"> <img src="" alt="mammalian cells cells2"style="height:200px;"> <img src="" alt="mammalian cells cells2"style="height:200px;"> <img src="" alt="mammalian cells cells2"style="height:200px;">

Protocol for Determining Optimum Seeding Cell Density

<p>

METHODS

Preparation
1. Warm medium (DMEM (1x) + GlutaMAXTM-I + 10% FBS), PBS and TE solution in a water bath @ 37ºC
2. Spray tubes, bottles, racks and everything that goes inside the hood with 70% ethanol

Taking off the medium

3. Take off almost all the medium inside the flask

Washing the cells

4. Wash gently the cells with PBS

Trypsinization

<p>
5. Cover the flask with 3-5 mL with Trypsin/EDTA solution
6. Incubate for 3 - 5 minutes @ 37ºC
7. Disperse the cells by hitting the flask (once)
8. Watch in the microscope to confirm trypsinization step
9. Add 5 mL medium to inactivate trypsin
10. Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)
</p>

Adding new medium

<p>
11. Remove medium + TE without touching the pellet
12. Add 5 mL of medium to the centrifuge tube with cells




13. Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)
</p>

Cell fixation


14. Fix the cells in 4% paraformaldehyde for 15 min @ RT
    
15. Wash with PBS three times (3 times, 5 minutes each)

<p>Cell permeabilization for intracellular biomarkers

16. Incubate with PBS + 0.1% Triton X-100 for 10 minutes @ RT
17. Wash with PBS three times (5 minutes each)
</p>

<p>Blocking

18. Block with PBS + 3% BSA for 1 hour @ RT
19. Wash with PBS (3 times, 5 minutes each)
</p>

<p>DAPI/Hoechst addition

20. Incubate the cells on 1:3000 DAPI for 5 minutes @ 4 ºC
21. Wash with PBS (3 times, 5 minutes each)
</p>

<p>Imaging

22. Cells were visualized by confocal microscopy. Images were aquired at fluorescence peak excitation and emission wavelengths of 360 nm and 460 nm, respectively.

Results

Column: Cell Count (Cells per ul)

50000	25000	12500	6250	3125	1563
<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">
781	391	195	98	49	Negative Control
<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">	<img src="" class="counttable">

<p><img src=""></p>

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

<p>

1 hour

<img src="" id="count"> <img src="" id="count"> <img src="" id="count"> </p>

<p>

2 hours

<img src="" id="count"> <img src="" id="count"> <img src="" id="count"> </p>

<p>

3 hours

<img src="" id="count"> <img src="" id="count"> <img src="" id="count"> </p>

<p>

4 hours

<img src="" id="count"> <img src="" id="count"> <img src="" id="count"> </p>

<img src="" style="position: relative; width:100%; top: 0; margin-top: 40px;"/>

<img src="" style="width: 100%;"/>

<p><img src="" style="width:50%;"></p>

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

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