Part:BBa_K1598005
pYear-RBS-TPH1-6xHis-Terminator
This is a composite part consisting of the PyeaR promoter, an RBS, the TPH1 expressing gene and a double terminator. The subparts in the biobricks BBa_K381001 and BBa_K1598002 have been tested by iGEM UCL 2015.
Contents
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) [3]. It was shown that microbial colonization of the gut is essential for maintaining normal levels of tryptophan hydroxylase and serotonin in the blood [4]. 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; thereby creating not just an anti-depressant/anxiolytic, but also a complete mental health regulation system.
Testing
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 UCL’s very own Dr. Marques’ revolutionary bulging bioreactor mechanism. 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. The PDMS chip was made up of 2 microfluidic channels separated by a flexible PDMS membrane. Mammalian cells were grown in the top channel, and medium was flowed through both channels. Increasing the flow of medium through the bottom channel periodically caused the PDMS membrane to rise, thereby increasing the distance between the mammalian cells and reducing their distance from the inner wall at the top. Thus, peristalsis was recreated in the chip, along with other gut conditions. This chip is a cheaper, improved version of the original.
Mammalian Cell Culture
Due to a lack of time, we were unable to use Caco-2 (small intestinal epithelial cells) as the mycoplasm testing results of the newly obtained cells did not arrive on time; therefore, CV-1 cells (Monkey Kidney Fibroblasts), which are similar to epithelial cells and can still give us a realistic representation of gut cells, were used in the following experimentation instead. Furthermore, some of the parts (pipes and connectors) we ordered for the microfluidics device weren't delivered on time, so we had to use 96 well plates, which have a volume similar to our device. Hence, preliminary experimentation was carried out to establish conditions and demonstrate an initial proof of concept.
Mammalian Optimum Cell Seeding Density Determination
To determine the optimum seeding density for the mammalian cells, the cells were grown at different initial concentration in a 96 well plate (approximately the same size as the chip) for 24 hours, and the final concentration of the cells was determined by staining them with hoechst, and calculating the number of cells per mm^2 using ImageJ, after taking the pictures given below using a microscope at 20x magnification.
| 50000 | 25000 | 12500 | 6250 | 3125 | 1563 |
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| 781 | 391 | 195 | 98 | 49 | Negative Control |
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As illustrated by the graph above, 6250 cells/ul was the ideal cell seeding density as the cells were at a sufficiently high concentration just before stationary phase of cell growth, and the cells looked the healthiest as they didn’t have to compete with too many cells for nutrients. This is the concentration of mammalian cells that will be initially put in the Gut-on-a-Chip.
Mammalian Cell Adherence Time Determination
Cells were seeded at the optimum seeding density of 6250 cells/ul in 3 wells of 5 different 96 well plates. Starting at t=0h, one 96 well plate was removed from incubation every hour, and the no. of viable cells adhered was measured by staining them with hoechst, and calculating the number of cells per mm^2 using ImageJ, after taking the pictures given below using a microscope at 20x magnification, just as done before.
1 hour
2 hours
3 hours
4 hours
The adherence time was established as 3 hrs because it had the highest number viable adhered cells like indicated in the graph above. Medium will have to be flowed in the microfluidics device after 3 hours at least. We cultured bacterial cells on mammalian cells in the chip to simulate the gut environment and measured the bacterial cell density
Bacterial Optimum Cell Seeding Density Determination
One of our main aims was to determine the optimum concentration of bacteria that can live in the chip without negatively affecting the mammalian cells already growing in it. This can establish a safe concentration of bacteria that can be administered through our probiotic without causing much damage or negative impact in the gut and to its natural microbiome.
This was done by inoculating mammalian cell cultures with different concentrations of E. Coli containing this part. The concentrations used were 9 tenfold dilutions of an overnight bacterial cell culture in a 96 well plate.
The graph above was used to determine the cell density for each dilution. Upon looking at each well under the microscope after 16hours, it was noted that the mammalian cells only survived when the bacterial cell seeding density was 1.29E+08 CFU/mL. Thus, the optimum bacterial cell seeding density and ideal probiotic dosage to administer were determined.
Bacterial and Mammalian Cell Interactions
We grew our genetically engineered E Coli. Nissle with the mammalian cells to study the rate at which our probiotic grows and colonizes the gut. We also studied the interactions between the 2 cells, and established that they symbiotically co-exist at constant cell densities after over 16hours in real-world conditions.
As illustrated by the graph above, mammalian cell density rapidly declines from approximately 1400 cells/mm2 to 700 cells/mm2 in the 1st 2 hours, when our bacteria are introduced into the device at t=0. The CFU/mL of bacteria also falls significantly from 1.29X1008 to 2X1007. This is because the microenvironments of both types of cells has significantly changed due to the introduction of bacteria, and they’re now competing for nutrients.
After 2 hours, both bacterial and mammalian cell densities begin to recover as they get used to the new conditions and begin comfortably coexisting. In around 6 hours, the mammalian cell density reaches it peak of 700 cells/mm2 and stays constant at the same level for over 16 hours. The same occurs with the bacterial cell density as it reaches its maximum of approximately 9.5X1008 CFU/mL, and stationary phase at around 11 hours. Hence, our system has shown that both cell types have found an equilibrium and can happily co-exist in balance in simulated real world gut conditions.
Since mammalian cells are more delicate than bacteria, we can extrapolate that our probiotic will not have any negative impact on the gut’s natural microbiome. This also helps us address an issue raised at the UK iGEM Meet-Up. There was a concern that the introduction of our probiotic may significantly alter the balance of the natural gut microbiome, and have several side-effects. However, as established through our experiment, our probiotic prototype’s bacterial cell density growth plateau’s after a while, leaving the other cells healthy, although there is an initial drop in cell density. In conclusion, our probiotic prototype takes around 11hours to colonize the gut, and we also determined an optimum bacterial cell density for probiotic dosage.
Characterisation of PyeaR Promoter: PyeaR
Characterisation of TPH1 Expression: TPH1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- [1] M. Berk et al. So depression is an inflammatory disease, but where does the inflammation come from? BMC Medicine 2013, 11:200 (accessed:http://www.biomedcentral.com/1741-7015/11/200)
- [2]Brian Leonard, Michael Maes Mechanistic explanations how cell-mediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression in Neuroscience & Biobehavioral Reviews Volume 36, Issue 2, February 2012, Pages 764–785
- [3]S.M. O’Mahony et al. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis in Behavioural Brain Research Volume 277, 15 January 2015, Pages 32–48 Special Issue: Serotonin
- [4]Yano JM et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. in Cell. 2015 Apr 9;161(2):264-76. doi: 10.1016/j.cell.2015.02.047.
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