Team:UCL/Effectors
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are an emerging class of probiotics that provide benefit to psychological health of patients by secreting compounds which affect nervous system. We have applied synthetic biology to incorporate the natural neuroregulatory properties of gut bacteria into synthetic genetic devices.
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Background
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Gut-brain communication involves biochemical signalling between intestinal microflora and host's central nervous system. These interactions are bidirectional: gut microbiome is influenced by our mental state and, at the same time, is capable of affect our mental well-being. The term psychobiotics describes an emerging class of probiotics that positively affect patient's mental health by producing neuroactive compounds, including neurotransmitters. The molecules associated with mammalian neurotransmission that have been isolated from gut bacteria include gamma-butyric acid (GABA), serotonin, dopamine, and acetylcholine Lyte, M. and Cryan, J. Microbial Endocrinology: The Microbiota-Gut-Brain Axis in Health and Disease
Advances in Experimental Medicine and Biology Volume 817, 2014, pp 221-239 [Link][1]. When secreted into intestinal lumen, these molecules can modulate the neural signalling within the enteric nervous system which communicates with brain via the vagus nerve, therefore influencing brain’s biochemistry and host’s behaviour.
“It is well recognized that some bacteria within the human GIT have the capacity
to produce many neurotransmitters and neuromodulators. For example, Lactoba
cillus spp. and Bifidobacterium spp. have been reported to produce GABA;
Escherichia spp., and Bacillus spp. have been reported to produce norepinephrine;
Streptococcus spp., Escherichia spp. and Enterococcus spp. have been reported to
produce serotonin; Bacillus spp. have been reported to produce dopamine, and
Lactobacillus spp. have been reported to produce acetylcholine and histamine. [...] Other bacterially-produced metabolites with proven neuroactive func-
tions include short chain fatty acids (SCFAs) and long chain fatty acids such as
conjugated linoleic acid (CLA). “
Microbial Endocrinology: The Microbiota-Gut-Brain Axis in Health and Disease
Advances in Experimental Medicine and Biology Volume 817, 2014, pp 221-239
Probiotics are live microorganisms that, when ingested in adequate amounts, exert a health benefit on the host Report on Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria, 2001. [Link][2]. The studies on mice indicate that several natural probiotic strains exhibit psychobiotic properties: treatment with Lactobacillus rhamnosus has been shown to regulate emotional behaviour and influence the mRNA expression of GABA receptor in host’s brain Bravo, Javier A, Forsythe, Paul, Chew, Marianne V, Escaravage, Emily; Savignac, Hélène M et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proceedings of the National Academy of Sciences of the United States of America vol. 108 (38), 2011, p. 16050-5 [Link][3], while another study has shown that administration of Bifidobacterium infantis results in increased concentration of tryptophan, a serotonergic precursor, in blood plasma and shows antidepressant properties as determined by forced swim test Desbonnet, L., Garrett, L., Clarke, G., Bienenstock, J., & Dinan, T. G. The probiotic Bifidobacteria infantis: An assessment of potential antidepressant properties in the rat. Journal of Psychiatric Research, 43(2), 2008, p. 164–174 [Link] [4]. Based on the current understanding of the role of commensal microbiota in regulation of host brain biochemistry and behaviour, we hypothesize that by engineering bacteria for precisely controlled neurochemical production we can develop probiotic strains that could potentially act as new strategies for mental health treatment.
Our constructs
The genes for our constructs were chosen based on the analysis of metabolic pathways involved in synthesis of neurotransmitter compounds, including tryptophan pathway, glutamate pathway, and acetylcholine pathway. The design of the constructs is explained below. The experimental protocols can be accessed here.
Our Synthetic Super Serotonin System
Serotonin is an essential metabolite, key neurotransmitter in mood regulation and a target of vast majority of antidepressant drugs. The majority of serotonin in the body is produced by enterochromaffin cells located in the gut Manocha, M. and Khan, W. Serotonin and GI Disorders: An Update on Clinical and Experimental Studies. Clinical and Translational Gastroenterology, 2012, 3(4), p.e13.
[Link][6]. Metabolomics study has revealed that serotonin levels in blood plasma are 2.8 fold higher in conventional as opposed to germ-free mice Wikoff, W., Anfora, A., Liu, J., Schultz, P., Lesley, S., Peters, E. and Siuzdak, G. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proceedings of the National Academy of Sciences, 2009, 106(10), pp.3698-3703.
[Link][7], suggesting that the interactions between host and gut microflora play crucial role in regulating the production of serotonin and functioning of serotonergic system.
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) O’Mahony, S., Clarke, G., Borre, Y., Dinan, T. and Cryan, J. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis. Behavioural Brain Research, 2015, 277, pp.32-48.
[Link][8]. It was shown that germ-free mice exhibit decreased expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, in enterochromaffin cells and decreased level of serotonin in the colon and the blood and that both recolonization of the gut and supplementation with 5-HTP, a product of TPH, restore the serotonin levels Yano, J., Yu, K., Donaldson, G., Shastri, G., Ann, P., Ma, L., Nagler, C., Ismagilov, R., Mazmanian, S. and Hsiao, E. Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis. Cell, 2015, 161(2), pp.264-276.
[Link][9].
With the aim to address serotonin deficiency and dysregulation of gut-brain communication in patients with mental health disorders and we have developed and characterized the construct that overexpresses human tryptophan hydroxylase for 5-HTP production.
BBa_K1598001 - the basic TPH1 part. The consensus sequence of human TPH1 was obtained from UniProt (accession no: P17752). The sequence was codon-optimized for E.coli and adapted to BioBrick format. The construct was synthesized by IDT in a form of gBlock gene fragment and cloned into PSB1C3 using traditional digestion-ligation cloning.
BBa_K1598002 - the IPTG-inducible construct containing the Lac expression cassete (BBa_413103) with the TPH1 gene (BBa_K1598001) - Lac expression cassette was obtained from the iGEM distribution. The parts were assembled using the gel extraction method.
The functioning of our serotonin-enhancing system relies strongly on the tryptophan availability in the gut. Several species present in gut microflora, including Escherichia spp. express tryptophanase, which performs degradation of tryptophan into indole, ammonia and pyruvate Chimerel, C., Emery, E., Summers, D., Keyser, U., Gribble, F. and Reimann, F. Bacterial Metabolite Indole Modulates Incretin Secretion from Intestinal Enteroendocrine L Cells. Cell Reports, 2014, 9(4), pp.1202-1208.[Link][10]. We propose to use antisense RNA complementary to tryptophanase-encoding gene as a strategy for downregulating the breakdown of tryptophan in the gut environment and, thus, optimization of 5-HTP synthesis.
In E. coli tryptophanase is encoded by tnaA gene. We have designed two synthetic regulatory small RNAs and investigated their applicability and sensitivity in silencing gene expression.
Our Amazing Artificial Acetylcholine Apparatus
Acetylcholine is a versatile neurotransmitter that has mediates signalling in central nervous system and brain as well as peripheral nervous system. Acetylcholine has been implicated in cognition, memory, and learning functions Klinkenberg, I., Sambeth, A. and Blokland, A. Acetylcholine and attention. Behavioural Brain Research, 2011, 221(2), pp.430-442.[Link][11]. Acetylcholine is also a principal neurotransmitter in vagal nerve, which connects brain to digestive system Lleó, A., Greenberg, S. and Growdon, J. Current Pharmacotherapy for Alzheimer's Disease. Annual Review of Medicine, 2006, 57(1), pp.513-533.[Link][12]. Acetylcholine released at the efferent vagus nerve endings decreases the excessive production of inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor-α (TNF-α) [13]. These inflammatory cytokines are elevated in patients with major depression Dowlati, Y., Herrmann, N., Swardfager, W., Liu, H., Sham, L., Reim, E. and Lanctôt, K. (2010). A Meta-Analysis of Cytokines in Major Depression. Biological Psychiatry, 67(5), pp.446-457. [Link][14] and have been shown to induce depressive-like behaviour in mice Larson, S. and Dunn, A. Behavioral Effects of Cytokines. Brain, Behavior, and Immunity, 2001, 15(4), pp.371-387.[Link][15] and humans Capuron, L., Ravaud, A., Neveu, P., Miller, A., Maes, M. and Dantzer, R. Association between decreased serum tryptophan concentrations and depressive symptoms in cancer patients undergoing cytokine therapy. Molecular Psychiatry, 2002, 7(5), pp.468-473.[Link][16].
The synthesis of acetylcholine is catalysed by choline acetyltransferase, CHAT. In humans, CHAT expression requires microbiota-dependent signalling and does not begin until microbial gut colonization after birth. Reardon, C., Duncan, G., Brustle, A., Brenner, D., Tusche, M., Olofsson, P., Rosas-Ballina, M., Tracey, K. and Mak, T. Lymphocyte-derived ACh regulates local innate but not adaptive immunity. Proceedings of the National Academy of Sciences, 2013, 110(4), pp.1410-1415.[Link][17]. Hence, imbalances in gut microflora can lead to disrupted acetylcholine activity, which has been strongly linked to depression. We propose to explore the acetylcholine-producing probiotics as a solution for restoring the proper functioning of cholinergic system in patients with depression and other psychiatric diseases. We have created a functional genetic device for inducible expression of human CHAT.
BBa_K1598003 - basic CHAT construct. The consensus sequence of human CHAT was obtained from UniProt (accession no: P28329). The sequence was codon-optimized for E.coli and 6xHis tag was added on C-terminal. BBa_B0034 RBS and BBa_B0015 terminator were added upstream and downstream of the gene, respectively. The construct was synthesized by IDT in a form of 2 gBlock gene fragments (1013bp and 1182bp with 30bp overlap) and cloned into PSB1C3 using Gibson assembly.
Our Great GABA Gears
GABA is the main inhibitory neurotransmitter in the brain and decreased GABA levels have been linked to depression and anxiety Kalueff, A. and Nutt, D. (2007). Role of GABA in anxiety and depression. Depression and Anxiety, 24(7), pp.495-517.[Link][18]. Several species of gut bacteria have been shown to produce GABA Cryan, J. and Dinan, T. (2012). Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nature Reviews Neuroscience, 13(10), pp.701-712.[Link][19] and metabolomics analysis has shown that the gut luminal GABA levels in ex-germfree mice are considerably higher than those observed in germfree mice Matsumoto, M., Kibe, R., Ooga, T., Aiba, Y., Kurihara, S., Sawaki, E., Koga, Y. and Benno, Y. (2012). Impact of Intestinal Microbiota on Intestinal Luminal Metabolome. Sci. Rep., 2.[Link][20] . The role of microbiota-derived GABA in gut-brain signalling can be supported by the presence of GABA receptors in gut epithelial cells B Li, Y., Xiang, Y., Lu, W., Liu, C. and Li, J. (2012). A novel role of intestine epithelial GABAergic signaling in regulating intestinal fluid secretion. AJP: Gastrointestinal and Liver Physiology, 303(4), pp.G453-G460. [Link][21].
The positive effects of GABA-producing species in the gut on the emotional behaviour of the host have been previously documented. In one study, administration of milk enriched in GABA-producing Lactobacillus brevis FPA 3709 had antidepressant effect on mice Ko, C., Lin, H. and Tsai, G. (2013). Gamma-aminobutyric acid production in black soybean milk by Lactobacillus brevis FPA 3709 and the antidepressant effect of the fermented product on a forced swimming rat model. Process Biochemistry, 48(4), pp.559-568.[Link][22] . Another study has shown that ingestion of Lactobacillus rhamnosus alters GABA mRNA expression in the host's brain and reduces the stress-induced and depression-related behaviour Bravo, J., Forsythe, P., Chew, M., Escaravage, E., Savignac, H., Dinan, T., Bienenstock, J. and Cryan, J. (2011). Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proceedings of the National Academy of Sciences, 108(38), pp.16050-16055.[Link][23] .
GABA is synthesized from glutamate in the reaction catalyzed by L-glutamic acid decarboxylase (GAD). We have created the device for overexpression of GAD and we suggest to investigate the therapeutic potential of administration of GABA-overexpressing probiotics to patients with decreased GABA levels.
BBa_K1598004 - the basic GAD part. The consensus sequence of human TPH1 was obtained from UniProt (accession no: Q99259). The sequence was codon-optimized for E.coli and 6xHis tag was added on C-terminal. BBa_B0034 RBS and BBa_B0015 terminator were added upstream and downstream of the gene, respectively. The construct was synthesized by IDT in a form of gBlock gene fragment and cloned into PSB1C3 using traditional digestion-ligation cloning.
Pregnenelone
Theoretical Overview
Also called Cytochrome P450scc, Cholesterol Side Chain Cleavage Enzyme (P450scc) is a mitochondrial enzyme that converts cholesterol into pregnenelone, which is a neurosteroid as well as the precursor to a variety of steroid hormones and neurotransmitters. The gene that encodes it is CYP11A1.
This is the reaction it catalyses:
cholesterol + 3 NADPH + 3 H+ + 3 O2 ⇄ pregnenolone + 4-methylpentanal + 3 NADP+ + 3 H2O
Furthermore, the catalysis involves 3 monooxygenase reactions, which requires two electron transport proteins: ferrodoxin (FDX1) and ferredoxin reductase (FDXR).
Our biobrick consists of the iGEM prefix followed by the J23101 constitutive promoter, RBS, FDXR gene, RBS, FDX1 GENE, RBS, CYP11A1 gene, rrnb double terminator, and the iGEM suffix. This will be ligated onto a PSB1C3 backbone.
DNA Assembly
Our biobrick (excluding the vector) has been split into 5 parts for synthesis. Once they arrive, they will be assembled together using Golden Gate Assembly by the experts at Synthace, one of our sponsors. Sap1I will be the enzyme used according to the standard Golden Gate assembly protocol.
- Day 1: PCR Amplification and Purification of parts, followed by Digestion.
- Day 2: Gel Purification, Golden Gate Assembly, and Transformation using DH5 alpha cells.
- Day 3: Picking of colonies and Inoculation
- Day 4: Minipreps, Digestion using ECOR1 and Pst1, Ligation with PSB1C3, and Transformation
- Day 5: Picking of Colonies, and Inoculation
- Day 6: Creation of Glycerol Stocks and Characterization using HPLC.
The protocols for all the steps describe above can be found on the protocols section of this wiki.
Golden Gate Assembly Protocol:
For assembling parts that are in Golden Gate standard, we used the following protocol:
| Components | Amount (μl) |
| Sap1(15 U) | 0,75 |
| T4 Ligase (400 U) | 1 |
| DTT (10 mM) | 1 |
| ATP (10 mM) | 1 |
| G-Buffer (10x, Fermentas) | 1 |
| parts | 40 fmoles each |
| ddH2O | Fill up to 10 |
| Total Volume | 10 |
Thermocycler programme:
- 37°C, 5 min
- 20°C, 5 min
- repeat (1. and 2.) 50 times
- 50°C, 10 min
- 80°C, 10 min
Characterization Assay (HPLC): The steps were followed according to the protocol given in this paper.
Relief of Side-effects from Depression/Anxiety drugs:
Participant A, a long term depression patient we interviewed, suggested that our novel probiotics will be more accepted if they had reduced the side-effects when compared to conventional drugs. She also mentioned how drugs gradualy became ineffective overtime, and the dosage needs to be constantly increased, which is a major financial burden. Well, the administration of Pregnenolone and its derivates prevents the development of tolerance, and augments recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice. Benzodiazepines, such as Valium, is one of the most popular antidepressant/anti-anxiety drug in the world! Basically, this removes the need to constantly increase the dosage, and gets rid of many side effects and withdrawal symptoms, associated with treatments that act on the GABA receptor. Hence, combining it, pregnenolone, with our biobricks creates a complete system, which not only treat mental illness but also reduce side effects of treatment! Hence we have expanded on our silver medal Human Practices activity by demonstrating how we have integrated the investigated issues into the design and/or execution of your project. [reference 1]
Pregnenolone-pretreated subjects showed significantly less sedation following diazepam (p<0.03); this effect was clinically apparent. Diazepam's amnestic effects were non-significantly attenuated, and ratings of anxiety were unaffected. Chronically administered pregnenolone antagonizes certain acute effects of benzodiazepines and may enhance arousal via antagonist or inverse agonist actions at the benzodiazepine/GABA(A) receptor complex. Pregnenolone was generally well-tolerated but, by itself, had no significant effects on mood, memory, self-rated sleep quality or subjective well-being. [reference 2]
·Depression/Anxiety:
Neurosteroids derived from Pregnenolone modulate several neurotransmitter systems such as gamma-aminobutyric acid type A (GABA(A)), N-methyl-D-aspartate (NMDA) and acetylcholine receptors. As physiologic consequences, they are involved in neuronal plasticity, learning and memory processes, aggression and epilepsy, and they modulate the responses to stress, anxiety and depression. There is evidence for a common mechanism of action between neurosteroids and sigma1-receptor ligands and focus on the potential therapeutic interests of such interaction in the physiopathology of learning and memory impairments, stress, depression and neuroprotection. [reference 3]
·Schizophrenia Treatment:
Patients receiving pregnenolone demonstrated significantly greater improvements in SANS scores (mean change=10.38) compared with patients receiving placebo. Adjunctive Pregnenolone Significantly Reduces Negative Symptoms. Serum increases in pregnenolone in the group randomized to this neurosteroid are significantly correlated with cognitive improvement. Pregnenolone was very well tolerated in this study and showed no untoward effects on weight, blood pressure, pulse, glucose, cholesterol, prolactin, or other monitored laboratory parameters. This is a very good read, and explains all the mechanisms, too. [reference 4]
Fifty-two participants (86.7%) completed the trial. Compared to placebo, adjunctive pregnenolone significantly reduced Positive and Negative Symptoms. Thus, add-on pregnenolone reduces the severity of negative symptoms in recent-onset schizophrenia and schizoaffective disorder, especially among patients who are not treated with concomitant mood stabilizers. [reference 5]
Emerging preclinical and clinical evidence suggests that pregnenolone may be a promising novel therapeutic candidate in schizophrenia. Pregnenolone is a neurosteroid with pleiotropic actions in rodents that include the enhancement of learning and memory, neuritic outgrowth, and myelination. Further, pregnenolone administration results in elevations in downstream neurosteroids such as allopregnanolone, a molecule with neuroprotective effects that also increases neurogenesis, decreases apoptosis and inflammation, modulates the hypothalamic-pituitary-adrenal axis, and markedly increases GABA(A) receptor responses. In addition, pregnenolone administration elevates pregnenolone sulfate, a neurosteroid that positively modulates NMDA receptors. There are thus multiple mechanistic possibilities for pregnenolone as a potential therapeutic agent in schizophrenia, including the amelioration of NMDA receptor hypofunction (via metabolism to pregnenolone sulfate) and the mitigation of GABA dysregulation (via metabolism to allopregnanolone). [reference 6]
Participants randomized to pregnenolone (n = 56) demonstrated greater improvements in functional capacity compared to placebo. Pregnenolone was also superior to placebo in the communication subscale. Neurosteroid changes correlated with functional improvements in female participants. Pregnenolone was well-tolerated. [reference 7]
·Aging, Memory, and Cognition:
Studies in animals demonstrated that the neurosteroids pregnenolone (PREG) and dehydroepiandrosterone (DHEA), as sulfate derivatives (PREGS and DHEAS, respectively), display memory-enhancing properties in aged rodents [reference 8]
In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. [refrence 9]
Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging. [reference 10]