Difference between revisions of "Part:BBa K1598004"

Latest revision as of 15:33, 9 October 2022

RBS-GAD-6xHis-Terminator

This part consists of B0034 RBS, human glutamate decarboxylase (GAD) gene with C-terminal His tag, and B0015 terminator.

Usage and Biology

GABA is synthesized from glutamate in the reaction catalyzed by L-glutamic acid decarboxylase (GAD) catalyzes decarboxylation of glutamate to gamma-aminobutyric acid (GABA). 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.

GABA is the main inhibitory neurotransmitter in the brain and decreased GABA levels have been linked to depression and anxiety [1]. Several species of gut bacteria have been shown to produce GABA [2] and metabolomics analysis has shown that the gut luminal GABA levels in ex-germfree mice are considerably higher than those observed in germfree mice [3] . The role of microbiota-derived GABA in gut-brain signalling can be supported by the presence of GABA receptors in gut epithelial cells [4].

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 [5]. 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 [6].

Sequence and Features

References

[1] Kalueff, A. and Nutt, D. (2007). Role of GABA in anxiety and depression. Depression and Anxiety, 24(7), pp.495-517.

[2] 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

[3] 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.

[4] 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.

[5] 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.

[6] 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

Added by LZU-HS-Pro-A

We need to verify that the strain can successfully express the required substance, γ-aminobutyric acid. We first examined the fluorescently transfected strains under a fluorescence microscope. To further accurately detect the expression level, ELISA (immunoassay (IA)) technique was used. ELISA technology through the solid phase of antigen or antibody and antigen or antibody enzyme labeling, adding the substrate of enzyme reaction, the enzyme will catalyze the substrate into color products, observe the number of products for qualitative or quantitative analysis. Therefore, we chose this technique for further detection of expression.

Figure 1 Successful expression of γ-aminobutyric acid in nissle 1917 (The amount of GABA is given in mg/L units and to verify expression, both post-cleavage strains and pre-cleavage strains were used for comparison)

The experimental results showed that our engineered strain could effectively produce GABA