Project Description

Introduction

The goal of our 2017 iGEM project is to genetically modify E. coli Nissle 1917, a probiotic, in such a way as to allow this organism to produce γ-Aminobutyric Acid (GABA). The three key words of our project are: GABA, GAD, and E. coli Nissle 1917.

I. GABA

GABA is a four carbon non-protein amino acid that functions as an inhibitory neurotransmitter in the brain, affecting one’s personality and stress management ability. AM et. al tested sixty-three adults with capsules containing 100 mg of GABA or dextrin as a placebo, and found that after 30 min, the experimental group had a decrease in the alpha and beta band waves compared to the control group, indicating that GABA might have alleviated the stress induced by mental tasks during the experiment. Moreover, GABA may be able to treat diabetes, according to Watanabe M, 2017. Clinically, GABA is used for treating sleeplessness, depression, autonomic disorders, chronic alcohol-related symptoms, and stimulation of immune cells ,(Siragusa S et al., 2007). Besides these uses, GABA is also sold on the market as an over the counter dietary supplement.

Though GABA is beneficial to human beings in many ways, it is not allowed to be produced through chemical process if the chemical is sold as an edible product since there may be harmful impurities during the production. Therefore, it becomes meaningful for us to use synthetic biology methods to produce food grade GABA.

II. Glutamate Decarboxylase (GAD)

Many microorganisms, including some Escherichia coli and Lactobacillus strains, can produce GABA (Dhakal et al.). Among these microorganisms, Lactobacillus brevis NCL912 has shown the highest production of GABA: 3.57 g/l. According to Dhakal et al., the difference in productivity of GABA in different strains is caused by the difference in their GAD enzymes’ properties, suggesting that the strain Lactobacillus brevis NCL912 may possess a unique GAD system, incorporating a unique GAD gene.

Li et al. characterized the components of GAD operon in Lactobacillus NCL912 in 2013: gadC, gadA, and gadR. GadA is the glutamate decarboxylase that catalyzes the chemical reaction from glutamate to GABA, while gadC functions as a glutamate/GABA antiporter, an exchanger of GABA and glutamate. Typically, gadC is only present in the genomes of limited strains indicating their strain-specific characteristic of GABA production (Wu et al., 2017). GadCA forms an operon and is regulated by gadR, which had much higher expression levels (Li, Li, Liu & Cao, 2013).

For the outstanding GABA producing ability of the GAD system in Lactobacillus brevis NCL912, we picked proteins from this system as components of our device.

III. E. coli Nissle 1917

E. coli Nissle 1917, a harmless strain of E. coli isolated by Alfred Nissle, is a commonly used probiotic in treating Ulcerative colitis, chronic constipation, and Crohn's disease. We chose this strain as our genetic engineering project due to its safety and its controllability. Since many researches have shown that E. coli Nissle 1917 is edible, we are able to derive our product, GABA containing milk, directly from the genetically modified species. Moreover, E. coli Nissle can be transformed and cultured using the same techniques as that of other E. coli strains.

Thus, by inserting the sequence of unique GAD system found in Lactobacillus brevis NCL912 into E. coli Nissle, we designed multiple parts and tested their individual abilities.

Design

I. Big Picture

We intend to put the GABA producing proteins in E. coli Nissle 1917, creating a probiotic GABA supplement that functions in human intestine, with milk as the switch to open or close the production. In this way, the production of GABA will be simplified since E. coli Nissle is edible.

Fig. 1 First Approach: BBa_K2326003

We chose the pTac promoter, a hybrid of Lac and Trp promoters, induce GABA production in E. coli Nissle in the human intestine in the presence of lactose from consumed milk. The promoter can be induced by both lactose and IPTG, where LacI represses transcription of up stream genes in this part, which are gadR, gadC, and gadA. The ribosomal binding site (RBS) for gadRCA with a relative strength of 300, 300, 3900 respectively are calculated by RBS Calculator from Salis Lab.

Fig. 2 Second Approach: BBa_K2326004

Because E. coli Nissle has not been used as an engineered strain until recent years, it cannot express large amount of foreign genes from a plasmid and failed to execute all the designs we had on our first attempt. So we cut our original design, leaving the most important enzyme, gadA, alone, which catalyzed GABA transforming from glutamate. We also changed the RBS of gadA into B0034, the most consensus sequence for E. coli. Moreover, we added GFP at the end of the circuit to simplify the process of assaying protein production.

II. Devices

Fig. 3 Third Approach: BBa_K2326005 & BBa_K2326006

The second device we designed enabled us to see an estimated amount of gadA expressed, but the negative result from the Amino Acid Analyzer (see demonstration) suggest the GABA produced in our project may flow back to the TCA cycle. As a result, we designed the third and fourth device as shown above. For BBa_K2326005, we added a His tag after gadA so we can extract the enzyme out and prevent it from being subjected to endogenous processes, and then produce GABA from the isolated protein. BBa_K2326006 enabled us to quantify the expression rate of gadA since the protein is synthesized with GFP.

Attrbutions

The competition was organized at Beijing National Day School by Ms. Xiaoling Yao, Dr. Ruoting Tao, and Zhongxiu Hu with the help from Haoqian Zhang and Yihao Zhang. Yantong Wang, Kuo Zhang, Yiyang Li, Yifei Yu, Hongliang Xiong, Zhuoyi Yang, Hao Wang, Yixin Huang, Kaiwen Dou, and Xilin Yang were recruited in March, 2017.

The topic of our project was a result of numerous group discussions. Every member played a role in determining a part of our project. Parts are designed by Zhongxiu Hu, Zhuoyi Yang, Xilin Yang, and Yantong Wang. More specifically, Zhongxiu Hu is in charge of designing the function of circuits; Xilin Yang and Zhuoyi Yang help with finding Ribosomal Binding Sites (RBS); Yantong Wang helps with adding protein analyzing elements on circuits. Yantong Wang designed all experiments involved in protein detection, and Zhuoyi Yang was in charged of the majority testing GABA concentration via Amino Acid Analyzer (AAA).

Every member had conducted experiments (more detail, Notebook), wrote wiki, and helped with presentation. The detailed allocation of wiki writing is shown below:

Our wiki drafts were written in Word and Kaiwen Dou translated them into html later. The overarching frame work of our wiki was designed by Hongliang Xiong.

Yifei Yu was in charge of designing the mathematic model that optimize our enzyme’s performance. He was also our accountant, while Zhongxiu Hu booked hotels for the team and Hao Wang ordered reagent.

Collaboration

With PKU

All experiments were conducted at Peking University with team PKU. Team members from Peking University provided us many beneficial suggestions on experiment designing, inspiring us to solve problems. Without their help, we would not be able to complete all of our experiments with the same efficiency.

With UCAS

We lent the lab shaker to team UCAS since our campuses are very close to each other.

Special Thanks

There are many institutes and people that helped us overcome many obstacles.

The microbiology technology company, Bluepha, has provided us technical support. Especially Haoqian Zhang, Bluepha’s CTO, leads us to a more mature circuit design as well as a logical presentation. He also lent us a HPLC though we did not use it to measure the final result.

National Institute of Metrology lent us the Amino Acid Analyzer that allowed us to measure the concentration of GABA. Their instructions enabled us to measure GABA with higher precision and efficiency.

Our instructors, Miss Xiaoling Yao, Dr. David Brackett, Dr. Ruoting Tao, and Mr. Yihao Zhang have given us many great suggestions on project design. Mr. Zhang helped us specially on experiment conduction and problem solving. Dr. Brackett proofed read all of our wiki contents and helped us with academic writing. Miss Yao and Dr. Tao gave us many suggestions on our presentation.

We are also thankful for School TV Station and Imaginist, the school magazine for facilitating us promoting iGEM and our team.

References

Abdou AM, Higashiguchi S, Horie K, Kim M, Hatta H, Yokogoshi H. (2006).Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans [abstract]. BoiFactor. 26(3):201-8. doi: 10.1002/biof.5520260305

Dhakal, Radhika., Bajpai, Vivek K., Baek, Kwang-Hyun. (2012, Jun 7). Production of gaba (γ - Aminobutyric acid) by microorganisms: a review. Brazilian Journal of Microbiology, 1230-1241. doi: http://dx.doi.org/10.1590/S1517 -83822012000400001

Li H, Cao Y, Gao D, Xu H. (2008, Dec.). A high γ-aminobutyric acid-producing ability Lactobacillus brevis isolated from Chinese traditional paocai. Ann Microbiol, 58, 649–653. doi: 10.1007/BF03175570

Li H, Li W, Liu X & Cao Y. (2013 Nov. 6). gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation. FEMS Microbiol Lett, 349 (2013) 108–116 doi: 10.1111/1574-6968.12301

Siragusa S., De Angelis M., Di Cagno R., Rizzello CG., Coda R., Gobbetti M. (2007 Nov.). Synthesis of γ-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. Appl Environ Microbiol, 73(22), 7283–7290. doi: 10.1128/AEM.01064-07.

Wu Q, Tun HM, Law YS, Khafipour E, Shah NP. (2017 Feb.). Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH. Front Microbiol. 2017; 8: 206. doi: 10.3389/fmicb.2017.00206