Team:NCKU Tainan/Design


Overview

  With our goal in providing an alternative solution to chronic stress-induced depression (CSID), we designed a probiotic that can catalyze the production of taurine in response to the increase of stress biomarkers in the intestine.

  We engineered E. coli Nissle 1917 to have two sensing systems, which are induced by stress biomarkers interferon-gamma (IFN-γ) and reactive oxygen species (ROS). When the concentration of these two biomarkers increase, enzymes required for the synthesis of taurine will be expressed and coordinately transform L-cysteine to taurine.

  With regard to biosafety, we chose to use E. coli Nissle 1917, a nonpathogenic E. coli strain. We removed the resistance gene to eliminate the concerns regarding the spread of antibiotic resistance.

  To make our engineered E. coli more accessible for public use, we decided to package our engineered bacteria inside a bubble named Menbles. We made a microfluidic simulation device named f(int) to predict how our bacteria will behave once it reaches the intestine.

Fig. 1. Overall design of the two sensing systems

Taurine Production Enzymes

  Taurine is a semi-essential amino acid appreciated for its role as a neuroprotective agent. Based on current research suggesting taurine’s potential in countering depressive symptoms, we engineered E. coli to utilize existing taurine production pathways to increase taurine levels in the body in response to high stress levels.

  The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].

Fig. 2. Taurine pathways in E. coli
  1. The L-cysteine sulfinic acid pathway includes the L-cysteine dioxygenase (CDO1) and L-cysteine sulfinic acid decarboxylase (CSAD) enzymes. First, CDO1 oxidizes L-cysteine naturally found in the intestine into L-cysteine sulfinic acid. Afterwards, CSAD catalyzes the decarboxylation of L-cysteine sulfinic acid into hyportaurine, which spontaneously oxidizes into the final product, taurine[1].
  2. The L-cysteine sulfonic acid pathway includes the L-cysteine sulfonic acid synthase (CS) and CSAD. CS converts O-phospho-L-serine into cysteine sulfonic acid. The pathway converges with the L-cysteine sulfinic acid pathway at CSAD, which converts cysteine sulfonic acid into taurine[1].
  3. In the JJU-CoaBC pathway, JJU converts L-cysteine, the same substrate as the L-cysteine sulfinic acid pathway, into L-cystate. The second enzyme CoaBC then catalyzes the production of taurine from L-cystate[2,3].

How did we test it?

  To confirm the function of the taurine production enzymes, we conducted high-performance liquid chromatography (HPLC) to determine the taurine production yield of in vivo and in vitro tests. The enzymes of the pathway that produce the highest amount of taurine are chosen to be incorporated into the biobrick of the oxidative stress and interferon-gamma (IFN-γ) sensing systems. More information can be found in Proof of Concept.

Sensing Systems

Oxidative Stress Sensing System

  Oxidative stress, such as reactive oxygen species (ROS) and their derivatives, plays an important role in numerous neurodegenerative diseases, including depression[4]. Chronic intestinal inflammation is strongly related to increased ROS, including superoxide radical (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (OH), which may lead to depressive symptoms if not treated.

  To make E. coli express the taurine-producing enzyme under oxidative stress in the intestine, we decided to utilize the soxRS regulon because it is the most efficient and sensitive promoter compared to others. Targeting the small changes of oxidative stress in human intestines is crucial for the production of taurine synthesis enzymes.

  In E. coli, SoxR, which mainly responds to superoxide[5], serves as a transcription factor that stimulates the expression of various antioxidant genes, including soxS[6]. The soxS gene is a component of the soxRS regulon, and oxidized SoxR (BBa_K3371100) can induce soxS expression by distorting the soxS promoter (BBa_K3771048)[6]. We added the soxR promoter and the coding sequence of SoxR in the reverse direction in front of the soxS promoter, the same as it is located in the E. coli chromosome. As more SoxR is produced, the soxS promoter facilitates the higher expression of CSAD (BBa_K3771003).

Fig. 3. Mechanism of the SoxRS system

How did we test it?

  To measure the strength and sensitivity of the soxS promoter, we constructed the soxRS regulon in a plasmid and replaced the soxS gene with a reporter, sfGFP. Hence, we can utilize the ELISA reader to detect the expression level of sfGFP (BBa_K1321337) and quantify the strength of the soxS promoter (BBa_K3771048). SDS-PAGE and western blot were performed to confirm the expression of the enzyme.

  As for the oxidant, we have chosen paraquat to address the abnormal functioning of ROS in stressed populations. Various molecules such as superoxide, hydroxyl radical, and hydrogen peroxide can cause oxidative stress to cells[7]. Paraquat is commonly used as an agent to induce oxidative stress for bacteria. It can react with oxygen to produce superoxide, hydrogen peroxide, and hydroxyl radical, etc.[8], which is used to simulate abnormal oxidative stress in the intestines of stressed individuals.

Interferon-gamma (IFN-γ) Sensing System

  To allow E. coli to detect IFN-γ in the human gut, we constructed an OmpA/OprF chimeric protein designed by Aurand and March[9]. The OmpA/OprF chimeric protein (BBa_K3771009) consists of OmpA protein from E. coli and a smaller section of OprF protein from P. aeruginosa.

  Outer membrane protein A (OmpA) (BBa_K3771010) is a transmembrane protein in E. coli that is responsible for maintaining the stability of the bacterial membrane. Serving as the main structure of the OmpA/OprF chimeric protein, the β-barrel conformation of OmpA is composed of extracellular loops that help play a role in the detection and binding of extracellular molecules[10]. On the other hand, OprF is the most common outer membrane porin in P. aeruginosa and has the ability to bind to IFN-γ[11,12]. Since OmpA and OprF are homologous in structure, extracellular loops from each type of protein can be exchanged and constructed into OmpA/OprF chimeric proteins of different amino acid sequences.

Construction of OmpA/OprF Chimeric Protein

Fig. 4. Construction of OmpA/OprF chimeric protein

  To construct our OmpA/OprF chimeric protein, we replaced Loop 1 (AA 38-54) of OmpA from E. coli with Loop 5 (AA 198-237) of OprF from P. aeruginosa[9], which resulted in the final OmpA/OprF chimeric protein (Fig. 4). The new extracellular peptides of the OmpA/OprF chimeric protein allow for binding of IFN-γ to the E. coli outer membrane.

Activation of the Psp System

  Binding of extracellular IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria[13].

Fig. 5. Mechanism of the Psp System[14]

  The mechanism of the signal transduction pathway of the OmpA/OprF chimeric protein to Psp system (Fig. 5). Binding of extracellular IFN-γ to the OmpA/OprF chimeric protein initiates signal transduction from the outer membrane to the inner membrane proteins PspB and PspC[9]. Subsequently, PspB and PspC bind to PspA protein, releasing PspF transcription factor[14]. As a result, free PspF activates the pspA promoter (BBa_K3071013), initiating the production of the enzyme required for taurine synthesis.

How did we test it?

  Experiments were conducted to test whether the two main components of the IFN-γ sensing system, the OmpA/OprF chimeric protein and the pspA promoter (BBa_K3071013), function effectively. Western blot was performed to confirm the expression of the OmpA/OprF chimeric protein. To determine the inducibility of the pspA promoter, various concentrations of human IFN-γ were added to E. coli bacterial culture with our constructed plasmid (PpspA-ofp-PompA-ompA/oprF) (BBa_K3771018), and the expression of the reporter, OFP (BBa_K156009), was recorded using a microplate reader. More information can be found in Proof of Concept.

Menbles

  In order to make our solution appealing to people suffering from depression and the public, we decided to put our bacteria inside a bubble named Menbles. They were made of alginate gel, which was added into calcium chloride to strengthen and form the bubble. These bubbles are acid resistant[15], and therefore these bubbles can protect the bacteria inside when going through the stomach, where gastric acid will pose little effect[16]. The bubbles will then be broken down in the intestine, where pH conditions and combined action of acid and trypsin will cause the alginate to break down[16] and release the bacteria to its surroundings.

  We also noticed that due to difficulty in swallowing among patients of mental illnesses[17], we are also challenged to make our bubbles as tiny as possible, so these patients do not chew and break our bubbles too soon before it reaches the intestine. To achieve a smaller-sized bubble, we dropped the alginate into the calcium chloride drop by drop. We experimented with three different sizes of bubbles, which are the 14.14 (Large), 4.19 (Medium), and 1.77 (Small) mm³ bubbles, to see which size is most effective in protecting the bacteria inside the bubble in different acidic environments.

Fig. 6. (A) Volume 14.14 mm³ (Large) bubble, with bubble diameter approximately 3 mm. (B) Volume 4.19 mm³ (Medium) bubble, with bubble diameter approximately 2 mm. (C) Volume 1.77 mm³ (Small) bubble, with bubble diameter approximately 1.5 mm.

How did we test it?

  Experiments were conducted to test the recovery rate of the bacteria in the bubble in slightly acidic conditions emulating the environment in milk tea to ensure that the bacteria will be protected until its final destination, the intestine. We used CFU quantification to measure the recovery rate of our bubble. Check out more on the Experiments and Results page.

Device

Microfluidics for Bacterial Retention Ratio

  When consuming probiotics, we often do not pay attention to the amount of bacteria that can stay for a prolonged time in the intestine. To address this issue, we developed a microfluidic chip named f(int), which is able to provide information regarding how much engineered E. coli Nissle stays in the jejunum. It can also serve as a platform for other researchers to further improve its functions in the future.

Fig. 7. f(int) microfluidic chip

Functional Test

  In order to visualize the bacterial retention ratio in the jejunum, we designed four kinds of channels with different internal structures. All microfluidic channels are serpentine-shaped, with one design being a smooth channel without any internal structures. The second design includes geometrically ordered circular folds along the channel walls. The other two have additional villi per circular fold, and two villi per circular fold. We also compared them at different conditions, such as with and without additional hyaluronic acid (which represents intestinal mucus [18]). By performing this experiment, we hope to gain a better understanding of how the internal villi structures affect the retention ratio of bacteria inside the intestine. For more information, check our Hardware page!

References

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e-mail : igem.ncku.tainan@gmail.com
phone : 06-2757575