Team:IISER Bhopal/Design

iGEM 2020 || IISER_Bhopal Design


The BIG PIE is a biological delivery system created to allow insulin production in-vivo within the body. The objective of BIG PIE is to compensate for the decreased/defective β-islet cells in the pancreas of diabetes patients by reprogramming the gut cells into β-islet cells and eliminating the need for external sources of insulin. We intend to package our modified bacteria as a capsule which creates our final product iβeta.

The design of this project has been refined constantly to create a delivery system which is effective, reliable and most importantly safe by design.

Click on the individual parts of this Bacteria to know more!

Representative Bacteria

Representative bacteria shows all constructs we intend to clone into our bacteria delivery system.

  • Plasmid1(left) : Polyprotein sequence with 3 effector proteins and protease fusion sequence have been cloned for reprogramming into β cells.
  • Plasmid2(right): Kill switch construct needed to kill bacteria outside the body.
  • Needle Like T3SS injection for penetrating and attaching to gut cell membrane.
  • VHH antibodies specific to the target cell.

Project Principle

Our project is based on the principle of “transdifferentiation”. We intend to reprogram a set of the newly differentiated intestinal crypt cells in the duodenum (gut) to β-islet-like cells, capable of glucose-responsive insulin secretion.

(Left) Pluripotent cell undergoing dedifferentiation then transitioning to another cell type. (Right) Stem cell directly transdifferentiating into another cell type without an intermediate step.

Previously transdifferentiation has been performed in human organoids and mice models with the aid of adenovirus vectors: a viral delivery system. Due to the pathogenic nature of viruses and the risks associated with them (mutagenesis, oncogenesis and immunogenicity), we decided to replace the viral vector with a probiotic bacterial vector that is safely accepted into the host.

Keeping in mind these aspects of our project, our design stands on 4 pillars which form the backbone of our work.

Vector for Transport and Delivery

Our project iBETA is using an FDA approved probiotic bacteria E.coli Nissle 1917. E.coli Nissle is a nonpathogenic Gram-negative strain used in the therapies of many gastrointestinal disorders including diarrhoea, irritable bowel syndrome etc and can reach the crypt cells in the duodenum of the small intestine.

E.coli Nissle is classified as a probiotic due to its lack of defined virulence factors along with the expression of fitness factors such as microcins, and iron uptake systems, and proteases which may support its colonization of the human gut. EcN exhibits a semi-rough lipopolysaccharide (LPS) phenotype and serum sensitivity and does not produce known toxins. EcN colonizes the intestine within a few days and it remains as colonic flora for months after administration.

Our literature reviews showed us that several cell types in the stomach, gut, liver could be transformed into beta islet-like cells capable of producing insulin. But we chose the gut because it is easily accessible. Our bacterial drug can reach the gut by oral consumption via capsules. The well-characterised environment of the intestine can easily host our bacteria and will show a long term result to our treatment. The crypt cells in the gut were part of the epithelium which made it relatively easy to access, most importantly they are young and developmentally flexible stem cells, that can be easily reprogrammed into beta islet-like cells by our bacterial delivery system using the principles of transdifferentiation.

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Priscilla Pie the E.coli is our mascot. Represents a probiotic bacteria E.coli Nissle, our chosen delivery vector capable of transforming the gut cells into β cells.
Attachment and Injection of Proteins
Representation of Type 3 Secretion System (T3SS) derived from pathogenic strain of E.coli. The T3SS tip penetrates the host cell membrane and transports effector proteins into the gut cell.

We intend to use a novel delivery mechanism : Type III Secretion System (T3SS)8 -‘molecular syringe’ found on certain Gram-negative bacteria consisting of a basal body and filament . The T3SS cloned in E.coli Nissle will transport the linear polypeptide sequence consisting of effector proteins’ - PDX, MAFA and NGN3 into the crypt cells in the gut.

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Construct with Polyprotein Sequence consisting of effectors - PDX1, MAFA and NGN3 and another sequence coding Tobacco Etch Virus(TEV) protease.

Studies have demonstrated that overexpression PDX1, NEUROG3, and/or MAFA, can reprogram the fate of various non-β cell types into glucose-responsive insulin-producing cells. Here’s an explanation of every part of our plasmid construct.

MAP 20
Mitochondria associated protein is a 20 amino acid long export signal peptide sequence for T3SS, which helps guide the protein sequence through the T3SS pore.

TMV Protease Substrate
Protease cleavage sites are required to separate and activate the transcription factors in the cytosol of the effector cells.

is a homeodomain transcription factor and is required for early embryonic development of the pancreas and differentiation of endocrine cell types including β cells. It plays a critical role in maintaining β-cell function.

is a member of the basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development.

also known as RIPE3b1, is a member of the MAF family of basic leucine zippers. It is a transcription factor that binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. For regenerative approaches, MAFA’s expression is also important to regenerate functional and mature β cells from pluripotent stem cells.

Interaction among PDX1, NEUROG3, and MAFA Transcription factors PDX1, NEUROG3, and MAFA are also mutually interacted/regulated during the pancreatic developmental process. PDX1 regulates the expression of NEUROG3 and MAFA in the development and reprogramming processes.

Represents the interaction of PDX1 with MAFA and NGN3 in regular β cells and how that will be mimicked in gut cells.

As per literature the proteins PDX, MAFA and NGN3 have been able to survive in human organoids and gut cells in mice. There is no protease action on these proteins as they are pre-existent in the gut, in lower concentrations. The presence of these transcription factors at high concentrations will influence the regulatory pathways to produce insulin in the reprogrammed gut cells while ensuring survival of the transcription factors as they are not considered foreign in the gut cells.

Targeting System

There were a number of strategies thought of for the targeting system. But we finally decided on a three-step targeting system. To ensure absolute accuracy and specificity in the attachment of our delivery vector to the target cell. It was imperative that transdifferentiation occurs specifically and only in the undifferentiated crypt cells of the duodenum.

While drug delivery design is not directly connected to the project design. It is essential to our targeting system prior to cell binding. Our therapeutic is enclosed in a capsule that forms an essential part of our targeting system to ensure the efficient and safe delivery of the bacteria to our target cells. Our bacteria are enclosed in an HPMC capsule which is pH sensitive and prevents the release of the bacteria in the stomach while our microspheres allow targetted entry into the crypt regions present below the epithelial mucous. To find out more about our capsule targeting system visit the implementation page.

Image I: Cannot resolve img-desc ~~ Prior to binding

Antibody specific to Crypt Cells We intend to display a Single domain Antibody (SdaB) with a β intimin base attached to E.coli Nissle surface. The SdaB corresponds to the Lgr5 antigen exposed on the crypt cells(CBCs) in the gut. The antibodies ensure that the delivery vector binds to the right type of cells. The single-domain antibodies from camelids (nanobodies or VHH) bind to β intimin a surface protein derived from EHEC. The display of the VHH on intimin guarantees a stronger affinity for the cell-specific antigens. The sdABs also survived better on the E.coli surface with very little proteolysis or cell toxicity. Thus this antibody display on E.coli surface specific to antigens on crypt cells is the first step that ensures accurate binding of the delivery vector to the crypt cells.

The strategies we are currently exploring involve displaying antibodies against cell-type-specific markers on E. coli’s surface10, displaying a cell-specific protease-sensitive T3SS inhibitor on E. coli’s surface, and fusing our effectors to C-terminal cytoplasmic retention signals downstream of the cleavage site of a cell-specific protease.

TEV Protease attached to β catenin cleaves the polyprotein sequence of PDX1, MAFA and NGN3 separating the transcription factors which are then transported to the nucleus due to their nuclear localisation signal and show their effect of reprogramming the cell.

Our final regulation step takes advantage of a pre-existing Wnt signalling pathway present mostly in the crypt base cells which is our target cell. We intend to clone a protein protected by the Wnt signalling pathway, β-catenin along with the protease TEV Protease (S219N) can cleave our polyprotein sequence and seperate the Transcription factors PDX1, MAfa and NGN3 in the cell cytosol.
In most cells β catenin(and attached protease) is degraded by the environment and hence the polyprotein sequence will not be active. However, in the crypt cells, the Wnt signalling pathway protects the β catenin, and the tagged protease can survive.
Since β catenin is a transcription factor usually found in the nucleus. We are also placing an NES(Nuclear Export Signal) at its C-terminus to retain it in the cytosol of the effector cell. This allows the cleavage of the polyprotein sequence in the cytosol, transport of the transcription factors to the nucleus and activation of downstream signalling required for cellular reprogramming.

Bacteria bind to crypt cells with Lgr5 receptors on their surface. T3SS injection penetrates the cell. CesT Chaperone unfolds the polyprotein and MAP20 guides the sequence across the T3SS pore into the gut cell where the 3 transcription factors act in the nucleus and reprogramme the cell to create β-islet like cells.
Biosafety Mechanism
(1) anti-toxin binds to toxin rendering it inactive in the functional state of the bacteria. (2) the low phosphate conditions beginning in the colon cause the PphoA promoter to activate translation of the repressor protein. The repressor binds to the promoter upstream of anti-toxin. This stops anti-toxin production. Excess toxin in the environment eventually kills the bacteria.

Safety has been a primary concern while creating Genetically modified organisms (GMOs). GMO usage has led to several claims and arguments about its unwarranted impacts on both the consumer and the surroundings. Keeping these safety aspects in mind, we have made some design considerations in our project which will ensure the safety of the gut microbiome, the host and the environment in which the pill contents will be released.

The construct shows the expression of cI repressor under a phosphate promoter active at low Pi concentration. The cI repressor binds cI lam promoter upstream of anti-toxin. Blocking anti-toxin formation. This allows free expression and increased concentration of toxin eventually killing the bacteria under low Pi as found outside the body.

To avoid GMO release into the environment we propose a single gate phosphate regulated kill switch that is active at low phosphate concentrations and kills our probiotic bacteria by the time it is egested and enters the environment (which has low phosphate conditions).

To kill the engineered probiotic, the Toxin - Colicin E2 DNase domain "miniColicin" (BBa_K1976048), is expressed under a constitutive promoter Pbad. This toxin can degrade host DNA whilst posing little threat to the gut microbiota. In the bacteria’s functional state, the toxin activity is inhibited by expressing the anti-toxin - immunity protein IM2 (BBa_K1976027) -which has a high affinity for miniColicin’s DNAse domain and prevents the toxin from functioning. The anti-toxin is expressed under promoter cI lam (BBa_R0051).

Killing Mechanism
The kill switch is activated at low phosphate conditions. At high phosphate concentrations in the small intestine, the PphoA promoter is inactive, allowing the bacteria to function. At low phosphate concentrations post egestion of the bacteria and removal from the body, PphoA promoter is activated and transcribes a repressor protein called cI repressor derived from E.coli phage lambda. This repressor binds to the cI lam promoter and renders it inactive, thus stopping the translation of anti-toxin inside the cell. The constitutively expressed toxin increases in concentration over time. It kills the bacteria which dies in the low phosphate environment by the time it is egested and released into the environment.

The human gut is colonised by millions of bacteria. A major concern with our proposed probiotic THE BIG PIE, is its release into the small intestine, which could cause in horizontal gene transfer, resulting in lab made plasmids entering the other natural bacteria present in the gut microbiome.Keeping this in mind, we propose to use chromosomal integration of our parts in the final probiotic bacteria that we will introduce into the pills. The integration of our genetic circuits and cassettes into the chromosome will remove any possibility of horizontal gene transfer.