Composite Parts Synthesised for THE BIG PIE
All cassettes are cloned into pGGA plasmids part of NEB’s Golden Gate Assembly Kit. The plasmid has a BsaI sequence in opposing directions, while the fragments are flanked by BsaI sequences on either side, flanked by complementary custom overhang generators. Digestion with BsaI creates overhangs on either side of the cassette and in the plasmid, and the corresponding overhangs bind together, allowing ligation to take place, with the final ligation product lacking intact BsaI restriction sites.
Transcription factor-mediated “reprogramming”, or “trans-differentiation”, approaches have been used to regenerate beta-like cells in vitro and in vivo. The studies have demonstrated that the overexpression PDX1, NEUROG3, and MAFA, can partially reprogram the fate of various non-beta cell types into glucose-responsive insulin-producing cells.
PDX1 : Also known as insulin promoter factor 1, is a homeodomain transcription factor. PDX1 is required for early embryonic development of the pancreas and the differentiation of endocrine cell types, including beta cells. In mature beta cells, depletion and reduction of PDX1 induces glucose intolerance, which suggests the critical role of PDX1 in maintaining beta-cell function. PDX1 also binds to the regulatory elements and increases insulin gene transcription.
NEUROG3 : This is a member of the basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development. Despite several studies, it remains unclear whether NEUROG3 is absolutely required for beta-cell development in humans.
MAFA : 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. Hence, the expression levels of MAFA are critical for proper beta-cell maturation. For regenerative approaches, MAFA’s expression is also important to regenerate functional and mature beta cells from pluripotent stem cells.
Cassette to be Cloned
TF Polyprotein Sequence
Map20 -Linker -
TEV Protease Substrate: We’ve added a cleavage site after the secretion tag here, because β-Catenin’s N-terminus is important for its interaction with the factors that degrade it, and we want to make sure that it’s undisturbed. Our protease evolved to separate polyproteins in cis, so we know it can do this. We’ve put an NES at the C-terminus to make sure that it stays in the cytoplasm, since β-catenin is a nuclear transcription factor.
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.
Cassette to be cloned
NH2 - Map20 - Linker -
The Delivery vector has a single domain antibody (sdAbs) from camelids(VHH). The antibody binds only to the LGR5 antigen present on the(crypt base columnar cells) CBCs at the base of the epithelial cell layer in the duodenum. However, to improve the antibody binding to the delivery vector. We are using β-intimin, a barrel-shaped protein, because the sdAbs bound to the β-domain of intimin displayed on E Coli show higher antigen-binding capacity. Thereby improving the antigen-antibody specificity and binding between the delivery vector and the target cell.
Cassette for Antibody
Our modified probiotic is required to be active only in the duodenum of the small intestine. To ensure this we have designed a kill switch that reduces the activity of the bacteria by the time it reaches the colon and kills it as soon as it is excreted out of the human body. We propose a single gate phosphate regulated kill switch that consists of a toxin, anti-toxin and repressor system.
Cassette for Kill switch
|1||Promoter||Pbad: Arabinose-Inducible Expression System||1279 nt|
|2||RBS||Ribosome binding site||12 nt|
|3||Terminator||Bidirectional Terminator consisting of BBa_B0010 and BBa_B0012||129 nt|
|4||Coding (NLS)||SV40 NLS: Polypeptide containing SV40 nuclear localization sequence||57 nt|
|5||Export Signal Tag||Map20: T3SS export signal peptide derived from Map gene||93 nt|
|6||Constitutive Promoter||Can be used to tune the expression level of constitutively expressed parts||35 nt|
|7||pH sensitive promoter||GadA-RBS is a pH sensitive promoter that has been shown to initiate transcription at low pH.||264 nt|
|8||Phosphate promoter||PhoA promoter is activated as a result of active binding of phosphorylated-PhoB, resulting in downstream expression of GFP. When a high concentration of extracellular phosphate ions is present, PhoR will dephosphorylate PhoB, and therefore inactivating it, and repressing the PhoA promoter for transcription.||85 nt|
|9||Colicin E2 DNase domain "miniColicin"||Colicin E2 is a peptide that is toxic to coliform bacteria. It is one of a group of proteins of this type, it being a nicking endonuclease. It has a corresponding activity inhibiting protein called immunity protein Im2, which is also in the registry under the name BBa_K1976027.||401 nt|
|10||Colicin E2 immunity protein (Im2)||This part encodes for the wild-type immunity protein inhibiting the nicking DNase Colicin E2. According to molecular dynamics simulations this protein inhibits also miniColicin (BBa_K1976048 and BBa_K1976049).||261 nt|
|11||cI lam Promoter||Lambda phage cI-repressible promoter. Facilitates high expression levels, while retaining the ability to shut off expression downstream of a desired signal.||49 nt|
|12||cI Repressor||Bacteriophage Lambda-derived transcriptional repressor. Binds to and inhibits transcription from cI lam promoter.||707 nt|
|13||Tev Protease||Tev Protease with a single S219V mutation in the internal cleavage site||729 nt|
|14||Coding (Degradation Tag)||Transcription factor and an effector of the Wnt signalling pathway. It is protected from degradation by the Wnt cascade, active in our target niche.||2343 nt|
|15||Coding (Protease Cleavage Site)||Contains two consecutive sites sensitive to cleavage by the Tobacco Etch Virus protease.||21 nt|
|16||Coding (Nuclear export Signal)||Mediates nuclear export when tagged to the C-terminus of a nuclear protein.||42 nt|
|17||Coding (TF)||Pdx1 is a homeodomain transcription factor and is required for early embryonic development of the pancreas and the differentiation of endocrine cell types including β cells.||852 nt|
|18||Coding (TF)||MafA is a transcription factor that binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose.||1062 nt|
|19||Coding (TF)||Ngn3 is a member of the basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development.||645 nt|
|20||Cell Surface Receptor||Intimin is an important virulence factor of enteropathogenic E. coli (EPEC) and mediates intimate adhesion to the intestinal epithelium of the host.||1977 nt|
|21||Coding||Lgr5 Crypt cell specific antibody||Pending|
|22||Composite||Encodes a system designed to deliver PDX1, MAFA and NGN3 into targeted human cells via the T3SS.||4840 nt|
|23||Composite||Encodes a device designed to selectively process the effector polyprotein in our target cells.||4768 nt|
|24||Composite||Encodes a display system that biases the binding of our chassis towards crypt base columnar cells.||Pending|
|25||Composite||Encodes a phosphate-sensitive kill switch based on the E2-IM2 toxin-antitoxin system.||1986 nt|
|26||-||Ensures adequate spacing between the SD site and the start codon.||6 nt|
|27||Coding||Separates different domains on a synthetic fusion protein.||33 nt|
|28||Coding||Enables analysis on a Western blot.||48 nt|
|29||Coding||This part encodes for the coding sequence of Tev Protease with a single S219V mutation in the internal cleavage site. (BBa_K1639008)||729 nt|
|30||Coding||This part encodes for a signal peptide derived from the map gene from the Enteropathogenic E. coli strain E22.||60 nt|
|31||Coding||This part encodes for the cI repressor derived from the Escherichia coli phage lamda (Bacteriophage lamda).||714 nt|