Parts related to ...
Our parts collection consists of five groups of parts:
... the secretion of exendin-4
Our basic part, Exendin-4 (BBa_K3096030) is a GLP-1 analog with increased resistance to degradation by the peptidase DPP-IV. In vivo, Exendin-4 should increase Insulin secretion from the pancreas. In order to make its secretion by bacteria possible, a secretion tag (BBa_K1012004) was fused to the amino terminus of Exendin-4. Moreover, we added an RBS (BBa_K3096000) and a terminator (BBa_B0014) necessary for a correct biosynthesis of the part. Additionally, a cell penetrating peptide called Penetratin (BBa_K3096013) was fused to Exendin-4 C-terminally to enable the delivery through cell membranes. In total, the composite part BBa_K3096042 encodes for an ameliorated Exendin-4 which can be secreted and contains a cell penetrating peptide. The addition of a Tet repressible promoter (BBa_K3096045) and combination with our glucose sensor (see section iii)) yields the part BBa_K3096044.
... the CRISPR/Cas 3 system
Cascade (BBa_K3096007), is a basic part representing CasA-CasB-CasC-CasD-CasE-Cas1-Cas2-CRISPR repeats which belong to the CRISPR cassette of Cas3. Together with the Cas3 basic part (BBa_K3096001), they form a type 1 CRISPR system used to degrade foreign DNA sequences which are targeted through guide RNA arrays. The Cascade and Cas3 bricks were linked to pBAD (BBa_K3096054, BBa_K3096055) in order to connect them with our regulatory system. Additionally, we designed an Mnt responsive promoter (BBa_K3096050) and combined it with our CRISPR array targeting the genomic DNA (BBa_K3096014) controlling its expression (BBa_K3096051), which can be linked to the N-Acetyl Glucosamine-sensing system. The expression of a second CRISPR array (BBa_K3096052) targeting Exendin-4 is controlled by an LsrR-regulated promoter (BBa_K3096053). It is thus linked to the availability of fatty acids.
... our regulatory systems
To refine our kill switch and allow for glucose-dependent secretion, we designed multiple regulatory composite parts. First of all, we designed a catabolite repression-responsive promoter without a LacI binding site, which should therefore allow for glucose-sensing (BBa_K3096011, modified from BBa_R0010). Subsequently, we added an RBS (BBa_K3096000), a Tetracycline repressor protein (BBa_C0040) and a terminator (BBa_B0014) to this part in order to produce a glucose-dependent Tetracycline repressor (BBa_K3096012). In our project, this part was meant to control the glucose-dependent secretion of Exendin-4.
Another composite part we designed, BBa_K3096002, consists of an RBS (BBa_K3096000), AraC (BBa_K1088005) and a terminator (BBa_B0014). It was designed to build a basis for AraC expression, with the possibility of adding a preferred promoter to the 5’ end.
The AraC part was then put under the control of the of the Clt promoter by combining it also with a constitutive Clt expression (BBa_K608351). This yielded the final temperature responsive system (BBa_K3096040).
We added a FadR promoter (BBa_K851060) 3’ of a constitutive FadR generator (BBa_K86105), which contains a constitutive promoter, an RBS and FadR. Downstream of the FadR promoter, we also added an RBS (BBa_K3096000). Altogether, the designed composite part BBa_K3096005 constitutes to fatty acid (Acyl-CoA) concentration-dependent activation of the downstream promoter. This can be used for the fatty acid-dependent expression of a desired protein. In our project, we used the composite part for the fatty acid-dependent activation and inactivation of our CRISPR/Cas3 system by regulating the Lsr (BBa_K091001) expression with it. The whole fatty acid responsive system is summed up in BBa_K3096046.
The part BBa_K3096020 is composed of a NagC generator, a constitutive promoter (BBa_J23100), a, RBS (BBa_K3096000), nagC (BBa_K3096018) and a terminator (BBa_B0014). NagC is a protein sensing the concentration of N-acetyl-Glucosamine 6-phosphate (GlcNAc 6P) which, in the presence of the substrate, inhibits the promoter pNagC.
Additionally, we modified BBa_K1937003, an mRFP generator controlled by the pNagC promoter for our personal needs by deleting the mRFP, yielding BBa_K3096006. Moreover, we added the Mnt repressor BBa_C0072 and the terminator BBa_B0014 3’ of the biobrick, altogether forming our composite part BBa_K3096008. These parts are the basis for the regulatory element of Glc-NAc sensing, which is summed up in BBa_K3096048.
Generally, we used BBa_I13453, the pBAD promoter, BBa_K091001 and the LsrR gene for the design and cloning of our constructs. Moreover, we re-synthesised BBa_K608351 since we were not able to transform our bacteria with the construct provided from the distribution kit. The Biobrick contains part of the temperature sensing system, which allowing for the expression of a desired protein at temperatures above or below 37°C.
Reporters
We designed a pMnt-RFP reporter (BBa_K3096025) to measure the expression of MntR and thus the sensing of GlcNAc. If GlcNAc is sensed, the reporter should lose its ability to express RFP.
Parts from our CPP collection
For our modelling, we designed multiple cell penetrating peptides (CPPs) with a His-tag (BBa_K3096019) for purification and antibody detection as well as a GFP with linkers (BBa_K3096023), making cellular visualisation possible.
CPPs are used for the transport of proteins through cellular membranes. We characterized them as biosafety level 1. In order to optimize the transport of Exendin-4 into the circulatory system, we designed various cell penetrating peptides: YTA2 (BBa_K3096010, composite: BBa_K3096026), Tp10 Transportan 10 (BBa_K380004, composite: BBa_K3096029), SynB1 (BBa_K3096017, composite: BBa_K3096035), Penetratin (BBa_K3096013, composite: BBa_K3096037)), pVec (BBa_K3096015, composite: BBa_K3096036) and TAT-LK15 (BBa_K3096016, composite: BBa_K3096038).
Please visit the registry to learn more about our parts.
