UPF-SkinBait 2024 IGEM Team is developing an innovative platform designed to treat and prevent skin parasitic infections by leveraging the human skin microbiome. Our initial focus is on combating scabies.
To address this issue, our team has engineered 11 fundamental parts, which are combined to create 12 new composite parts. These components are grouped into two main functional blocks:
The first block includes components specifically designed to combat scabies through the expression of Cry proteins (Cry3A and Cry4Ba). To regulate their expression, we selected the BBa_J23119 promoter (designated MG_010), which functions strongly in both E. coli and C. acnes, and MG_026, which is active only in C. acnes.
In the second block, we first focused on ensuring that the engineered microbiome, intended for use as a lotion, could effectively release its therapeutic components. To achieve this, we incorporated the C. acnes signal peptide RoxP, which aids in protein export. However, it remains uncertain whether the cry proteins will be successfully exported. Therefore, the next step for this block was focused on incorporating a RNA thermometer, which is a temperature-sensitive non-coding RNA molecule that post-transcriptionally regulates the expression of a C. acnes-specific endolysin (CAP 10-3 endolysin).
At lower temperatures, the RNA adopts a stable secondary structure that blocks the ribosome-binding site, preventing translation. As the temperature increases, this structure melts, exposing the ribosome-binding site, thereby allowing translation and leading to endolysin production, which induces cell lysis, thereby releasing the cry proteins.
To evaluate the functionality of the RNA thermometer, we coupled it with fluorescent markers, such as sfGFP and mCherry, and tested its response under different temperature conditions. Our team also wanted to characterize the behavior of the RNA thermometer and was first going to be coupled with two different fluorescent proteins, sfGFP and mCherry, in order to evaluate its behavior when exposed to different temperatures.
Finally the parts can be grouped with the following tables: Basic Parts and Composite Parts.
| DNA Parts | Registry | Length (bp) | Description |
|---|---|---|---|
| MG_010 | Part:BBa_J23119 | 35 | Codes for the strong promoter MG10 from the Anderson’s collection, shows expression on E. coli but also on C. acnes.1 |
| MG_026 | Part:BBa_K5311000 | 194 | Codes for the strong promoter MG26, originating from one of the natural promoters in C. acnes that regulates the 16S ribosomal RNA region, doesn’t work on E. coli. |
| RBS cell free MJ | Part:BBa_K5311004 | 29 | Codes for the Ribosome binding site applied on the Cry proteins parts to ensure expression. |
| Cry3Aa | Part:BBa_K5311002 | 2105 | Codes for the Cry3Aa toxin, actuator which we aim to demonstrate its efficiency on Sarcopter Scabiei.2 |
| Cry4Ba | Part:BBa_K5311003 | 3557 | Codes for the Cry4Ba toxin, actuator which we aim to demonstrate its efficiency on Drosophila.3 |
| RoxP | Part:BBa_K5311001 | 69 | Codes for the RoxP, which is a signal peptide used on C. acnes to detect the expression for the Cry proteins. |
| RNA Thermometer | Part:BBa_K115002 | 52 | Codes for the RNA thermometer FourU, biosafety mechanism that allows expression of our protein of interest when exposed to a temperature near to 37ºC. 4 |
| CAP 10-3 Endolysin | Part:BBa_K5311006 | 858 | Codes for the recombinant endolysin from bacteriophage CAP 10-3, biosafety mechanism that has lytic activity against C.acnes.5 |
| sfGFP | Part:BBa_K5311007 | 738 | Codes for the sfGFP, variant of the Green Fluorescent Protein (GFP) engineered for enhanced folding efficiency, used for the proof of concept of the RNA thermometer on C. acnes. |
| mCherry | Part:BBa_K5311008 | 708 | Codes for the mCherry, a red fluorescent protein derived from the coral Discosoma, used for the proof of concept of the RNA thermometer on C. acnes. |
| DNA Parts | Registry | Length (bp) | Description |
|---|---|---|---|
| MG_010 + RBS cell free MJ + Cry3Aa + RoxP + His-tag | Part:BBa_K5311010 | 2268 | Genetic construct created to produce Cry toxin 3Aa constitutively in C. acnes, it can be also expressed in E. coli. |
| MG_026 + RBS cell free MJ + Cry3Aa + RoxP + His-tag | Part:BBa_K5311011 | 2419 | Genetic construct created to produce Cry toxin 3Aa constitutively in C. acnes. |
| MG_010 + RBS cell free MJ + Cry4Ba + RoxP + His-tag | Part:BBa_K5311012 | 3712 | Genetic construct created to produce Cry toxin 4Ba constitutively in C. acnes, it can be also expressed in E. coli>. |
| MG_026 + RBS cell free MJ + Cry4Ba + RoxP + His-tag | Part:BBa_K5311013 | 3871 | Genetic construct created to produce Cry toxin 4Ba constitutively in C. acnes. |
| MG_010 + RBS cell free MJ + CAP 10-3 Endolysin + His-tag | Part:BBa_K5311014 | 936 | Genetic construct created to produce the CAP 10-3 Endolysin constitutively in C. acnes, it can be also expressed in E. coli. |
| MG_026 + RBS cell free MJ + CAP 10-3 Endolysin + His-tag | Part:BBa_K5311015 | 1095 | Genetic construct created to produce the CAP 10-3 Endolysin constitutively in C. acnes. |
| MG_010 + RNA thermometer FourU + sfGFP + His-tag | Part:BBa_K5311016 | 839 | Genetic construct created to produce the fluorescence protein sfGFP when the temperature reaches 37ºC in C. acnes, it can be also expressed in E. coli. |
| MG_026 + RNA thermometer FourU + sfGFP + His-tag | Part:BBa_K5311017 | 998 | Genetic construct created to produce the fluorescence protein sfGFP when the temperature reaches 37ºC in C. acnes. |
| MG_010 + RNA thermometer FourU + mCherry + His-tag | Part:BBa_K5311018 | 809 | Genetic construct created to produce the fluorescence protein mCherry when the temperature reaches 37ºC in C. acnes, it can be also expressed in E. coli. |
| MG_026 + RNA thermometer FourU + mCherry + His-tag | Part:BBa_K5311019 | 968 | Genetic construct created to produce the fluorescence protein mCherry when the temperature reaches 37ºC in C. acnes. |
| MG_010 + RNA thermometer FourU + CAP 10-3 Endolysin + His-tag | Part:BBa_K5311020 | 959 | Genetic construct created to produce the CAP 10-3 Endolysin when the temperature reaches 37ºC in C. acnes, it can be also expressed in E. coli. |
| MG_026 + RNA thermometer FourU + CAP 10-3 Endolysin + His-tag | Part:BBa_K5311021 | 1168 | Genetic construct created to produce the CAP 10-3 Endolysin when the temperature reaches 37ºC in C. acnes. |