Team:OUC-China
Parts
Overview
This year,OUC-China constructed many new parts that can help increase the production of engineered bacteria.We hope our parts will be adaptive to every microbial fermentation factories.Our parts include genes related to quorum sensing,ribozyme riboswitch and transport RNA.
Quorum sensing
In our platform when the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the transcription of production gene. We transformed our fragments into the genome of our engineering bacteria and measured the intensity of SSRE promoter.It is worth mentioning that, in order to better characterize the SSRE promoter, we optimized the codon of the part BBa_E0040 and expressed it in Aureobasidium melanogenum, expanding the application chassis range of this part. The sequence optimized of the codon also can be a suitable part for the team committed to developing the potential of Aureobasidium melanogenum.
| Part | Description | Type |
| BBa_K4193004 | SSRE Promoter | regulatory |
| BBa_K4193005 | AtlPT4 | coding |
| BBa_K4193006 | AtCRE1 | coding |
| BBa_K4193018 | PTP2 | coding |
| BBa_E0040 | GFP | coding |
| BBa_K4193026 | Qs system(production) | composite |
| BBa_K4193027 | Qs system(validation) | composite |
Ribozyme riboswitch
To make sure our platform always maintains high production, our team used a ribozyme riboswitch to identify the product molecule. And through further engineering, we used the aptamer to construct a kill switch that only lets the engineered bacteria live when the product molecule is produced in high intensity.To construct different riboswitches with different threshold,we change few 5’ bases to regulate activities of helix-slipping-based ribozyme switches.
| Part | Description | Type |
| BBa_K4193002 | Ribozyme riboswitch | RNA |
| BBa_K4193020 | Ribozyme riboswitch | RNA |
| BBa_K4193021 | Ribozyme riboswitch | RNA |
| BBa_K4193022 | Ribozyme riboswitch | RNA |
Transport RNA
There are also some other parts used in our project
In order to transport the products out of the engineering bacteria in time and break through the limitation of limited types of protein transporters, OUC-China decided to apply the aptamer to the transmembrane transport of products. This RNA molecule with transporter function consists of three strands of two different RNA. Among them, one RNA is responsible for targeting the cell membrane structure, and one RNA is responsible for specifically binding products.
| Part | Description | Type |
| BBa_K4193003 | RNA10 | RNA |
| BBa_K4193013 | RNA9 | RNA |
| BBa_K4193014 | RNAcomplex(RNA9:RNA10=2:1) | Composite |
Other parts
There are also some other parts used in our project.It is worth mentioning that GABA-1 aptamer was de novo designed by using the secondary structure of the aptamer and the specific binding with the ligand to the aptamer and molecular docking and dynamic simulation for testing.But it didn’t work as we expected.So we built the new RNA aptamer GABA-2. After obtaining experimental data of GABA-1, we learned experience and de novo designed an new aptamer with stronger binding ability on the basis of GABA-1.
| Part | Description | Type |
| BBa_K4193000 | GABA RNA aptamer1 | RNA |
| BBa_K3286216 | GABA RNA aptamer2 | RNA |
| BBa_K4193016 | electrcompeter plasmid | Plasmid |
