Team:FAFU-CHINA/Parts
Overview
In order to construct a photocontrol system in the co-culture of E. coli and yeast, we need to introduce photocontrol elements in eukaryotes.
In eukaryotic cells, there is a ubiquitination pathway for protein degradation. By combining light sensors with this pathway, light signals could be used to regulate protein abundance. In order to quickly detect the expression of the target gene, we also created a matching detection element.
Basic Parts
| Name | Type | Description | Designer | Length(bp) |
|---|---|---|---|---|
| BBa_K3732000 | Protein_Domain | photosensitive degron (psd) module. | Jiehui Ye | 1248bp |
| BBa_K3732002 | Reporter | mRFP1 | Jiehui Ye | 675bp |
| BBa_K3732001 | Translational_Unit | kanMX | Jiehui Ye | 1356bp |
| BBa_K3732004 | Regulatory | CMV | qinyu chen | 204bp |
| BBa_K3732005 | Regulatory | CMV enhancer | qinyu chen | 304bp |
BBa_K3732000
★LOV2
The light-receptor domain LOV2 binds flavin mononucleotide(FMN) as a cofactor, which is the main photoactivated compound. Excitation of the FMN cofactors by blue light results in the formation of a bond between FMN and cysteine in the LOV2 domain. This induces structural rearrangement of the LOV2 domain, leading to the unfolding of the α -helix at the carboxyl terminal.
★Protein structure of the LOV2 domain and light-induced structural changes during the photocycle
The green strip shows the β -folding of the LOV2 core, while the rod-like structure highlights three key residues in FMN and signal transduction, Cys966, Phe1008 and Gln1029.
★LOV2 activation stage and experimental test time
The dynamics of the chromophore mainly occur on the ultra-fast time scale, and the protein matrix changes occur after this, and the time constant of the entire Jα helix unwinding is 313µs. Compared with the light state Jα helix, corresponding to the residues of the whole protein including the Jα helix and the Jα helix alone, the RMSD vs. time is plotted as shown below.
★cODC1
In ODC, the carboxy-terminal 37 amino acids (cODC) function as a degron that is directly recognized by the proteasome.
By fusing cODC1 to the end of LOV2, we have an element that uses light signals to regulate protein abundance.
When LOV2 is activated by light，which makes the cODC1 degron accessible for recognition by the proteasome and ubiquitin - independent degradation of the whole construct.
BBa_K3732002
MRFP1 is a codon optimized RFP, which is suitable for expression in SACCHARomyces cerevisiae. It is an important reporter gene for detecting the performance of photocontrol elements.We will observe the intensity of red luminescence in the sample through an electron microscope to test the basic function of the light control element.
BBa_K3732001
It is a complete transcription unit that makes yeast resistant to KanR and is used for screening transformants.
BBa_K3732004
Human cytomegalovirus(CMV) immedicate early promoter.
BBa_K3732005
Human cytomegalovirus immediate early enhancer.
Composite Parts
| Name | Type | Description | Designer | Length(bp) |
|---|---|---|---|---|
| BBa_K3732003 | Reporter | Test element for optical control element | Jiehui Ye | 3293bp |
BBa_K3732003
This is a composite component composed of BBa_K3732002, BBa_K3732000 and BBa_K3732001.
We fused the DNA sequence of BBa_K3732000 with the 3 'end of the DNA sequence of the red fluorescent protein in the plasmid to study the biological function of BBa_K3732000 in the early stage. Meanwhile, BBa_K3732000 and kanMX were connected in series in the plasmid. After the element was tested, primers with upstream and downstream homologous arms of the termination codon of the target gene were designed to clone 5 'mRFP1 to kanMX 3' for homologous recombination. As a result, BBa_K3732000 and KanMX were added to the loci of the target gene.
