Team:NYMU-Taipei/Parts
Parts
Overview
This year, our parts can be divided into categories of three, including plasmids for basic constructs, plasmids for constructs of detection assay and also plasmids for constructs of spike cleavage design.
Plasmids for basic constructs
This category includes the parts of original proteins or protein fragments, ranging from RBD of hACE2, pepP protease(for spike protein cleavage), and the bacterial expression vector we use to express the proteins
Plasmids for constructs of detection assay
This category includes the modified plasmids which are finally translated into protein complex that is used in spike protein detection assay, e.x. red fluorescent protein
Plasmids for constructs of spike cleavage design
This category includes the plasmid for the spike protein cleavage design, which will finally be translated into the construct of “Protease-RBD of hACE2” and spike fragments(No.1-4)
Basic Parts
| Name | Description | Type | Designer | Length |
|---|---|---|---|---|
| BBa_K3682000 | Bacterial expression vector | Backbone | You-Cheng,Lee | 1733bp |
| BBa_K3682001 | pepP sequence | DNA | Yan-Syun,Huang | 1323bp |
| BBa_K3682002 | hACE2 RBD sequence | DNA | You-Cheng,Lee | 2238bp |
| BBa_K3682003 | RFP sequence | DNA | Yu-Fan,Lin | 675bp |
| BBa_K3682004 | cleavage site 1 on spike protein sequence | DNA | You-Cheng,Lee | 63bp |
| BBa_K3682005 | cleavage site 2 on spike protein sequence | DNA | You-Cheng,Lee | 63bp |
| BBa_K3682006 | cleavage site 3 on spike protein sequence | DNA | You-Cheng,Lee | 63bp |
| BBa_K3682007 | cleavage site 4 on spike protein sequence | DNA | You-Cheng,Lee | 63bp |
Composite Parts
| Name | Description | Type | Designer | Length |
|---|---|---|---|---|
| BBa_K3682008 | Bacterial expression hACE2 RBD | Plasmid | You-Cheng,Lee | 3979bp |
| BBa_K3682009 | Bacterial expression pepP protease | Plasmid | Yan-Syun,Huang | 3056bp |
| BBa | Bacterial expression red fluorescent protein | Plasmid | Yu-Fan,Lin | N/A |
| BBa | Bacterial expression pepP-hACE2 RBD | Plasmid | Yan-Syun,Huang | 5294bp |
| BBa | Bacterial expression spike protein fragment 1 | Plasmid | You-Cheng,Lee | 1799bp |
| BBa | Bacterial expression spike protein fragment 2 | Plasmid | You-Cheng,Lee | 1799bp |
| BBa | Bacterial expression spike protein fragment 3 | Plasmid | You-Cheng,Lee | 1799bp |
| BBa | Bacterial expression spike protein fragment 4 | Plasmid | You-Cheng,Lee | 1799bp |
| BBa | Bacterial expression Spike protein monomer | Plasmid | You-Cheng,Lee | 5552bp |
Improvement of an Existing Part
We improved “Red Fluorescent protein” (BBa_K2040119) to “His Tag inserted Red Fluorescent protein” (BBa_J23100) . We introduced the modification of improving purification efficiency by adding 6x-His tag to C-terminus. In our experiment, we made a construct which contains Red Fluorescent Protein with his tag on C terminal. we do this to test our gel covered with Cu2+ that can capture his tag. With rfp, we can immediately check whether it binds on the gel with naked eye. Since there is no available part before, we hope this new part we made can bring convience to the teams in the future. We chose to improve BBa_K2040119 because we have the rfp construct from previous team. We can use the sequence as fragment without buying the rfp sequence.We were dedicating to observing binding affinity with his tag within a short amount of time; as a rusult, the appearance of visibly red fluorescence protein is important. We added 6x-His tag to the C-terminus of an existing part. In this way, rfp is available to protein purify simply by using Ni-NTA beads. With our new part, in the future, if there is team want to visualize protien construct binding affinity, they can use our part doing so.
To test whether we have successfully made the part, we firstly run the gel see the kda of plasmid is on the right site. Next, we tansform the plasmid into DH5a and culture in BL21 to mass produce our target protein. Finally, we purified this product by Ni column. And then run the SDS page(Fig.2) Shown in the Fig.1 below, we succeeded in purifying RFP with his tag by using Ni column.
