Team:ZJU-China/Newparts
Newparts
Overview
This year, we designed 5 parts, and all of them had been submitted to the iGEM Registry following the BioBricks assembly standard.
Here is a brief description of our parts. For more detailed information about our parts, we strongly recommend you visit the corresponding parts pages on the iGEM BioBricks Registry.
Parts Table
| Name | Type | Description | Length(bp) | Designer |
|---|---|---|---|---|
| BBa_K3398000 | T7 | Modified T7 promoter 1 | 64 | Mingxiao Wei |
| BBa_K3398001 | T7 | Modified T7 promoter 2 | 64 | Mingxiao Wei |
| BBa_K3398002 | T7 | Modified T7 promoter 3 | 64 | Mingxiao Wei |
| BBa_K3398003 | coding | mamC_ZZ fusion protein | 771 | Mingxiao Wei |
| BBa_K3398004 | coding | mamC_ZZ fusion protein with GST tag | 1464 | Mingxiao Wei |
| BBa_K3398005 | coding | scFv_Fc fusion protein | 1497 | Xiner Ying |
| BBa_K3398006 | coding | scFv_Fc protein with FLAG tag | 1524 | Xiner Ying |
Improved T7 Promoters
BBa_K3398000, BBa_K3398001, BBa_K3398002
As a widely used promoter for expression of heterogenous protein in some E.coli strains such as BL21 (DE3) for its high efficiency, we improved T7 promoter by introducing some point mutations distributing in the range from +25 to +31 of the T7 promoter sequence. We chose EGFP as a reporter to test the function of our modified T7 promoters.
Parts expressed by E.coli BL21 (DE3)
These parts are coding sequences of mamC and ZZ fusion protein. And for separation from other proteins at the purification step, we introduced a GST tag at the N-terminus and a thrombin site between the fusion protein and tag for cleavage after purification. To maximize the expression of a functional protein, they were subcloned into gene expression vector pGEX-2TK and transformed into E.coli BL21 (DE3) to express. These parts can also be expressed by magnetotactic bacteria such as MSR-1 to produce magnetosomes modified with ZZ protein on the surface as a self-assembly platform for polymer varied antibodies.
Parts expressed by E.coli SHuffle® T7 Express Cells
These parts are coding sequences of scFv and Fc proteins. And for separation from other proteins at the purification step, we introduced a 10× histidine tag at the N-terminus which was followed by an enterokinase cleavage site for cleavage after purification. To maximize the expression of a functional protein, they were subcloned into gene expression vector pET19b and transformed into E.coli BL21 (DE3) to express. As the redox potential in the cytoplasm of general E.coli such as BL21 is too high, the disulfide bond in scFv will be reduced into thiol, which may not express the functional fusion proteins. So we chose E.coli SHuffle® with a reduced cytoplasmic environment as the chassis organism to express scFv-Fc fusion protein. The part of scFv-Fc is ideal molecule which can bind HER2 cells strongly and specifically, by adding report genes at either C- or the N-terminus, they can be used for the detection of HER2 cells.
