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Revision as of 11:13, 24 October 2020
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.
| 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 |
Optimum concentration of IPTG for expressing
The transformed cells were grown until the OD600 reached 0.6, at which time protein expression was induced by a gradient from 0 to 2mM of IPTG. The cells were then incubated at 30°C for an additional 24 h.
Optimum induction time for expressing
The transformed cells were grown until the OD600 reached 0.6, at which time protein expression was induced by 2mM IPTG. The cells were then incubated at 30°C for a gradient from 0 to 24 h. For negative control, none inducer was introduced.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer (TIANGEN,Beijing,CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5% stacking gel, 8% separating gel). Gels were run at 110V for 15min and then at 160V for ~50 min in running buffer.
Coomassie staining
Following SDS–PAGE, gels was stained with Coomassie brilliant blue (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% acetic acid) for 2 h at room temperature. Destain in destaining buffer (5% ethanol, 10% acetic acid) overnight.
Western-blotting of proteins
Following SDS–PAGE, samples were transferred to nitrocellulose membrane (Merck) by semi-dry transfer (Bio-Rad). Membranes were blocked 1 h at room temperature in 5% (w/v) nonfat milk in TBST, probed with a rabbit anti-FLAG antibody (Abcam) overnight at 4°C, washed with TBST, probed with a goat anti-rabbit HRP-conjugated antibody (Sangon) for 1 h at room temperature, washed with TBST, and subsequently imaged using ChemiDoc (Bio-Rad).Particularly, the blocked membranes were directly probed with a rabbit anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary antibody.
Co-immunoprecipitation
The supernatant of cell lysate of mamC-ZZ or purified mamC-ZZ was incubated with purified FLAG-tagged scFv-Fc overnight at 4°C. Afterwards, the mixture was incubated with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. The FLAG-tagged proteins scFv-Fc and the interacted mamC-ZZ from the lysate were then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted with acid elution buffer. The elution was then digested by thrombin to cut off GST region. The product was then analyzed by SDS-PAGE and western blotting. For negative control, only purified scFv-Fc was used as input.
Cell culture
MDA-MB-453, MDA-MB-231, MCF-7, and MDA-MB-468 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere with 5% CO2 at 37°C. All cells were passaged at 80% confluency.
Targeted cell uptake evaluation of scFv-Fc using flow cytometry
MDA-MB-453, MDA-MB-231, MCF-7, and MDA-MB-468 cells at 90% confluence were collected, respectively, and seeded in a six-well plate. After overnight incubation, the media were replaced with the fresh media containing 30 μg/mL of scFv-Fc and further incubated for 1, 3, and 6 h. The cells were then collected by centrifugation after treatment using 0.5% trypsin-EDTA and washed thrice with PBS. In the end, the cells were resuspended in 500 μL PBS, and the Alexa Fluor® 488 fluorescence of the cells was immediately analyzed using a flow cytometer (CytoFLEX, Beckman Coulter Inc., USA).
Culture of Magnetotactic bacteria
Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose Magnetospirillum gryphiswaldense strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation Center(Taiwan, China).
The culture medium(BCRC medium no.611) of M. Gryphiswaldense MSR-1 is prepared as follows.
| Magntospirillum medium | |
|---|---|
| KH2PO4 | 0.68g |
| Na-thioglycolate | 0.05g |
| L(+)-Tartaric acid | 0.37g |
| Succinic acid | 0.37g |
| Trace elements solution | 5ml |
| Resazurin | 0.5mg |
| Distilled water | 1000ml |
| NaNO3 | 0.12g |
| Na-acetate | 0.05g |
| Vitamin solution | 10ml |
| Fe(III) quinate solution | Hyundai |
| Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N2 gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add. | |
| Trace elements solution | |
|---|---|
| Nitrilotriacetic acid | 1.5g |
| MgSO4·7H2O | 3g |
| MnSO4·2H2O | 0.5g |
| NaCl | 1g |
| CaCl2·2H2O | 0.1g |
| ZnSO4·7H2O | 0.18g |
| CuSO4·5H2O | 0.01g |
| Kal(SO4)2·12H2O | 0.02g |
| H33BO3 | 0.05g |
| Na2MoO4·2H2O | 0.01g |
| Distilled water | 1000mL |
| FeSO4·7H2O | 0.1g |
| CoSO4·7H2O | 0.18g |
| NiCl2·6H2O | 0.025g |
| Na2SeO3·5H2O | 0.3mg |
| Na2WO4·2H2O | 0.4mg |
| Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH). | |
| Ferric quinate solution, 0.01 M | |
|---|---|
| FeCl3·6H2O | 0.45g |
| Quinic acid | 0.19g |
| Distilled water | 100mL |
| Ferric quinate solution:Dissolve and autoclave at 121℃ for 15 min. | |
In the incubation period, aerobic environment is conducive to the growth of M.gryphiswaldense strain MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack or AnaeroPack was purchased from Mitsubishi Gas Chemical(Tokyo, Japan).
Reference
[1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L. et al. Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity. Mol Biotechnol 62, 18–30 (2020).