Difference between revisions of "Part:BBa K4159000:Design"
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| − | + | Description: GFP DARPin construct | |
| + | Background: The main component of the GFP-binding DARPin construct is the specific GFP-binding DARPin part BBa_K4159005. The part has also the T7 promoter (BBa_K4159002) and the specific RBS sequence (BBa_K4159001). The spacer is also one of our teams registered basic parts BBa_K4159003, and is derived from the pBx1-VHH_template3-3XMyc-Spacer plasmid from Chen et al. (2021). | ||
| + | Characterization: In vitro transcription and translation was performed according to the eponymous lab protocol. BBa_K4159005 were transcribed and transformed and stored at - 80 °C. | ||
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| + | The 96-well plate (not flat-bottom due to a mix-up) was washed 3 times with TBS, air-dried and 100 μL of 66 nM GFP. The dilution of GFP was done with 4.53 µl in 9.99 mL of TBS. The 96-well plate was incubated overnight at 4 °C on a shaker. The target was further immobilised according to the protocol (see our experiments page to see more details). | ||
| + | |||
| + | After reverse transcription of the mRNA, the cDNA was sent for sequencing. | ||
Revision as of 17:44, 30 September 2022
Description: GFP DARPin construct Background: The main component of the GFP-binding DARPin construct is the specific GFP-binding DARPin part BBa_K4159005. The part has also the T7 promoter (BBa_K4159002) and the specific RBS sequence (BBa_K4159001). The spacer is also one of our teams registered basic parts BBa_K4159003, and is derived from the pBx1-VHH_template3-3XMyc-Spacer plasmid from Chen et al. (2021). Characterization: In vitro transcription and translation was performed according to the eponymous lab protocol. BBa_K4159005 were transcribed and transformed and stored at - 80 °C.
The 96-well plate (not flat-bottom due to a mix-up) was washed 3 times with TBS, air-dried and 100 μL of 66 nM GFP. The dilution of GFP was done with 4.53 µl in 9.99 mL of TBS. The 96-well plate was incubated overnight at 4 °C on a shaker. The target was further immobilised according to the protocol (see our experiments page to see more details).
After reverse transcription of the mRNA, the cDNA was sent for sequencing.