Difference between revisions of "Part:BBa K4159000:Design"

Revision as of 15:45, 1 October 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.

References: Hansen S, Stüber JC, Ernst P, Koch A, Bojar D, Batyuk A, Plückthun A. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. Sci Rep. 2017 Nov 24;7(1):16292. doi: 10.1038/s41598-017-15711-z. PMID: 29176615; PMCID: PMC5701241.

https://www.ncbi.nlm.nih.gov/protein/5MA6_B

Chen X, Gentili M, Hacohen N, Regev A. A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies. Nat Commun. 2021 Sep 17;12(1):5506. doi: 10.1038/s41467-021-25777-z. PMID: 34535642; PMCID: PMC8448731.

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