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

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This part consists of the T7 promoter, RBS34, His-tag, coding sequence for a GFP-binding DARPin [1] (designed ankyrin repeat protein), and a spacer of 217bp [2]. The part does not include a terminator as the sequence has been used in ribosome display. The spacer sequence for ribosome display should preferably be about 100 amino acids in length.
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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).
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Characterization:
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We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.
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In vitro transcription and translation was performed according to the eponymous lab protocol. The BBa_K4159000 DARPins 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).
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After reverse transcription of the mRNA, the cDNA was sent for sequencing. Below is picture 1. where one can see the DARPins that were reverse transcribed before sending to sequencing.
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[[File:BBa K415000 1.png|200px|thumb|left|Picture 1. Gel picture of our ribosome display DAPRins. ]]
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The sequencing results showed that there was some affinity between a few of our AIP binding DARPins and for this GFP-binding DARPin, which shows that the construction works, and all basic parts of this composite part are sufficient for performing ribosome display. More information of the sequencing results and the quality of the results visit our results page https://2022.igem.wiki/aalto-helsinki/results.
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References:
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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.
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https://www.ncbi.nlm.nih.gov/protein/5MA6_B
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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.

Latest revision as of 17:17, 1 October 2022

This part consists of the T7 promoter, RBS34, His-tag, coding sequence for a GFP-binding DARPin [1] (designed ankyrin repeat protein), and a spacer of 217bp [2]. The part does not include a terminator as the sequence has been used in ribosome display. The spacer sequence for ribosome display should preferably be about 100 amino acids in length.


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:

We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.

In vitro transcription and translation was performed according to the eponymous lab protocol. The BBa_K4159000 DARPins 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. Below is picture 1. where one can see the DARPins that were reverse transcribed before sending to sequencing.

Picture 1. Gel picture of our ribosome display DAPRins.


The sequencing results showed that there was some affinity between a few of our AIP binding DARPins and for this GFP-binding DARPin, which shows that the construction works, and all basic parts of this composite part are sufficient for performing ribosome display. More information of the sequencing results and the quality of the results visit our results page https://2022.igem.wiki/aalto-helsinki/results.


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.