| Parts | Pages in Lab Manual |
|---|---|
| Part 5.6: Clonal analysis of potential target binders | 132–136 |
Pre-Lab & Post-Lab Instructions
Supporting Files
Day 19 Protein Alignment Worksheet (PDF)
Day 23 Post-Lab Notebook Tips and Reminders
Follow all instructions and answer all six questions below for your Day 23 post-lab:
You have sequenced the binder variant gene from seven different miniprep plasmids (i.e. independent clones each isolated from a separate transformed bacteria colony). The DNA sequence results for these seven binder genes (Binders 1–7) are in the binder sequences file (linked above).
The DNA sequence of the “wild-type” (WT) binder gene is also in this file. This WT sequence encodes the original scaffold protein (which presumably would not have any natural binding affinity to the SARS-CoV-2 target on its own) that the surface display library is based on. To create the original library (e.g., for use in the initial magnetic bead sorting), random mutations were introduced into this scaffold protein to generate genetic diversity. The seven binder gene clones you isolated after your FACS screening are all derived from the randomly mutated variants of this original WT scaffold gene.
- Translate the DNA sequence for each of the seven binder genes (Binders 1–7) and the original WT scaffold gene into their corresponding amino acid sequence. Perform a protein alignment for all eight proteins (Binders 1–7 and the WT scaffold) using either SnapGene or NCBI COBALT (refer back to the Day 19 post-lab worksheet, linked above) if you need instructions on how to perform the alignment). Take a screenshot of this protein alignment and insert the JPG of that alignment into your Day 23 post-lab on LabArchives.
- How many different unique binder proteins are represented in the seven clones that you sequenced?
- How many variable amino acid residues are there among the seven binders with respect to the WT scaffold?
- Which region(s) of the scaffold protein most likely make up the binding interface between the binder protein and the SARS-CoV-2 target? Explain your reasoning.
- Of the variable amino acid residues you counted from Question 3, how many of those are identical in all seven of the binders you sequenced? What might be the significance of these residues? Pick any one of these identical residues and suggest a hypothesis about why that particular residue was changed from the WT scaffold sequence in all seven binders isolated.
- Of the variable amino acid residues you counted from Question 3, how many of those show variances within the seven binders you sequenced? What might be the significance of these residues? Pick any one of these residues and suggest a hypothesis about how that particular residue may be affecting the interaction of the binder protein to the SARS-CoV-2 target.
