RobWarden:Lab1

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  • Would you expect the phage to tolerate…
    • P8 Modifications:
      • Neutral C-Terminus: No, looking at the p8 protein structure (PDB 1NH4) I would expect the charged c-terminus amino acid to be necessary in interacting with another p8 to form the coat.
      • CTG -> CTA: Yes, the phage should tolerate this, because it will not change the output protein at all. E. Coli’s codon bias lies much more toward CTG than CTA, though, so it will slow down phage growth.
      • Double Size: Yes, provided when you design the longer protein the hydrophobic transmembrane region is left alone, and the hydrophilic ends are what is elongated. These elongated regions must also follow the hydrophobic/philic pattern of the natural structure, which keeps hydrophobic residues on one side of the helix, and hydrophilic residues on the other.
    • P3 Modifications:
      • Neutral C-Terminus: No, Studies have found that the phage can tolerate fusions to the n-terminus, which tells me that the c-terminus is buried/attached to p6. If this is the case, making this neutral instead of charged could destroy an interaction and make the protein unstable or unattachable.
      • CTG -> CTA: Yes, the phage should tolerate this, because it will not change the output protein at all. E. Coli’s codon bias lies much more toward CTG than CTA, though, so it will slow down phage growth.
      • Double Size: Yes, There have been successful fusions to the n-terminus, so I don't see that doubling the protein on that end would be very problematic.
    • Transcriptional terminators that are:
      • 2X Stronger: Because termination is signaled by a hairpin structure and the M13 genome is so compact, a stronger transcriptional terminator may make it difficult to transcribe the following gene. 2X may be able to be tolerated, since it is not that much stronger.
      • 100X Stronger: Probably not, for reasons listed above.
      • 2X Weaker: The worry with weaker transcriptional terminators is that you may transcribe a gene that shouldn't be expressed yet, although a slightly weaker (2X) terminator may not cause too much harm.
      • 100X Weaker: Probaly not, for the reason listed above.
  • M13 Relatives - Other Filamentous Viruses (fd and f1)
    • Sequences almost completely conserved
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