User:Janet B. Matsen/never forget

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* DTT is a reducing agent which will help keep the Cys thiol groups from getting oxidized
* DTT is a reducing agent which will help keep the Cys thiol groups from getting oxidized
* Tris-HCl and Tris are NOT the same!  (2012/11/14)

Revision as of 21:53, 14 November 2012


Significant Figures

  • If you have a measurement like 12.34, and you are showing the error, would you write
    • (a) 12.34 ± 1.56
    • (b) or do you include the number of significant figures in your measurement: 12.34 ± 1.567?
    • Mary: I would go with (a). That is certainly how all of the journals I publish in handle this.
  • If you are using a lac promoter and you add IPTG, arent you also inducing expression of LacZYA?
    • I think so. But if you have a high copy plasmid, you are making much much more of your enzyme. :w


  • DTT is a reducing agent which will help keep the Cys thiol groups from getting oxidized
  • Tris-HCl and Tris are NOT the same! (2012/11/14)


Why are bacteriophages a big problem for bacterial biotech, but viruses in mammalian biotech not?

Ans (from dialogue with Mary Lidstrom 8/2012)

  • Phage can be encapsulated in tough capsids. However, viruses infecting multicellular organisms, they evade the immune system by coating themselves with a portion of the cell membrane, but that makes them fragile outside a host.
  • That doesn't happen for yeast, but the yeast viruses seem to be transmitted through the yeast mating process and don't infect the host cells from the outside. I don't know why that would be, except perhaps because it is a safe mechanism for transfer and yeast do mate at a high frequency. That means of course that even if an infection occurs, sterilization to get rid of the infected yeast gets rid of the virus also. That's much less problematic than having to scrub all the ventilation systems, walls, etc. to get rid of phage.

Protein Modeling/Fusion Proteins

Can you use a protein structure from a database to preduct whether you can use that protein as part of a fusion protein?

Ans: from 2012_09_11 summary from e-mail from Justin about visualizing FLS &

  • You may have the structure file, but you are going to run into two issues:
    • "First, the floppy ends of the proteins are not in the structures. At the N and C terminal, you will often see a discrepancy between the actual sequence and what you see in a crystal. This is because you can only see parts of the protein that are essentially fixed in space and not moving around much. So where exactly to overlay the proteins won't be totally clear.
    • Second, as long as the ends are floppy you really won't be able to predict how it looks and the units interact. The only reason to look at a structure for fusions is to know if the ends are highly structured and an integral part of the protein (i.e. in a sheet or helix). If this is the case it is best to avoid fusing those sections. However, if the ends are "floppy" or "unstructured" (as in not a helix or sheet, and not an integral part of the protein... this is for sure the case if you can't see the actual ends in the structure) you can most likely make a functional fusion. Now... there may be empirical differences between how well an N vs C terminal function, but their will really be no sound way to predict that. This is generally speaking, there are special cases, such as a protein that ends in a helix and a fusion made starting at a helix so the two proteins are very specifically oriented relative to each other. But these cases are extremely rare."
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