User:Brian P. Josey/Notebook/2009/10/13

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PEG

I need to have a few quick notes about polyethylene glycol. Polyethylene glycol (PEG) is an linear organic compound with terminal ends composed of alcohol functional groups and a chain of ether groups between then ends. It tends to have a molar mass under 20,000g/mol, but this varies because it is a polymer and specific lengths and masses can be generated at will. PEG is soluble in water, benzene, methanol, and dichloromethane. However, it is not soluble in ether or hexanes. If we attach the PEG to a hydrophobic compound we would create a surfactant, which would lower the surface tension of a liquid, and in the process facilitate the easier spreading of the compound. Most importantly however, it can be used to apply osmotic pressure to a biochemical system, which is very important to our investigations.

Andy has a lipid from Avanti Polar Lipids that has two hydrophobic tails going one way, and a PEG tail going in the opposite direction.

How to Make Kinesin

For our experiments, we use kinesin that is produced by the bacteria E. coli. While we do not personally make the kinesin that we use, I thought it might be useful to have the general process of how to make it in my notebook. To create kinesin from E. coli, the E. coli has to be genetically modified to make the motor protein. Naturally, kinesin is not a part of the biochemistry of any prokaryote because it is a eukaryotic motor protein. After altering the DNA of the E. coli the resulting kinesin has to be isolated and collected. The general process of doing this is:

  1. First strands of DNA that includes a gene to create the kinesin and coding for a histidine tag have to be engineered for the the E. coli
    • Natural kinesin, like the ones that occur naturally in our own cells does not have a histidine tag. This tag is important for its collection in a later step and must be coded for in the modified DNA.
  2. These strands of DNA are then placed into the bacteria and serve as their plasmids. In prokaryotes the DNA creates a circular structure, called the plasmid, that serves the same purpose as the chromosomes in the nucleus of our own cells. With these engineered pieces of DNA, the E. coli begin to naturally produce the kinesin, which it obviously does not use on its own. In essence the bacteria serve as many little factories of kinesin.
  3. To remove the kinesin from E. coli the bacteria is chopped up into small portions, creating a concoction of dead cells and its components, including the kinesin.
  4. This concoction is then feed though a column extraction that contains Ni-agrose. The histidine tag has an affinity for the nickel and will interact and attach to it, remaining in the column while the other components pass through and are collected.
  5. To remove the kinesin from the column imidazole is poured through the column. The imidazole has a higher affinity for the nickel than the histadine tag, resulting in it replacing the tag and kinesin, allowing them to be collected and used.

We have some questions concerning this process, namely it is how the histadine tags are interacting with the Ni-agarose and the casein that we use to passivate the flow cells. We know that histidine has an affinity to interact with cysteine, another amino acid, that is prevalent on the structure of the kappa-casein. We would like to know how they interact, and if that is the key for why kappa-casein is so effective as as a coating for the flow cells. Another thing that we are interested in is how the histidine tag interacts with the nickel in the Ni-agarose. We know that it interacts, it is a crucial step in the collection of kinesin, but exactly how it interacts is a currently a mystery. If we can understand that more clearly, we could use this knowledge to advance our understanding of the questions centered on the kappa-casein. One idea is that we can convert the histidine tag into a protected group that will not interact with anything and then proceed with the usual gliding motility assays to see how that affects the motility.

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