to my ealier post about plant cell cultures, could anyone tell me a little bit about, or direct me to a good website about biotechnology? Specifically biotech involving using bacteria, plant, and animal cells to create useful chemicals and pharmaceuticals for us. I understand how the metabolic pathways are modified (sort of), and how the cells produce the proteins, but how do they produce the non-protein chemicals, and how are the chemicals (protein and non) separated from the biomass?
Welcome to Sciforums NenarTronian. Are you talking about genetic engineering? Recombinant DNA technology? Peace Please Register or Log in to view the hidden image!
Recombinant DNA technology The methods used in rDNA technology are fairly simple. We take, for example, the sentence (gene) for insulin production in humans and paste it into the DNA of Escherichia coli, a bacterium that inhabits the human digestive tract. The bacterial cells divide very rapidly making billions of copies of themselves, and each bacterium carries in its DNA a faithful replica of the gene for insulin production. Each new E. coli cell has inherited the human insulin gene sentence. How do we transfer the gene embodying the instruction for insulin production? One approach would be to cut the appropriate gene from human DNA and paste, or splice, it into plasmid DNA, a special kind of DNA that takes a circular form and can be used as a vehicle for this editing job. Our "scissors" are the class of enzymes called restriction enzymes. There are well over a hundred restriction enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule. With these scissors used singly or in various combinations, the segment of the human DNA molecule that specifies insulin production can be isolated. This segment is "glued" into place using an enzyme called DNA ligase. The result is an edited, or recombinant, DNA molecule. When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production. More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning. This highly simplified description of rDNA technology does not fully convey the enormous complexity and awesome economy and efficiency of genetic processes. in-depth look at rDNA technology
There are problems with getting many human proteins out of ecoli. First, their usage of different codons is very different and human protiens that use many infrequent codons in ecoli aren't expressed at high levels even if put under a very strong promoter (the part of a gene that determines how much mRNA is generated for directing protein synthesis - in this case a lot of mRNA would not effectively generate much protein because a lack of sufficient numbers of the tRNAs reduces the processivity of the translation reaction). Second there are a lot of post translational modifications (many proteins have sugars and long chain fatty acids stuck on them - some proteins are anchored to the membrane this way) we do that ecoli don't and these don't happen. Insect cell culture is used to get around some of them, but there are always processing differences. There are many different approaches that depend on what your goals are. Typically if a protein is the product of interest it is given a secretion signal sequence and then the protein is separated from the medium using standard two phase extraction techniques that depend on solubility and then purified further downstream. For metabolic products they just need to supply the pathways (possibly including importer/export proteins) and substrates and they then use appropriate purification techniques on the media. Typically in both cases the media is constantly renewed to remove wastes and provide the necessary goodies for growth etc. and this is done often with centrifuges that separate the media from the cells to keep the titer in the reactors high (some also remove dead cells). I know of no good websites or books tailored to your general question, though they probably exist. If you have more specifc questions, fire away and I'll try and give references and answers.
