David M. Truong 16:24, 30 January 2012 (EST):If anyone else has examples of Genes/proteins used today found by prospecting, please add it and put a sentence description
- Ben Slater 17:27, 3 February 2012 (EST):Under Cellulosic Biomass degrading genes found in Cow Rumen, you say "These microbes cannot be cultured in lab." Given that the vast majority of life can't be cultured in a lab, I was wondering about what potential exists for different culturing conditions that would allow this to change. What obstacles are blocking us from a wider array of culturable microbes, and is it possible to bypass these barriers? Increasing the pool of bioprospecting sources seems like it would be very helpful, as you never know what crazy thing nature has already invented that we can hijack.
- David M. Truong 20:23, 4 February 2012 (EST): Ben, that's an interesting question. Like I mentioned, there are many microbial species living in the vastness of the world's oceans. These microbes have often adapted to incredibly harsh conditions, such as different energy sources such as hydrogen sulfide and/or higher ocean pressures without the need for light. That's not even including organisms on land, that for whatever reason, don't grow well in lab. Although it is tempting to identify a new general culture protocol for these "other" organisms, it might be more prudent to focus on a case by case basis, purely by need. For instance, the recently infamous GFAJ-1 microbe (the arsenic microbe), is also an extremophile and was specially cultured in higher pH. It is interesting because, maybe it uses arsenic in it's DNA (most likely not). But if there is an interesting microbe found in another interesting enviroment, say Mars for example, I think people would definitely focus on that. This isn't the most straight-forward response, but let me get back to you on this.
- Jeffrey E. Barrick 11:56, 5 February 2012 (EST):Sometimes microbes require certain "growth factors" that might be vitamins or quorum sensing molecules produced by other microbe that they are "used to" living with. In certain cases supplementing agar plates with the compounds has enabled them to be grown in lab . Another reason that things are often "unculturable" is that they have adapted to grow very slowly (but efficiently) and we are not patient enough in lab, or they get overgrown by other bacteria int he same soil samples that are microbial "weeds" before they have a chance to form colonies.
- Jeffrey E. Barrick 11:56, 5 February 2012 (EST): I think Dave has a great point in his article that even with all the diversity in nature, evolution only produces things that are "good enough". While often much better than anything we can currently create in the lab de novo, they are not optimal. In many cases nothing may have evolved to do what we want at all.
- Jeffrey E. Barrick 11:56, 5 February 2012 (EST): Maybe you should point out that one difficulty with bioprospecting by the random cloning approaches is that it only works when one fairly short piece of DNA has all the genes required for a function. Often one has to computationally look for gene clusters that you can recognize (as in the case of polyketide synthase genes), amplify with degenerate primers specifically to those genes, and move multiple genes around and screen them to find new functions.
- Erik Quandt 15:26, 5 February 2012 (EST): Here is an informative paper on how to construct a metagenomic library from soil bacteria and how to use these libraries to screen for function . Interesting note on coverage: "It has been estimated that more than 10^7 plasmid clones (5 kb inserts) or 10^6 BAC clones (100 kb inserts) are required to represent the genomes of all the different prokaryotic species present in one gram of soil" but.. "These estimates are based on the assumption that all species are equally abundant. To achieve substantial representation of the genomes from rare members (less than 1%) of the soil community, it has been calculated that libraries containing 10,000 Gb of soil DNA (10^11 BAC clones) might be required"!
- Brian Renda 11:00, 6 February 2012 (EST): Not sure where to put this in the article, but a problem is also filtering out the interesting bacteria from the less interesting bacteria in a metagenome. When creating a library, it seems like it would be hard to maintain enough complexity to capture the plausibly rare and beneficial genes you're looking for. One solution to this might be to look into metagenomes where there is a huge selection pressure to have the genes you're looking for. Such an environment would have to be particularly hostile - something with very high concentrations of a given contaminant perhaps. While it doesn't allow you to capture the genomes of rare bacteria any better, it would increase the relative population of a bacteria with the physiology of interest. Here is an interesting paper that talks about some of the evolutionary dynamics of such an environment . It does create sort of a catch-22 though. Harsher conditions that lead to over representation of useful genes to cope with the environment also can have significantly lower overall concentrations of cells - on the order of 10,000/g of soil. By traditional library construction methods, this would require you to harvest dozens of kilograms of soil for extraction. From doing just 80g of soil at a time, I can't even imagine how problematic just 25kg could be. A solution from the literature is to use φ29 DNA polymerase to amplify low concentrations of environmental DNA for library construction , but I suspect it would be hard to demonstrate re-amplification bias without testing the process with a known and sequenced metagenome.
- David M. Truong 15:04, 6 February 2012 (EST): Great finds guys. Would you mind adding them into the wiki. The summaries you added here will be sufficient.
- James L. Bachman 13:20, 5 February 2012 (EST) Under Limitations you said that using emPCR on short-reads will introduce bias, what exactly do you mean by bias in this context and how is that physically done?
- David M. Truong 22:38, 5 February 2012 (EST):"In addition, many protocols utilize emPCR after nebulization of DNA, which can introduce sequencing coverage bias. Nebulization randomly fragments sample genomic DNA into various lengths, which can be of varying GC content and secondary structure. Since emPCR amplifies single fragments, this can often lead to underrepresentation of difficult to PCR fragments. For low copy templates, this can often mean low sequence coverage in important areas. "
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