Notes, thoughts, and news on synthetic biology by 
The first (and sometimes 3rd, 12th, 25th, 134th...) step of any genetic engineering experiment is often extracting DNA from some organism or another. While novel gene synthesis technology will likely make this procedure obsolete, these days it's still most economical to do it by hand. Extracting DNA from fruits like strawberries has also seen a popular resurgence thanks to groups like DIYbio, with instructions for making DNA shots available online for a fun and nerdy party activity. Today my iGEM team extracted RNA from strawberries and oranges to isolate the genes responsible for strawberry allergy and orange flavor as BioBrick parts.
We extracted RNA instead of DNA because many plant genes contain introns, which are pieces of DNA that don't end up in the final protein sequence. By isolating the RNA, which already has these bits spliced out, we can get just the sequence that codes for the protein we're interested in. The procedure is pretty similar either way--chop up and homogenize some of the fruit tissue (using liquid nitrogen!!!), add soapy, salty water to break up the cell membranes, precipitate the genetic material using alcohol, and then isolate and wash the DNA or RNA. I'm used to doing this with yeast or bacteria, and it was a lot of fun to play with food in the lab today!
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As a structural biologist, this is something I rarely get to see -- I typically only work with genes that have been conveniently inserted into a high-production plasmid. What is your strategy for isolating the desired gene from the pooled RNA? Do you intend to create a whole cDNA library, or do you have primers for specific PCR from a reverse-transcribed product? Did you have to create an RNAse-free zone in your lab for this?
Posted by: Sparky Clarkson | June 15, 2010 8:22 PM
We made a cDNA library by doing a reverse transcriptase reaction with oligo-dT primers that bind to the poly-A tail of the mRNA. Somewhere in there is hopefully the cDNA of the template we want! We probably should have made an RNAse-free zone, we just tried to be as sterile as possible, we'll see how it worked when we try the PCR later this week!
Posted by: Christina Agapakis
| June 15, 2010 10:00 PM
Oooh, making biobricks from eukaryotic genes hmm? Although the orange flavour genes could be very interesting ones too biobrick. Wish them the best of luck from me!
Posted by: Lab Rat | June 18, 2010 6:05 AM