Arking:JCAOligoTutorial5
Assembly
The methods for fabricating DNAs remain a cost and time-limiting aspect of synthetic biology research. One day, all you'll do is type the sequence into some machine and it will pop out the other end, and you won't really need to understand anything about it. Actually, that black box exists today--there are companies like DNA 2.0, Blue Heron, and Geneart which do "Gene synthesis". You can type in a sequence up to around 20,000 bp long into their websites, and 2 weeks later they'll mail you some plasmid containing that sequence. Unfortunately, it's pricey -- it costs around $0.39 per base, and what they're going to do to make it isn't wildly different from what you're learning to do in these tutorials. For an overview of methods, check out "Gene synthesis demystified" Trends in Biotechnology, 27(2):63-72, 2009 PMID: 19111926
In practice, folks who do a large volume of DNA manipulation, at least in academia, do things in two phases. The BioBrick approach to cloning (which we're learning in these tutorials) makes those two phases very concrete and specific: you make basic parts and then you assemble into composite parts.
Step 1: Identify the basic parts available
You need to establish what basic parts will be needed to make what you want. There's no automatic way of doing this, so I can't really tell you how to do it. Suffice it to say, look at the complete list of available basic parts and try to make a complete cassette out of it. If there is some "missing" function in the Biobrick library, make a basic part for it.
Step 2: Write out the design for your basic part
Once you've identified the appropriate basic parts, write them down in order. So, you might make something like this:
I0500.b0034.Bca1117.b0015.r0040.Bca1046.b0032.E0040.b0016.Bca1046.b0034.E1010.b0015 Pbad rbs Cre term Ptet Lox rbs GFP term Lox rbs RFP term
What this thing is supposed to do is constitutively produce GFP under normal growth conditions. When the cells are exposed to arabinose, the Cre protein gets made, the region between the two Lox sites would be excised, and the cells turn red. ...at least that's what it's designed to do.
Before you start making something like this, you need to analyze it a little more. There is a good chance that some of the substructures of the composite have already been made. For example, we already have 3 useful parts in our toolbox:
E0241: b0032.E0040.b0016 rbs GFP term
I13507: b0034.E1010.b0015 rbs RFP term
Bca9089: b0034.Bca1117 rbs Cre
Step 3: Minimize the Design
So, we can simplify our design as:
I0500.Bca9089.b0015.r0040.Bca1046.E0241.Bca1046.I13507 Pbad (cre) term Ptet Lox (gfp) Lox (rfp)
This is as simple a construction as could be designed based on the current set of available parts. Let's now abstract this design a little further as just:
A.B.C.D.E.F.G.H
Step 4: Parallel (or convergent) synthesis
In this part of the tutorial, I'm not going to tell you how you actually go about making the junctions between parts. It's pretty complicated and we're constantly updating the protocols. So, we'll cover this when you actually get to lab. For now, just keep in mind that you install each "." one at a time. So, the most efficient way of making the above part is to assemble it convergently. So, split it in half, then half again, and so on until you only have single pairs left.
Round 3 A.B.C.D.E.F.G.H Round 2 A.B.C.D E.F.G.H Round 1 A.B C.D E.F G.H
So, now we have a plan for putting the thing together. In round 1, we join A with B, C with D, E with F, and G with H. Each of these dimeric composite parts will be assigned a number like e0241. Next time that dimer is called for in a construction, you'll be starting one step ahead. In round 2, we joing AB with CD and EF with GH. In round 3, we joing ABCD with EFGH, and then we're done!
If you have any comments or want to report a potential error in the tutorial, please email me (Chris Anderson) at JCAnderson2167-at-gmail.com
