General Cloning Protocol
"Cloning" is the term used in molecular biology for the insertion of another organism's gene into a target host organism (eg. taking the Green Fluorescent Protein (gfp) gene from the A. victoria jellyfish and putting it in E. coli to get E. coli to glow green). Though this sounds simple, multiple in vitro DNA-assembly steps are involved that are not always simple in execution. One should remember that each of these in vitro steps is in essence a chemical reaction (often carried out by enzymes), which is subject to environmental conditions (salt concentration, temperature), quality and quantity of reagents (how much cut DNA, presence of dNTP's, etc), and theoretical yields. If the yield for any of these reactions in our series is too low, then we are unlikely to get the desired end product (usually our gene of interest inserted into our target plasmid). Therefore, we should always seek to optimize the yield of desired product for each of our reactions; this simply means use the HIGHEST quality reagents in the most OPTIMAL conditions you can.
Here is a highly generalized set of steps in cloning:
- Production of the linear insert. A linear piece of a desired DNA sequence can be obtained in many ways, including traditional PCR, Assembly PCR, cutting a piece out of an already existing vector (as in biobricks cloning), synthesis orders (through companies), primer extension reactions, and primer annealing.
- Cutting the insert and target vector with appropriate endonucleases. We use restriction endonuclease enzymes to cut specific sequences of DNA. The ends of these cut pieces of DNA will then stick together if their sequences are complementary, allowing us to ligate linear pieces of DNA together in a highly specific way.
- Ligating the linearized vector and insert together. Here, we simply combine our insert and vector in a reaction with the enzyme DNA ligase, which covalently links free ends of DNA together.
- Transformation of the completed vector and screening. E. coli will take up DNA through its membranes under certain conditions (heat shock, electroporation), which allows us to put our finished DNA vector into the bacterium. We plate our transformants on selective media, which will allow only those cells to grow that have successfully taken up our vector.
Depending on what one wants to build, the construction steps for cloning can look very different. Even for identical constructs, differences in style among researchers will lead to different cloning methodologies. If one chooses to stick to standardized systems of cloning such as BioBrick cloning, construction protocols can be identical across almost all situations. Such standardized systems can greatly simplify how one approaches cloning, but they are not always suited to every situation (eg. standard assembly in BioBrick cloning runs into problems when one wants to build fusion peptides). The take-home message is that there is rarely ONE WAY in which to clone something. How we build DNA constructs depends on what we need to build and how the individual researcher wants to assemble their DNA. Things people take into account are: cost, speed, reliability, accuracy of assembly. Arguably, synthesis technology takes the cake on speed, reliability, and accuracy, but at even 25cents/bp, it is still prohibitively expensive for most people's cloning needs.
A more detailed example:
What follows is a highly specific example of a cloning protocol. Let's say you want to assemble two genes, a and b, in series. When the construction is complete, you would like the products of genes lacZa(300bp) and gfp(800bp) to be the fused protein LACZa-GFP, with a short 2xGGGS linker in between the two domains. You also want to add: a medium-strength promoter (35bp) and a strong RBS (~10bp) upstream of the coding region, a terminator at the 3' end of your transcript (100bp), and cut sites that will allow you to insert the whole thing into a BioBrick plasmid (20bp flanking each side). First, let us work through the logic of planning the assembly:
First, let's consider assembly. The finished product will likely be in excess of 1300bp and so will likely cost >$1000. You're lab is running short on money, so you're not going to be able to afford a You have DNA that codes for b on hand in a BioBrick vector. The DNA for a, however, doesn't exist and is too big to make by either primer annealing (200bp w/ IDT Ultramer oligo synthesis; this is very expensive, anyway). You think you can make b by doing primer extension of two 170bp ultramers but quickly realize that the two Ultramers will cost >$400. Compared to a straight-up synthesis order from DNA2.0, this seems too costly and risky (the primers might just not work because of cryptic hairpinning or something weird).