Composite Parts

My earlier posts have been about basic parts. Now I want to divert your attention to composite parts. Putting basic parts together in new ways is a key area of research in the field of synthetic biology. In fact, putting basic parts together is really the whole point of parts-based synthetic biology.

Part assembly depends on a number of molecular biology tools. Among these are plasmids, which are short, circular pieces of DNA ranging from about 1000 to 10,000 basepairs in length maintained inside bacterial cells. For comparison, the E. coli genome contains around 4.5 million basepairs, and the human genome contains around 3 billion basepairs. Plasmids are used to maintain foreign DNA inside a bacterial cell and in assembly. A plasmid contains three components: an antibiotic resistance cassette, an origin of replication, and a cloning site. The antibiotic resistance cassette confers resistance to a particular antibiotic upon the host bacterial cell. This allows researchers to supplement cultures with the corresponding antibiotic to ensure that their bacteria faithfully maintain their plasmids despite the additional energy required to do so. Failing to include the antibiotic would select for the individuals that stop replicating their plasmids, and evolution would do away with your BioBrick parts as the "lazy" individuals outcompeted the others. An origin of replication, on the other hand, is required so that the cell's machinery copies the plasmids and they do not become diluted as the bacteria divide each generation.

A cloning site is a location on the plasmid containing known restriction sites where foreign DNA can be spliced into the plasmid. BioBrick plasmids use the standard BioBrick prefix and suffix as their cloning site. To assemble two basic parts, you must begin with two plasmids containing the two individual parts, one to be placed downstream from the other. Using restriction enzymes, the downstream part is cut out of its plasmid and the plasmid containing the upstream part is cut across the DNA immediately after its own part. The downstream part is then spliced into the plasmid after the upstream part, leaving you with a plasmid containing your new composite part that can be put into a bacterial cell. This cut-and-paste method becomes a bit more complicated when all of the details are included, but the overall principle is pretty simple.

This method can be repeated to build composite parts made of more than just two basic parts. The characteristics of the composite part depend on the type and order of constituent parts. By knowing a few biological principles, you can build a composite part that performs a predictable function. Such composite parts include expression cassettes, which contain a promoter, ribosome binding site, protein coding domain, and sometimes a transcriptional terminator. An expression cassette contains all of the parts required to instruct a cell to produce a particular protein. For example, the BioBrick part J04450 instructs a cell to produce red fluorescent protein, or RFP. RFP is very similar to GFP, which I described in my last post. Cells containing this part are visibly red and glow bright red under ultraviolet light. The image in this post shows a petri dish with E. coli that were carefully placed and allowed to grow and fluoresce. Each color corresponds to a group of E. coli cells carrying a slightly different expression cassette in a plasmid.

There are a huge number of potential composite parts because the number and types of basic parts are extensive. Thinking up new ways to put parts together for useful applications is an important and thriving area of research, and luckily it's accessible to undergraduates like me.

Image Credit: Nathan Shaner and Paul Steinbach (via TPM)

References and Further Reading:

Registry of Standard Biological Parts.