 |
| The simplest expression system(panel A) includes a promoter (curved arrow), |
| a ribosome binding site (RBS, oval), a gene (straight arrow) and a transcriptional terminator |
| (hexagon). A more complex architecture has an operon structure (panel B), where multiple genes, |
| with their own RBSs, are expressed by a single promoter; a two-gene operon is shown. |
| Individual or composite gene expression systems can be placed in a plasmid vector (panel C), |
| which is maintained by the host strain at a specific copy number per cell, depending on the |
| particular replication origin, or in the bacterial chromosome (panel D). Dashed line indicates the |
| genomic DNA of the host. |
| Promoters control the transcription of the downstream DNA. Their sequence determines the |
| information on transcriptional strength and regulation by other factors. Different promoter |
| classes are present in E. coli, according to the specific sigma factor that determines their |
| regulation. Other factors, such as activator/repressor proteins, which are widely used in synthetic |
| circuits, can provide an additional degree of control. |
| RBSs are small sequences that are placed upstream of a gene. They are transcribed into mRNA |
| but are not translated, since protein synthesis starts with the AUG codon of the coding sequence. |
| RBS sequence determines the binding affinity between ribosome and mRNA, thus affecting the |
| translation initiation rate. In particular, mRNA binding to ribosomes occurs at the |
| Shine-Dalgarno region of an RBS with the rRNA of the 30S ribosomal subunit of E. coli. |
| Transcriptional terminators of the rho-independent class exploit hairpin structures to stop RNA |
| polymerase activity during transcription. This class of terminators is commonly used in |
| synthetic circuits, as opposed to rho-dependent terminators that mediate transcription stop via |
| an ATP-dependent process, which has not been completely understood. |
| Genes encode the desired proteins, which can actuate disparate specific regulatory or |
| metabolic functions. Gene sequence can affect the mRNA folding, which may result in different |
| translation initiation rate and different decay rate of the transcript. Moreover, codon usage bias |
| determines preferences of specific codons over others for a given organism. For these reasons, two |
| identical proteins encoded by two different genes (designed exploiting genetic code redundancy), |
| can have extremely diverse synthesis rates. |
