Unit 4: Introduction to Parts
Introduction
There are coding and non-coding regions in a genome. Coding regions code for the proteins whereas non-coding regions regulates the expression in different ways, direction different process in central dogma. Non-coding sequences include promoter, RBS and terminator. Coding and non-coding work together to express a complete functional protein.
Promoter
Promoter is a DNA sequence to direct the start of the transcriptional machinery and lead to transcription of downstream genes. In short, promoter is a switch to turn on the transcription process. There are different kinds of promoters. Their action and functions are listed below.
Ribosome Binding Site (RBS)
RBS is the RNA sequence where ribosome binds to it to initiate translation. An RBS is an RNA sequence upstream of the start codon that affects the rate at which a particular Open Reading Frame (ORF) is translated. Various aspects of RBS design affect the rate at which the ORF is translated. You can use this information either to better understand how RBSs work, or to design new RBSs. Before designing a new RBS, you should first consider using existing high quality strong RBSs. You are advised to use constitutive prokaryotic RBS. A RBS is place upstream of each protein doing sequence.
Protein Domain /CoDing Sequence (CDS)
A coding sequence may contain one or more protein domains, which is the basic unit of a protein’s 3D structure. Each domain is folded and functioned independently. Putting different protein domains together can serve a different set of functions. For example, a upstream protein may inhibit or promote the expression of a downstream gene. These are the most common applications of protein domains:
Terminator
Translation is stopped by terminators. The conventional model of transcriptional termination is that the stem loop causes RNA polymerase to pause and transcription of the poly-A tail causes the RNA:DNA duplex to unwind and dissociate from RNA polymerase. However, in general, most transcriptional terminators will not terminate transcription with 100% efficiency.
There are a few common types of terminators with different functions:
Plasmid backbone
Plasmids are circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. Plasmid DNA is easily purified from cells (by mini-prepping), manipulated using common lab techniques and incorporated into cells. Most BioBrick parts in the Registry are maintained and distributed in the form of plasmids. Thus, construction of BioBrick parts, devices and systems usually requires working with plasmids. Plasmid backbone contains replication origin and selection marker (an antibiotic resistance gene). This facilitates the uptake of the gene of interest into the host cell and the propagation of gene inside the host cell. The restriction enzyme sites upstream and downstream along the coding sequence allow restriction, extracting only the coding sequence for further experimentation. In the figure below, BioBrick part represents the protein coding sequence, i.e. gene of interest.
Prefixes and Suffixes
The prefix and suffix contain restriction enzyme cut sites, they should be inserted to all constructs in iGEM at the beginning and the end of sequences respectively. Using restriction enzymes EcoRI, XbaI, SpeI and PstI, these prefix and suffix enable easy transfer and assemble your parts.
Part design
Basic parts are put together to form a composite part to serve certain programed functions. Every complete functional composite part is composed is a Promoter - RBS - CDS - Terminator (in this oder). When ordering, add prefix before promoter and suffix after terminator. Below are one elementary and one more complex composite parts as examples.
Elementary example:
Complicated example (Hong_Kong_UCCKE 2017 team):
