Translational units are DNA sequences that encode mRNAs that can be translated into a polypeptide chain which then folds into a 3 dimensional structure called a protein. It includes both an RBS and the DNA sequence that encodes the protein, called the protein coding sequence.
Translational units consist of at least three parts, a Translational start (which encompasses both an RBS and Head domain), one or more Internal Domains including Special Internal Domains, and a Tail Domain. For more details, see the Protein domains page. Thus, the translational unit starts with the RBS, the site of ribosome binding and translational initiation, and ends with the stop codon, the site of translational termination.
Before you design a new translational unit, you might check if your translational unit already exists in the Registry.
Here are some basic things to consider when designing translational units.
- A translational unit contains one or more Internal domains. Design the internal domains so that the codon usage is optimized for the chassis in which the part will be used.
- If you also want to control where the protein is located in the cell, you might consider a localization sequence as the Head domain.
- If you also want to purify or quantify the protein later, you might add an affinity tag as either the Head domain or the Tail domain.
- The Tail domain of most translational units is made up of just a double stop codon TAATAA.
- If you want to control the degradation rate of the protein in E. coli, you might add a degradation tag as the Tail domain.
- Make sure that the translational unit doesn't have any BioBrick sites in it (EcoRI, XbaI, SpeI, or PstI). If it does, you'll need to remove them.
- If you are planning on synthesizing the translational unit via commercial gene synthesis, design your translational unit sequence to remove useful restriction enzyme sites. See a list of suggested sites for removal at OpenWetWare.
- After you do an initial design of your translational unit sequence, we suggest you run the beginning of the sequence (first 200-300 bp) through a secondary structure prediction algorithm like mFold to check if any hairpins form. In particular you should look for any hairpins that form between the RBS or start codon and the rest of the protein coding sequence that might inhibit translational initiation. If you find a hairpin, try to make some silent mutations in the coding sequence to disrupt the stability of the secondary structure.