Ribosome Binding Sites/Construction


Ribosome Binding Sites (RBSs) are typically short sequences, often less than 20bp. That means that it is often cheaper and faster to order the DNA for an RBS to be chemically synthesized rather than prepping a plasmid carrying the sequence from cells or attempting to amplify the RBS sequence via PCR. This tutorial briefly outlines the steps involved in making an RBS from de novo synthesized DNA.

1. Design your RBS part BCArrow.png 2. Order oligos BCArrow.png 3. Double-stranding

1. Designing the oligos needed to make an RBS

  1. Obtain the desired sequence for the RBS from the registry RBS collection, or from another source. If your RBS sequence comes from another source, now is a good time to enter your sequence in the registry as a new part.
  2. Append the BioBrick prefix and suffix to your RBS sequence. The prefix is 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' and the suffix is 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'. You can find a good tutorial explaining where these prefix and suffix sequences come from [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here].
  3. Don't forget that you'll want your RBS to be double-stranded so once you have the sequence you want, you'll need to make the complementary sequence using one of many available [http://www.google.com/search?client=safari&rls=en&q=reverse+complement&ie=UTF-8&oe=UTF-8 tools]. The oligo is called the reverse oligo and the strand with your RBS sequence is called the forward strand.
  4. If your find that your RBS sequence with the appropriate prefix and suffix is >100bp then it might be difficult to order the entire RBS as a single oligo. The strategy in this case is to order the 5' end of the RBS with the BioBrick prefix on the forward strand and then order the 3' end of the RBS with the BioBrick suffix on the reverse strand. As long as there is at least a 20bp overlap between forward and reverse strand, youll be able to construct the full length RBS and prefix and suffix from the forward and reverse oligos (see the next section).

2. Ordering your oligos

  1. You can order your forward and reverse oligos from companies such as [http://www.invitrogen.com Invitrogen] or [http://www.idtdna.com IDT].
  2. These services are cheap and if you order early enough in the day, you can often get your DNA by the next day.

3. Constructing a double-stranded RBS

  1. Once your single-stranded oligos have arrived, you'll need to anneal them to make a double-stranded RBS ready for use in an assembly
  2. First, you'll need to resuspend your oligo's since they typically arrive as dehydrated DNA. You can read a protocol for resuspending oligos that is recommended by Invitrogen [http://openwetware.org/wiki/Reconstituting_primers here].
  3. Once your primers are resuspended, you need to anneal them. You can find some protocols for annealing primers [http://openwetware.org/wiki/Annealing_primers here]. If your RBS and prefix/suffix were too long to order on single oligos and instead you ordered the 5' end on the forward strand and the 3' end on the reverse strand with >20bp overlap, you can construct the full length double-stranded molecule via [http://openwetware.org/wiki/Annealing_and_primer_extension primer annealing and extension].
  4. Once your primers are annealed, you can digest them just like you would a plasmid, the digested RBS can be used in any BioBrick assembly.