Adding a synthetic RBS to a biobrick coding sequence=
Since the Registry requires coding sequences to be provided without ribosome binding sites, you will often need to add one. For this purpose we have designed three oligonucleotides:
rbs1f1 and rbs1r1 are the two strands of a synthetic strong ribosome binding site for E. coli. The outline procedure is as follows:
1. Mix rbs1f1 and rbs1r1 and perform 1 cycle in a PCR machine to allow annealing.
- Water: 37 microlitres
- Oligo rbs1f1: 2 microlitres
- Oligo rbs1r1: 2 microlitres
Perform I cycle of denaturation and annealing and halt at the end of the annealing step. Add 5 microlitres buffer E and 2 microlitres each EcoRI and SpeI. Incubate this for 2 hours at 37˚C. Heat treat at 65˚C for 15 minutes to remove enzyme activity (glass bead purification may not work well with such small DNA fragments). Store this at -20˚C. There is a stock in the freezer, so you may be able to skip this step.
2. Digest the biobrick coding sequence with XbaI. Purify and ligate to the rbs (standard ligation, 4 microlitres each biobrick and rbs plus one microlitre each buffer and ligase).
3. Use one microlitre of this ligation as a template for PCR with Pfu, using primer rbsclonf1 as forward primer and either bbinsr1 or the specific biobrick reverse primer. Clone the PCR product as a new biobrick.
4. Since the rbs is so small, you will probably not be able to detect it on a gel just as an increase in insert size. For this reason, we have included a SacI site in the forward rbs primer. This should not be present in the coding sequence forward primer. Thus plasmid minipreps can be screened using SacI with either SpeI or PstI and looking for excision of the insert. Alternatively, PCR screening can be performed using rbsclonf1 and a suitable reverse primer.
Note: the SacI site also means that the product with rbs will be an Edinbrick (provided that the coding sequence contains no SacI sites). Thus it can be cloned using SacI/SpeI in an Edinbrick vector if desired.