Cfrench:bbprimerdesign
1. Designing Primers to Make a New Biobrick
This is well covered in the [Registry notes (link to be added here). More will be added here later, but what I mainly want to point out is the possibility of designing a primer with a SacI site (GAGTCT) overlapping the XbaI site of the biobrick prefix, so that the prefix runs:
nnnngaattcgcggccgcttctagag ctc nnnnn
Note that this does not in any way interfere with the required prefix sequence; biobricks incorporating such a SacI site are fully compliant (but note that this cannot be done for coding sequences, for which the prefix must overlap the ATG of the start codon).
What is the advantage of this? Mainly that you don't have to include either a full prefix or a full suffix on your forward and reverse primers, provided that the sequence you intend to clone contains no internal SacI sites. Your prefix can be as short as: nnGAGCTC and your suffix as short as nnACTAGTA. You can then digest your PCR product with SacI/SpeI and insert it into a vector which will provide the rest of the required sites to make a fully compliant biobrick. We have prepared a series of such vectors based on pSB1A2, containing marker genes which can be excised and replaced with the PCR product. These are:
- Edinbrick1: contains lacZ', which generates a blue pigment in lacZ-delta-M15 hosts such as JM109 in the presence of IPTG and Xgal (best for general use)
- Edinbrick2: contains xylE, which generates a yellow pigment in the presence of catechol (for use when your biobrick has LacZ activity)
- Edinbrick3: contains idoA, which generates a blue pigment with no chromogenic substrate required (in the current version activity is so low that it takes 2 days for colour to develop, so not that useful, but we may make a better version, which will save on Xgal and not require a special host)
- Edinbrick4: contains both lacZ' and xylE.
Another potential advantage is that the SacI sites are preserved during the Biobrick combination process. This means that if you have a complicated composite biobrick made up of parts with SacI sites, you can digest with SacI and run on a gel to generate a ladder of the individual parts, to check sizes. This provides an easy way of checking that you have what you think you have without sequencing (eg, if you have just revived a clone from the freezer and want to check that it really is what it says on the label).
Edinbrick2 has already been deposited in the Registry as BBa_J33204, so should be on the iGEM2007 plates. We will be happy to supply any or all of the others on request. Next step is to make better versions in pSB1A3 and other suitable vectors.
