Silver: BB Strategy
From OpenWetWare
Making a New BioBrick Part
- Create an insert containing the new part flanked by BioBrick ends.
- For parts that are smaller than ~85 bp, then the part can be make using an oligonucleotide insert.
- For parts that are between ~ 85 - 150 bp, then the part can be make by using overlapping oligonucleotide inserts.
- For parts that are larger than ~85 bp and are based on an existing DNA fragment, then use PCR amplification of the existing DNA.
- Digest the insert and the BioBrick vector.
- Ligate the insert into the vector.
- Verify the new part.
Combining Two Parts
- If possible, use a suffix insertion (insertion of the added part behind the existing part), over a prefix insertion (insertion of the added part in front of the existing part).
- Gel extraction of fragments less than 200 bp is rather difficult, so plan your strategy so that the insert part is larger than 200 bp.
- Isolating one of two very similarly sized DNA fragments by gel extraction is difficult, so avoid doing a digest which produces an insert which is within ~700 bp of the size of the cut vector. If necessary, use a triple digest (typically ApaLI is the third restriction enzyme) to cut the vector in an additional location, while leaving your insert intact.
prefix (insert) | suffix (vector) | prefix (vector) | suffix (insert) | |
Enzyme 1 | EcoRI | EcoRI | SpeI | XbaI |
Enzyme 2 | SpeI | XbaI | PstI | PstI |
Buffer | 2 | EcoRI | 2 | 3 |
- Prefix 5'-gaattcgcggccgcttctaga-(part)-actagtagcggccgctgcag-3' Postfix
- In separate tubes, digest both vectors containing the parts to be combined using the scheme above.
- Mix:
- 700 ng BioBrick vector
- 1 µL 10x BSA
- 1 µL 10x NEB buffer x
- 0.1-0.2 µL Enzyme 1 (20 units/mL)
- 0.1-0.2 µL Enzyme 2 (20 units/mL)
- distilled water to final volume of 10 µL
- Mix:
- Incubate overnight at 37 °C.
- To the vector digestion mix only, add 0.1 µL CIP next morning and incubate for 1 hr. at 37 °C.
- Purification of vector and insert
- Run an agarose gel of the insert (and optionally, the vector).
- Cut the insert out of the agarose gel and extract it using Qiagen's Gel Extraction Kit (and optionally, the vector).
- If the vector was not gel extracted, use Qiagen's PCR Purification Kit to remove the small, undesired DNA fragments.
- Ligate the insert into the cut vector to combine the parts.
- Transform the new BioBrick vector into E. coli.
- Verify the new part.
Making a Final Part and Incorporating it into a Yeast Shuttle Vector
- Final parts can be simply incorporated into a Sikorski vector by isolating the part and ligating it into an appropriately cut Sikorski vector.
- Perform preparative digests of the Sikorski vector, and the full part per the following.
Sikorski vector | insert & vector digest |
pRS304* (TRP1) | EcoRI, SpeI |
pRS305 (LEU2) | XbaI, PstI |
pRS306 (URA3) | EcoRI, SpeI |
- Alternatively, the final construction step of the BioBrick part can be combined with incorporation into a Sikorski vector (a vector which allows the part to be integrated into the yeast genome) by performing a triple ligation.
- Perform preparative digests of the Sikorski vector, the forward part, and the back part per the following.
Sikorski vector | vector digest | forward part digest | back part digest |
pRS304* (TRP1) | EcoRI, Not I | EcoRI, SpeI | XbaI, NotI |
pRS305 (LEU2) | NotI, PstI | NotI,SpeI | XbaI, PstI |
pRS306 (URA3) | EcoRI, Not I | EcoRI, SpeI | XbaI, NotI |
- Ligate, transform, and verify the part.
- Note:
- If this is the only construct to be incorporated into the 580a strain, then the final BioBricks part should be incorporated into the URA3 Sikorski vector (pR306; Silver collection pPS750).
- If this is not the only construct that will be integrated into a single yeast strain, then you must decide which auxotrophic marker will be used for this particular construct.
- The orientation of the BioBrick part should be opposite that of the auxotropic gene once incorporated into the Sikorski vector.