Part:BBa_K1807000:Design
Protein generator device suitable for blue-white screening and Gibson Assembly.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Source
Escherichia coli; Registry of Standard Parts
Design Notes
Above is a diagram of our Adapter BioBrick in the pSB1C3 vector. Our design incorporates a SmaI restriction digestion step and subsequent use of the cut Adapter as a vector in a Gibson Assembly Reaction or any other assembly method based on flanking overlapping sequences (as depicted below).
BBa_K1807000 was designed as a blue-white screening device that would also be easily used in Gibson Assembly.
Surrounding the lacZ alpha coding sequence are two SmaI restriction sites (CCC/GGG). SmaI is a blunt-end endonuclease- we used it to simulatenously linearize our vector and remove the lacZ alpha coding sequence. Our BioBricks contained overhangs that make them compatible with the SmaI-digested BBa_K1807000.
The overhang sequences used to make this part are as follows:
BBa_K1807000 Assembly 5'end Overhang (complementary to pSB1C3): cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGAG
BBa_K1807000 Assembly 3'end Overhang (complementary to pSB1C3): TACTAGTAGCGGCCGCTGCAGtccggcaaaaaagggcaag
The subparts of the device are as follows: BBa_R0011, BBa_B0034, BBa_E0038, BBa_B0015.
How to Use this Part
The Splice-In Flanking Sequences (30bp each) that allow any Protein Coding Sequence to be inserted in place of lacZ alpha:
UPSTREAM of CDS : 5' AGCACATACGAGAAAGAGGAGAAATACCCC 3' + Start Codon (ATG) + CDS
DOWNSTREAM of CDS: CDS + Stop Codon(s) + 5' GAGCCTTTCGTTTTATTTGATGCCTGGCCC 3'
iGEM York 2015 recommends that teams and researchers use the above-listed Splice-In Flanking Sequences together with BBa_K1807000 for Gibson Assembly or any other cloning or assembly method relying on complementary overhangs.
Cloning
The Splice-In Flanking Sequences can be incorporated in a pair of primers as 5' overhangs. Such a primer pair can be used to directly amplify a target CDS from genomic DNA extraction, purified plasmid DNA or genomic (plasmid) DNA from a colony (as in a Colony PCR).
If cloning from an operon or just a protein domain, for efficient translation it is important to include Start and Stop Codons. Those can be added to the overhang sequence as depicted above.
Synthetic Gene Design
If designing a G Block fragment or any other dsDNA that is to be chemically synthesized the Splice-In Flanking Sequences can be added to each end.