Difference between revisions of "Part:BBa K2030002:Design"

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For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and purified from gel. The PCR product of AckA was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into <i>E. coli</i> DH5&alpha; and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 1266 (<i>AckA</i>). The biobrick was then sent for sequencing for further verification.
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For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and the 2044 bp band purified from gel. The PCR product of AckA was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into <i>E. coli</i> DH5&alpha; and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 1266 (<i>AckA</i>). The biobrick was then sent for sequencing for further verification.
  
 
===Source===
 
===Source===

Revision as of 10:00, 17 August 2016


Acetate kinase (ackA) from Synechocystis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The complete sequence of the genome of Synechocystis sp. PCC 6803 (Genbank accession number NC_000911) was used to design primers for amplification of this gene.

FW primer: gccgcttctagATGAAATTCCTGATTCTCAATGC

RV primer: ttcttcctgcagcggccgctactagtaTTACGATTGCTTTCTCTGTCC

The primers were designed to anneal at 64 °C using Phusion High-Fidelity DNA Polymerase.


The uppercase nucleotides hybridizes with the template and the lowercase nucleotides contains the overhang. The overhang in the FW primer contains the last 13 bp of the prefix, which includes 6 protective bases before the XbaI site to ensure efficient cutting. The overhang in the RV primer contains the suffix with additional 6 protective bases to ensure efficient cutting with PstI.


For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and the 2044 bp band purified from gel. The PCR product of AckA was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into E. coli DH5α and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 1266 (AckA). The biobrick was then sent for sequencing for further verification.

Source

Genomic DNA from Synechocystis sp. PCC 6803

References