Part:BBa_K3697003:Design
Homology Arms for KanR integration in B. Subtilis
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Two major design considerations were made during the designing of this sequence. One, we had to make sure that each of the homology arms were at least 500 bp as they had to be of sufficient length to allow a recombination event in B. subtilis to occur. Two, we wanted to make sure that the sequence was something that would be easy for us to acquire and make, so we chose a portion of the pOpen yeast plasmid (pOpen Yeast is easy to acquire through Stanford Free Genes) that can be easily made and purified through a restriction digest of pOpen Yeast with AarI and MspA1I and then running on the products of that digest on an agarose gel (cutting and gel purifying the segment that is 1100 bp in length).
Source
These homology arms originally come from the pOpen Yeast plasmid and contain the portion of this plasmid that codes for the Kanamycin resistance gene. The design for this system were inspired by the work done by Josef Altenbuchner and Marian Wenzel [2]
[2] Wenzel, M. and Altenbuchner, J. (2015) Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate‐dependent phosphotransferase system. Microbiology (United Kingdom), 161(10), 1942–1949.