Part:BBa_K3697003

Homology Arms for KanR integration in B. Subtilis

This part is derived from a portion of the pOpen Yeast plasmid between the AarI and MspA1I cut sites. More information about pOpen Yeast and how to get it through Stanford Free Genes can be found on their website and at this link: https://stanford.freegenes.org/products/popen_build. This sequence was then divided into two homology arms (homology arm 1 and homology arm 2 as marked in the annotations for the part) which should flank the region in the genome where the user would like the recombination to occur. When incorporated into the B. subtilis genome, these two sequences (homology arm 1 and homology arm 2) become the two homology arms needed to trigger B. subtilis' natural process of recombination in response to fragment of DNA containing the same sequence (the sequence listed below without the ATG labelled as "gene/fragment to be flanked"). One thing to note about the recombination response is that it will be triggered by any sequence that includes both of these "homology arms". They could flank something that someone wants to integrate into the genome or there could be nothing between homology arms. In both cases, a recombination event will occur.

Usage and Biology

One important thing to note about these homology arms is that they are both 550 base pairs in length. This is important because when trying to trigger a recombination event in B. subtilis it is best to use 2 regions of homology of at least 500 base pairs (totaling at least 1000 base pairs of homology) flanking the region where you would like recombination to occur. Shorter regions of homology can be used, but some people have documented reduced transformation efficiency when doing this [1].

Having known homology arms in the genome can be helpful for a couple reasons. One, they could be incorporated into the B. subtilis genome so that any target sequence flanked by regions of high homology to these homology arms could be incorporated into the B. subtilis genome. Two, these homology arms could be incorporated into the B. subtilis genome flanking a sequence of interest, then if the target sequence is taken up by B. subtilis the sequence being flanked by these homology arms could be excised from the B. subtilis genome (an example of a system using this behavior is described in part BBa_K3697004).

Modelling its Effectiveness

Due to the fact that we did not have lab space due to the restrictions related to the COVID-19 pandemic. We did not have adequate time to test every metric of the part in lab, but we did establish computational models based on the literature surrounding this part and the system it was incorporated into. This modelling is summarized and explained below:

[1] Dubnau D. Sonenshein AL, Hoch JA, Losick R. Genetic exchange and homologous recombination, Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, 1993Washington, DCASM(pg. 555-584)

Sequence and Features

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]