Part:BBa_K2406013
M3 (Target site for Cre Recombinase)
Introduction
Cre/LoxP recombination is the most popular site-specific recombination system used in biotechnology [1]. It is particularly popular for creating conditional knock-outs in mammalian cells, but it can also function in other popular chassis, including yeast, E. coli, and in vitro [1]. In order to create multiple, distinct Cre-induced recombination events within one cell, it is required to go beyond the traditional Cre/LoxP system. This is because all LoxP sites can recombine with each other, so having more than two can lead to unintended and unwanted recombination events between several LoxP sites at different locations. A way to get around this issue is to use orthogonal Lox sites, i.e. Cre-recognised target sites that recombine with one another but not Lox sites of another class [1]. These sites are created through mutations in the spacer region between the two left/right arms recognised by Cre recombinase [1]. Essentially, in a few reported Lox mutants, spacer regions would be incompatible with one another, meaning recombination could not occur between these two orthogonal lox sites [1]. Here, we report the construction of a biobrick form of LoxM3that is known to be orthogonal to LoxP [1].
Results
Sequencing results (attached below) indicate that LoxM3 has been constructed successfully by annealing two oligos together and ligated annealed product into pSB1C3 backbone. All primers used were standard biobrick primer VF. We proceeded to test activity of LoxM3 in vitro. We incubated the linear annealed oligo product of LoxM3 with the full plasmid containing this target site. We then exposed cells to cell-lysate from cells that were induced to express Cre Recombinase BBa_K2406080. Results are shown in adjacent gel photo. We performed this assay in parallel with all other orthogonal target sites BBa_K2406008, BBa_K2406009., BBa_K2406010., BBa_K2406011., BBa_K2406012., BBa_K2406014., BBa_K2406015., BBa_K2406016., BBa_K2406017. All assays showed identical results. Lanes with a smear were treated with cell lysate. A faint band can be seen within the smear. Adjacent lanes indicate the circular plasmid DNA. Bright band in cell lysates lane ran more slowly, indicating recombination between sites occurred, as linear DNA of a higher molecular weight would be produced, which would run more slowly on the gel compared to control plasmid that did not undergo recombination.
Discussion
We have successfully assembled a Lox target site that is known to be orthogonal to LoxP and recombine with itself [1]. Therefore, sequencing serves as a primary verification for this parts identity and function. Preliminary in vitro assay results are positive. However, the assay was done with incomplete controls, e.g. no lanes run where plasmid was treated with cell lysate not expressing Cre recombinase. Furthermore, there is no control lane that demonstrates definitively how a linearized plasmid product that has undergone recombination would run on the gel. Due to time constraints, these further tests were not completed before the registry freeze. However, as stated, preliminary results are promising and sequencing confirms we have successfully assembled a useful biobrick part in LoxM3.
References
[1] Missirlis, P.I., Smailus, D.E., and Holt, R.A. 2006. “A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination”. BMC Genomics 7: 73.
Sequences
File below confirms sequence of all orthogonal lox sites used. Media:File:Sequencing Results Edinburgh UG.zip
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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