Part:BBa_K3814068:Design
fliK Landing Pad (100%)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1087
Illegal NheI site found at 1110
Illegal NheI site found at 2383 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2529
Illegal XhoI site found at 1219 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2555
Illegal NgoMIV site found at 2923
Illegal NgoMIV site found at 3083
Illegal AgeI site found at 1513 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
In USYD 2021's project, we have used recombineering to insert 5kb chunks of DNA into E. coli. The particular recombineering strategy we have employed in our design is the bacteriophage λ Red recombineering system, and we are inserting our gene clusters into the fliK gene.
- In a study by Juhas and Ajioka (2016), the fliK gene in the E. coli was shown to be an optimal location for recombineering, and 15kb was successfully inserted there in one iteration. - The bacteriophage λ Red recombineering system is described in the diagram below, and many strains of E. coli have these systems already in place (Sharan et al., 2009). We have decided to use the JM109 strain and the recombineering functions were going to be brought in by the pKD46 plasmid.
Figure 1. Recombineering system using the bacteriophage λ Red system. According to Sharan et al. (2009), the homology arms need to be only 50bp for successful recombination, Additionally, only three genes, gam, bet and exo, are involved. The gene product of gam “prevents an E. coli nuclease, RecBCD, from degrading linear DNA fragments”, which allows for linear DNA to survive in vivo for recombination. The roles of exo and bet are shown above, with the gene product of bet, Beta, being an “ssDNA binding protein” and exo having “5′ to 3′dsDNA exonuclease activity”.
We devised a strategy called Babushka Blocks. See below an images that showcase how the homology arm (red and purple) would help in inserting a section of DNA into the fliK gene:
Figure 2. Babushka block design. To insert Cluster 1 into the fliK landing pad, Cluster 1 must be first hybridised with the primer, which contains the red homology arm (Step 1). Afterwards, there will be two matching homology arms between the landing pad and Cluster 1: the red and purple arms. As a result, Cluster 1 is able to be inserted into the landing pad, and the end result has the genes of interest inside the landing pad (Step 2).
On top of this, we have designed an inducible promoter that combines the work of two researchers (Schell & Poser, 1989; Meyer et al., 2018). It uses the nahR gene and sal promoters to activate transcription in response to the inducer salicylate, and can with a few base changes in the promoter, can provide different promoter effects. See below a diagram of the design and the base changes that induce varying transcription level effects:
Figure 3. Inducible promoter design. According to Cebolla et al. (1997), nahR produces a transcription factor that controls the expression of genes regulated by sal promoters. In the presence of salicylate, expression of those genes is facilitated.
Figure 4. Sequences that generate different expression levels of a gene (RHS). Combined with the addition of TTC (Schell & Poser, 1989), this should generate a system that is tightly repressed and highly expressed in the absence and presence of salicylate.
We have designed two fliK landing pads, this one having 100% expression levels and another (K3814067) having 34%. On top of using these landing pads to insert gene clusters into, we aim to simultaneously use assays to determine the effectiveness of these base changes in changing expression levels. This will help inform the way in which we conduct the rest of the experiments.
Source
iGEM USYD 2021
References
Juhas, M., & Ajioka, J. W. (2016). Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-016-0571-y
Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204. https://doi.org/10.1038/s41589-018-0168-3
Schell, M. A., & Poser, E. F. (1989). Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. Journal of Bacteriology, 171(2), 837–846. https://doi.org/10.1128/jb.171.2.837-846.1989
Sharan, S. K., Thomason, L. C., Kuznetsov, S. G., & Court, D. L. (2009). Recombineering: a homologous recombination-based method of genetic engineering. Nature Protocols, 4(2), 206–223. https://doi.org/10.1038/nprot.2008.227