Part:BBa_K2329000
A non-reversible switch
Cre-lox recombination is recombinase system that introduces a method to control the expression of a gene. The orientation and location of the loxP site will make it possible for the Cre recombinase to rearrange the sequence, which is flanked by the two loxP sites. If the direction of the loxP sites are orientated in the opposite direction of each other, so the recombinase will invert the sequence in between them. The mutant recombination sites lox66 and lox71 are used instead of the wild-type ones, in order to create an inversion that does not flip back. This makes the whole system non-reversible and creates a memory of activation in the cells[1].
Usage and Biology
Using a strong constitutive promoter TEF1[2] within the two mutant loxP-sites creates a possibility to switch the direction of the promoter. This can be used to express different genes lying upstream or downstream of this construct.
The switch is an improvement of the biobrick part BBa_K740000. The modified loxP-sites (lox66 and lox71) allows the switch to become non-reversible. These modification correspond to a mutation in the end of the first and second wild-type LoxP sequences. The promoter PTEF1 is used instead of J23115, but the idea is the same. Figure 1 shows where the loxP-PTEF1-loxP is located on the plasmid, Cre-Cas9, which is partly used for the characterization explained below.
Characterization
A study of only the flipping of the promoter was made, by adding an inducible Cre-recombinase plasmid. The idea behind the construction of our plasmid Cre-Cas9 is that if the switch worked, the PTEF1 would flip and switch direction. Therefore, instead of expressing GFP it would express Cas9. One can then expect a drop in GFP expression. To read more about the idea of Cas9 and the whole plasmid please check out [http://2017.igem.org/Team:Chalmers-Gothenburg/Description Project description].
The inducible Cre-recombinase plasmid was added to combat the possible weak expression from our systems original FUS1 promoter. The PFUS1 activation is accomplished through the yeast pheromone pathway, which in our case gets activated by the GPCRs. The activation of the FUS1 promoter will lead FUS1 to expression of Cre-recombinase and recombine the LoxP-sites, but due to the potential aforementioned issues with PFUS1 another approach was made.
The expression of Cre-recombinase in the plasmid is activated in presence of galactose. The cell culture with the Cre_Cas9 plasmid and the Cre-recombinase plasmid, was grown in a Delft medium containing galactose. By monitoring fluorescence using microscope during the course of three days, it would be possible to draw the conclusion if the switch is non-reversible or not.
Three replicates were done, and all gave a similar result. The three days for one replicate are presented in Figure 2. The expression of fluorescence appears to decrease as time passes. During the first day it seems that the efficiency isn't 100 %, or perhaps that it takes time for the GFP to degrade.
The fluorescence image indicates that the flip of the promoter has worked, and stopped expressing GFP. To confirm that the promoter has switched direction, a colony PCR was run. Figure 3 shows the gel electrophoresis image . The upper run shows a band if the flip succeeded and the lower run shows a band at 1300 bp if it succeeded and one at 500 bp if it did not succeed. A negative control was run which showed no band at the upper run and a band at 500 bp in the lower run, which it supposed to do according to the primers used. In the gel image it is visible that one of the three replicates seems to have flipped correct. Worth noting is that the succeeded replicate seems also to show a band at 500 bp in the lower run, which might indicate that the system is not 100 % efficient, but one must also take into account that the DNA template used in the colony-PCR was taken from day 1, when some fluorescence was still visible. The other two seems to have a problem in the PCR, because no band was shown in the lower run as well, where a band should appear regardless of the promoter direction.
With this the conclusion can be draw that the non-reversible system worked, as the literature predicts[1]. From these results, an improvement of the previous biobrick part BBa_K740000 was done. The direction of the promoter did not flip back during these three days, compared to the BBa_K740000 which constantly recombined back and forth. To read more about the results check out [http://2017.igem.org/Team:Chalmers-Gothenburg/Results Team: Chalmers-Gothenburg Achievements: Project results]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 275
References
[1] Zhang Z. Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Research [Internet]. 2002 [cited 10 October 2017];30(17):90e-90. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
[2] Kitamoto N, Matsui J, Kawai Y, Kato A, Yoshino S, Ohmiya K et al. Utilization of the TEF1-a gene ( TEF1 ) promoter for expression of polygalacturonase genes, pgaA and pgaB , in Aspergillus oryzae. Applied Microbiology and Biotechnology [Internet]. 1998 [cited 10 October 2017];50(1):85-92. Available from: https://www.ncbi.nlm.nih.gov/pubmed/9720204
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