Latest revision as of 16:02, 24 October 2017

Constitutive promoter flanked by two loxP sites

This is a constitutive promoter (J23115) flanked by two opposite loxP sites so that it can flipped by Cre recombinase.

Usage and Biology

Cre-loxP is a site-specific system that can make a DNA sequence flipped. This part can make a constitutive promoter (J23115) flipped and it can work as a switch driven by Cre. Sequence and Features

Functional Parameters

We constructed K740001 to test this part. The expression of RFP depends on the direction of the promoter. We transformed BBa_K740000 and BBa_I718008 into EPI300, and used flowcytometer to get the number of cells that expressed RFP.

We can get from this figure that the efficiency of this part depends on the expression of Cre. We can easily get a 80% efficiency with a short inducing time, and better efficiency may be gotten by some control methods, for example, feed forward.

Improvement

The iGEM Team Chalmers-Gothenburg 2017 improved this part by creating a non-reversible switch with the two loxP-sites. The old loxP-sites were changed to lox66 and lox71 which both contain a mutation that make the flip non-reversible. A study over three days was done, which concluded that the switch system worked, further more on that in the characterization section, or check out the original part BBa_K2329000 page.

Characterization

Figure 2: The GFP expression over three days. The left pictures shows the bright-field images of the cells to give a input about the total quantity of cells and the right pictures show the fluorescence of these cells.

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 P_TEF1 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 P_FUS1 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 P_FUS1 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.

Figure 3: The gel electrophoresis result for the colony-PCR. The band visible is one control (C), and three replicates (1-3). A band at 1300 bp indicates a success in the flipping of the promoter, and a band at 500 bp shows a failure. The ladder used is GeneRuler 1kb.

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]