Regulatory
Switch

Part:BBa_K2329000

Designed by: Karl Alex Hedin   Group: iGEM17_Chalmers-Gothenburg   (2017-09-19)
Revision as of 14:50, 8 October 2017 by AlexHedin (Talk | contribs)


A non-reversible switch
The Cre-lox recombinase system introduce a technique 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 is orientated in the opposite direction the recombinase will result in inversion of the sequence. The mutant lox66 and lox71 site create an inversion that doesn’t flip back [1].

Usage and Biology

The strong promoter, PTEF1, within the two mutant loxP-sites, make it possible to switch the direction and therefor encoding different genes. The non-reversible switch is a modification version of BBa_K740000, by modifying the loxP-sites so the switch becomes non-reversible. Another promoter, TEF1 instead of J23115, is used as well but the idea is the same. The modification of the LoxP-site includes a mutation in the end of the first and second LoxP sequence. The mutations are put in place in order to make LoxP recombination non-reversible. In Figure 1 shows where the LoxP-PTEF1-LoxP is located on the plasmid, Cre-Cas9, which is party used for the characterization explained below.
Figure 1: The construct of the plasmid Cre-Cas9 with a clearly illustration of where LoxP-PTEF1-LoxP is located, in comparison with the other parts in the plasmid. The plasmid origin is from pRS416.

Characterization

Figure 2: The GFP expression over three days. The left pictures show i microscopic picture over the cells to give a input about the quantitative 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 the system plasmid (Cre-Cas9) was that if the switch system worked, the PTEF1 would flipped and switch direction. So, instead of coding GFP it would code for Cas9. Therefor losing theirs GFP expression.

The inducible Cre-recombinase plasmid was added due to the consider of the possible weakness of our systems original FUS1 promoter and the activation of it, so the Cre-recombinase wouldn’t be enough. The encoding of Cre-recombinase in the plasmid is activated in present of galactose. So, the cell culture, with the flipping system and Cre-recombinase plasmid, was grown in a Delft medium, containing galactose. By watch under fluorescence microscope under three days, it would be possible to draw conclusion if the switch is non-reversible or not, at least after three days.

Three replicates were done, and all gave a similar result. The three days are presented in Figure 2. A clear pattern is seen. The expression of fluorescence appears to decrease over the days. During the first day it seems that the efficiency isn't 100 %, or that it takes time for the GFP do degrade.

The fluorescent picture indicate that the flip of the promoter might worked, and stopped the encoding of GFP. To really make sure of it, and get an easy look on sequence level of the construct a colony-PCR was run. This will also make sure that the whole promoter flanked with loxP-sites haven’t been cut out as well, which is unlikely due to the LoxP-site is designed in opposite direction. Figure 3 show the gel electrophoresis picture. The upper run show only a band if the flip succeeded and the lower run show both a band if it succeeded, at 1300 bp, and if not succeeded, at 500 bp. A negative control was run as well, which showed no band at the upper run and a band at 500 bp in the lower run, which it supposed to do. In the gel picture, it is visible that one of three seems to have flipped correct. The other two seems to have a problem in the PCR, because no band was shown in the lower run as well, there it should appear a band regardless the direction of the promoter.

Figure 3: The gel electrophoresis result for the colony-PCR. A band at 1300 bp indicate a success in the flipping of the promoter, and a band at 500 bp show a failure. The ladder used is GeneRuler 1kb.

With this the conclusion can be draw that the non-reversible system worked.























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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 275


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Parameters
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