Part:BBa_K2269017

GP3 (PhiC31 RDF)

In order to increase the genetic control over the expression of any color inducing protein through a viral vector strategy, a modular and orthogonal AND gate was designed. In normal basis, plant would be constitutively expressing PhiC31 recombinase and the stress-inducible promoter would be in OFF state (i.e. in opposite direction to the desired CDS) . In this case, PhiC31 attachment sites have been already recombined (attR and attL), being PhiC31 uncapable of inversing them back by itself. To do so a helper protein is required, so called recombinase directionality factor (RDF). With this approach recombinase switch becomes more robust, stable and less leaky.

Biology and Usage

Figure 1. Dynamic performance between RDF and PhiC31. a) Graphic representation of the register assembly comprised by a promoter and a terminator with the recombined sites. It represents the negative control in the graphic. b) Genetic construct that allows a constitutively expression of PhiC31 under a weak promoter (OD* 0,01) c) Graphic representation of the genetic construct comprised by a promoter and a terminator with the attachment sites. It represents the positive control in the graphic. d) Graphic representation of the gp3 genetic construct. Its expression is under a strong promoter and was tested in both treatments (OD* 0,1 and OD* 0,2). e) Plot of the luciferase expression levels obtained over time. f) Bars chart representing the difference of luciferase expression levels obtained before and after RDF induction.

• Final Optical Density of Agrobacterium tumefaciens culture. e) Plot representing data obtained

Recombination directionality factor’s (RDF or gp3) ability to reverse the integrase actuation direction allowing the inducible stress-promoter to acquire the active state was demonstrated in this experiment. Following the experimental design (http://2017.igem.org/Team:Valencia_UPV/Experiments#GP3) we proved the effectiveness of the RDF in the recombination system.

As can be seen in Figure 9, the expression of gp3 together with PhiC31 trigger luciferase expression obtaining expression levels similars to a weak promoter (Pnos promoter). Interestingly, the highest level of luciferase expression (expression levels similar to a weak promoter — pNOS) was obtained with a low RDF expression (infiltrated at OD 0.1). Whereas, when the RDF was relatively high (infiltrated at OD 0.1, that is 2-fold in comparison with the other experimental treatment), the activation was lower than a weak promoter (pNOS) expression levels.

Conclusion: The ability of the recombinase directionality factor (RDF) to reverse the integrase action direction was demonstrated. Thus, postulating as the best way to control the direction of the stress-inducible promoter to express a color inducing protein.

RDF characterization

RDF:phiC31 ratios define re-inversion efficiency. Bearing that in mind, we resolved to demonstrate that the optimal ratio between RDF and PhiC31 is 20:1 using a weak promoter for the recombinase expression and a strong one for the RDF. However, several experiments were performanced in order to demonstrate this hypothesis.

Figure 2. Dynamic performance between RDF and PhiC31. a) Graphic representation of the register assembly comprised by a promoter and a terminator with the recombined sites. It represents the negative control in the graphic. b) Genetic construct that allows a constitutively expression of PhiC31 under a strong promoter (OD* 0,1) c) Graphic representation of the genetic construct comprised by a promoter and a terminator with the attachment sites. It represents the positive control in the graphic. d) Graphic representation of the gp3 genetic construct. Its expression is under a strong promoter and was tested in both treatments (OD* 0,05 and OD* 0,15). e) Line graph representing the data obtained of the level of luciferase expression over time for RDF module infiltrated at OD 0.05 and 0.15. f) Line graph representing the data obtained of the level of luciferase expression over time for RDF module infiltrated at OD 0.2 and 0.35. *Final Optical Density of Agrobacterium tumefaciens culture

As can be seen in Figure 2.E and 2.F, any of the RDF’s ODs assessed were able to trigger luciferase expression (i.e. re-inverting the promoter).

Conclusion: These results help to corroborate the unproductive complex hypothesis. When PhiC31 expression is under a strong promoter regulation, the formation of this complexes impedes the site-specific recombination event.

References
1. Keravala, A., Groth, AC., Jarrahian, S., Thyagarajan, B., Hoyt, JJ., Kirby, PJ., and Calos, MP. (2006). A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Molecular Genetics and Genomics, 276(2), 135–146

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 79
  Illegal BamHI site found at 816
  Illegal BamHI site found at 846
  Illegal BamHI site found at 932
  Illegal BamHI site found at 942
  Illegal XhoI site found at 773
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 13
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1303
  Illegal SapI.rc site found at 1628