Difference between revisions of "Part:BBa K2269017"

Revision as of 00:43, 2 November 2017

No part name specified with partinfo tag.

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
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 421
Illegal NheI site found at 591
Illegal NheI site found at 1633
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

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>BBa_K1742004 short</partinfo>
+<partinfo>BBa_K1742017 short</partinfo>
-PhiC31 is a site-specific serine recombinase derived from a ''Streptomyces phage'' [1]. The enzyme recognizes two different attachments sites called also attB and attP, and also excise a sequence flanked with attB and attP sites close to the promoter.
+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.
-https://static.igem.org/mediawiki/2015/a/af/Valencia_UPVPhiC31.png
-== Biology and Usage ==
+== Biology and Usage ==
-The ''Nicotiana benthamiana'' codon-optimized PhiC31 gene was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector.  The PhiC31 integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S).
-In order to see the activity of the integrase, Valencia_UPV 2015 designed a reporter element ([https://parts.igem.org/Part:BBa_K1742008 BBa_K1742008]) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attP:T35S:attB:omegaUTR) of PhiC31. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator.
+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.
-With a binary GoldenBraid assembly step we obtained a multigenic construct ([https://parts.igem.org/Part:BBa_K1742013 BBa_K1742013]) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into ''N. benthamiana'' plants for testing the expression of GFP.
+*Final Optical Density of Agrobacterium tumefaciens culture. e) Plot representing data obtained
-https://static.igem.org/mediawiki/2015/8/85/ValenciaUPV_PhiC31CodonOptimGFP.png
+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.
-<small><p><b>Figure 1. Expression levels of GFP in ''N. benthamiana'' leaves. A) Agrobacterium-mediated transformation with the multigenic construct ([https://parts.igem.org/Part:BBa_K1742013 BBa_K1742013]). B) Plant leaf transformed with the PhiC31 reporter element assembled with the P35S promoter without ATG, GFP coding sequence and the terminator T35S.</b></p></small>
-== Valencia_UPV 2017 characterization ==
+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.
-Parts used in the characterization:
-https://static.igem.org/mediawiki/parts/d/d8/Design2upvigem.png
+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.
-Figure 1. Graphic representation of Plant-Human genetic circuit with the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites. A) In presence of PhiC31 integrase, recombination occurs allowing the expression of a translation of a nonsense sequence or stuffer fragment. In this case, the circuit is in OFF state B) Graphic representation of the toggle switch in ON state only when PhiC31 and gp3 are expressed. Only when the promoter is activated under stress conditions, corresponding color protein will be expressed.
+RDF characterization
-These results showed a counterintuitive correlation between the recombinase and luciferase. The higher the expression of recombinase is, the lower the probability that inversion occurs is. That is, the more concentration of recombinase in plant cells, the fewer inversion efficiency is.
-Genetic parts and results:
-https://static.igem.org/mediawiki/parts/9/90/Resultscharactupvigem.png
-Figure 2. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a weak promoter. e) Transcriptional unit for the expression of the Firefly Luciferase under the control of a strong promoter. f) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter. g) Bars chart representing luciferase expression levels before and after induction with PhiC31.
+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.
-Method:
-An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 2a and Figure 2c) was 0,02 and the optical density of PhiC31 construct (Figure 2b) was 0,01. A triplicate sampling of different plants was performed in order to take account for biological variability due to unknown or uncontrollable conditions.
-TimeLine:
+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
-Register assembly constructs (Fig. 2a and 2b) and the controls (Fig. 2e and 5f) were agroinfiltrated and after 54h post-infiltration, leaves were sampled. After 48h, all leaves were also sampled. Overall, two points of samples were taken in the assay.
-After analyzing the data obtained from luciferase assay, it can be observed at Figure 2:
-)A significantly difference between the OFF and the ON state expression levels.
-)The recombinase expression under a weak promoter (pNOS promoter) shows the same luciferase expression that the constitutive positive control (genetic construct which is expressing constitutively luciferase protein under the strong promoter).
-) However, the recombinase expression under the strong promoter shows the same expression level of the constitutive weak control (genetic construct based on the constitutive expression of luciferase under a  pNOS weak promoter)
-The likelihood of producing the inversion event should be directly proportional to the luciferase protein concentration inside plant cell. Consequently, more quantity of phiC31 recombinase entails more probability of activating reporter gene expression. However, this experiment shows that once recombinase expression exceeds a certain threshold, the effect changes so the likelihood of up-regulating the reporter gene expression decreases.
-In order to provide robust evidences to the hypothesis explained above, a subsequent experiment was performed. In this case, PhiC31 behavior was analyzed while being regulated by a weak promoter (pNOS) together with an optical density (A.tumefaciens culture concentration) was 10-fold higher than the used in aforementioned experiment (Figure 3).
-In order to provide robust evidences to the hypothesis explained above, a subsequent experiment was performed. In this case, PhiC31 behavior was analyzed while being regulated by a weak promoter (pNOS) together with an optical density (A.tumefaciens culture concentration) was 10-fold higher than the used in aforementioned experiment (Figure 3).
+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).
-https://static.igem.org/mediawiki/parts/2/29/Resultscharactupvigem3.png
+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.
-Figure 3. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Plot representing PhiC31 behavior when infiltrated at OD 0,1.
-Conclusion: It was demonstrated the proper phiC31 functioning and determined its behavior under different protein concentrations and promoter strengths. Although the expected hypothesis may be that the more recombinase concentration the more inversion events, the obtained results suggest the existence of a threshold above which that assumption is not true. Therefore, a low recombinase expression is needed in order to maximize recombinase action.
-Our team has improved the characterization of the Phytobricks (PhiC31) documenting our experimental design and the results that were obtained. All data is also documented on the Main Page in the Registry.