Coding

Part:BBa_K1742004

Designed by: Ivn Casas Rodrigo   Group: iGEM15_Valencia_UPV   (2015-09-04)

PhiC31 Plant codon optimized

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. Valencia_UPVPhiC31.png

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 (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. With a binary GoldenBraid assembly step we obtained a multigenic construct (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.

ValenciaUPV_PhiC31CodonOptimGFP.png

Figure 1. Expression levels of GFP in N. benthamiana leaves. A) Agrobacterium-mediated transformation with the multigenic construct (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.

Valencia_UPV 2017 characterization

Parts used in the characterization:

Design2upvigem.png

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.

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:

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.

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: 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: 1)A significantly difference between the OFF and the ON state expression levels. 2)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). 3) 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).

Resultscharactupvigem3.png

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.


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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
n/aPhiC31 Plant codon optimized