Team:Evry/Biology/Transposons

From 2014.igem.org

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<div align="center">d) Integration of transposons in Pseudovibrio <i>denitrificans</i></div>
<div align="center">d) Integration of transposons in Pseudovibrio <i>denitrificans</i></div>
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Here, we tried to proved that there was actually a random integration of the transposon in the strain's genome.me.Pour cela la technique d'inverse PCR est utilisée.
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Here, we tried to proved that there was actually a random integration of the transposon in the strain's genome. To achieve this, we performed a reverse PCR.
<img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG"/><br>
<img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG"/><br>
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<div align="center">The transformation of Pseudovibrio <i>denitrificans</i> have been verified by the phenotype and the genotype. Now we know that transposas is fonctionel for our bacteria. c</div>
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<div align="center">The transformation of Pseudovibrio <i>denitrificans</i> has been verified by the study of its phenotype and its genotype. We therefore confirm the efficiency of the transposon system in our bacteria. c</div>
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<h4> <b>Creation of Transposon Plasmid </b></h4>
<h4> <b>Creation of Transposon Plasmid </b></h4>
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The second part consists in introduce our construction in this plasmid. The probleme is that our plasmid have some biobrick restriction site. So we decided to build our plasmid name Transposon plasmid without biobrick restriction site.  
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The second part of our work consisted in introducing our construction in this plasmid. A problem we had to face though was the presence of biobrick restriction sites in our plasmid. We decided to modify our Transposon plasmid so it would no longer have those restriction sites.  
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<br>Voici le schema de notre projet.
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<br>Here is the schema of our project.
   
   
<img src="https://static.igem.org/mediawiki/2014/a/a5/Schema_4bis.png"/><br>
<img src="https://static.igem.org/mediawiki/2014/a/a5/Schema_4bis.png"/><br>
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Il s'agit de construire un merge entre pSB1C3 et pNK2. Cela permettra d'amplifier notre plasmide de la transposas dans des cellules non pir, mais aussi d'envoyer notre plasmide sous format biobrick. Le plasmide pSB1C3 nous sert ici de backbone.  
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Our aim was to merged pSB1C3 and pNK2 into a single plasmid, in order to have a plasmid that would replicate in non-pir cells. Also, our goal was to have a plasmid matching the requirements of the biobrick format. Here we used the pSB1C3 plasmid as the backbone.  
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<br> Pour séparer les 2 plasmides ont pourra digérer le merge avec BglII et extraire sur gel notre Transposon Plasmid. Ce dernier plasmid ne contient que les éléments essentiel pour la transposition. Entre les 2 élèment transposeble iS10 nous intégrons le prefix et suffix iGEM afin de pouvoir intégrer dans le génome toutes les biobricks.  
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<br> To isolate the two plasmids it would be possible to digeste the merged plasmid with BglII and then extract by gel eletrophoresis our Transposon plasmid, which only contains the core elements needed for the transposition. Between the two transposable elements iS10 we will eventually integrate the biobrick prefix and suffix to be able to integrate any biobrick in a genome.  
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Premièrement il s'agira d'amplifier et d'assembler chaque éléments par Golden Gate.  
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As a first step, we have to amplify and assemble each element by the Golden Gate assembly method.
<img src="https://static.igem.org/mediawiki/2014/1/15/Schema_8_%281%29.png"/><br>
<img src="https://static.igem.org/mediawiki/2014/1/15/Schema_8_%281%29.png"/><br>
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<b><li> Mutation de l'oRiVR6Kgamma</b>
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<b><li> Mutation of oRiVR6Kgamma</b>
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Le premier soucis est que l'origine de réplication OriVR6Kgamma contient un site de restriction XbaI. Aussi avant de l'amplifier avec les overahang golden gate, il s'agira de muter ce site dans le plasmide directement par PCR mutagenese.  
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The first issue we encountered was the presence of XbaI restriction sites in the OriVR6Kgamma sequence. Thus, before amplifying it with Golden Gate overhangs, we had to induce mutations in that site of the plasmid using PCR mutagenesis.  
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<br> Le plasmid pNK2 est donc amplifier avec des primers permettant de modifier le site XbaI : 5' TCTAGA 3' en 5' ACTAGA 3'.
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<br> pNK2 plasmid was amplified with primers allowing us to modify the XbaI site : 5' TCTAGA 3' en 5' ACTAGA 3'.
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<br> Après amplification du plasmid, purification du produit de PCR et transformation plusieurs colonies sont obtenues. Plusieurs colonies sont mis en culture en milieu de sélection Kanamycine (50µg/mL) et le plasmide pNK2 est extrait. Ces plasmides sont digéré par XbaI afin de vérifier si le site a été muté.  
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<br> After amplification of the plasmid, we purified the PCR products obtained and transformed several colonies we got. Many colonies were inoculated in liquid cultures supplemented with kanamycin (50µg/mL) and the pNK2 plasmid was finally extracted. Those plasmids were digested by XbaI in order to verify the mutation of the XbaI site.
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Revision as of 03:00, 18 October 2014

IGEM Evry 2014

Biology - Transposons

Transposon


Transposons, known as mobile elements or transposable elements, are DNA sequences able to move randomly within the genome. This phenomenon is based on different transposition mechanisms. One of them, the cut-and-paste mechanism, requires an enzyme named transposase. Transposases bind to the end of transposons sequences, which consist of inverted repeats and then catalyze the excision and insertion of the transposons into the genome.

Fig. 1 General transposon mechanism
1. The transposable element, surrounded by IS sequence is "cut-and-paste" from a locus to another thanks to the transposase (arrow). 2. Insertion of the transposon occurs randomly in the genome

Transposase Tn10 / IS10


The complex Tn10/IS10 is involved in the non-replicative cut-and-paste mechanism. The transposable segment is excised at its ends and is then re-inserted randomly in a DNA site.

The Tn10 transposase protein is made of 402 amino-acids, which recognises inverted repeats insertion sequence; Is10-right and Is10-left. The Tn10 protein expression is strongly regulated by various positive and negative regulation mechanisms.

IS 10 is an insertion sequence composing the transposon Tn10. The two IS10 elements, IS10-Right and IS10-Left, contain all of the Tn10 encoded genetic determinants; as the coding region of the transposase protein Tn10. The two ends of IS-10 have a similar terminal inverted repeat of 23 bp, corresponding to the transposase binding site. However, some genetic drifts occurring between both of these sequences caused variation in their functionality. Indeed, IS10-right is fully functional while IS10-left is partially functional.



Our project



We tested different plasmids and methods to transform Pseudovibrio denitrificans but yet unsuccessfully. Thus, the integration in the genome was tested. Doctor Brian Jester gave us the pNK2 plasmid and DH5α pir cells. In fact, the plasmid pNK2 contains a particular origin of replication OriVR6Kgamma. This ORI VR6K gamma is controlled by the pi protein, which is encoded by the pir gene. Indeed, the pi protein allows the replication of the plasmid by binding a particular site in the ORI sequence. Hence, the oriVR6K gamma can only be replicated in a bacterial strain producing the pi protein. This ori was already used in the iGEM competition in 2009 by a french team. The gamma origin is adjacent to the pi protein binding site and other sites bounded by the host cell proteins involved in its own reproduction.



Fig. 2 Plasmid map of pNK2-CRPIIh
The plasmid is 6,356 bp long and annotated. It contains the transposase Tn10 and the origin of replication oriVR6K. The sequence including the antibiotic resistance cassette kanR, and the mGFP gene is surrounded by IS sites. Restriction sites used by the registry are encircled by red rectangle.


Insertion of transposon


  1. Transformation Pseudovibrio denitrificans with pNK2

    We tested pNK2 by transforming Pseudovibrio denitrificans bacteria with the plasmid by electroporation. The selection of cells was performed in 1X marine broth medium supplemented with kanamycine 50µg/mL. In fact, Pseudovibrio denitrificans can grow on medium with kanamycine 25µg/mL. (Sensitivity to antibiotics)


  2. Phenotypic verification



    Fig. 3 Image of Pseudovibrio denitrificans colonies in a petri dish containing kanamycin. Pseudovibrio denitrificans were previously transformed with the pNK2-CRPIIh plasmid.


    Fig. 4 Growth curve of Pseudovibrio denitrificans in M9 supplemented with casamino acids and 3% NaCl

  3. Genotypic verification

    a) Amplification of transposon

    The kanamycin gene is included in the transposon sequence of pNK2 and we thus amplify this gene for our verification PCR.
    We successfully obtained an amplicon corresponding to the kanamycin gene in our transformed cells. Considering the plasmids are not able to be replicated in an other strain than pir cells, we assumed the amplification we obtained were from ADNg and not from plasmids.

    As a first verification, we obtained this gel for 15 colonies of Pseudovibrio denitrificans.


    b) Sequencing of 16S

    The amplification of transformed cells' DNA with primers 16S, which amplify the sequence of the ribosome 16S, were purified and sent to sequencing.
    The sequence we obtained proves that we transformed actual Pseudovibrio bacteria with an integrated transposon.

    c) Amplification of specific sequence of Pseudovibrio denitrificans
    After the results of sequencing of Pseudovibrio denitrificans' genome, a sequence specific of this strain has been discovered.
    This sequence is the following (read of 1000 bp):


    GACGGTGTCATTGATCTGGATAATGTCAACGAGCAGACCGGATCTTATCAGTTTGTCGGTGATGATGGGTTTGATACGGCAGGGCGCAGTATCTCTTCAGCTGGTGATG
    TTGATGGTGATGGTAAGGATGATCTGCTCATCGGTGCTGCGAATGCTAATGGTAGTGGTGCCAACCAAGGATCCGCTTCAGGGGCTGCTTATCTGATGACGGCTTCTGCACTA
    GCAGCCGCTGATGCCGCTGACGGCACCACTGATGGTGTTATTGATTTGGGTAATGTCAATGAGCAGACTGGATCTTATCAGTTCAATGGTACAGAAGTAATGGACCAAGCCGG
    AACTCGTGTAACATCTGCAGGCGATGTGGATGGCGATGGCAAAGATGATGTCTTTATCAGCAGCATTTTTGCAGATGATGGCGGCTCCAGTTCTGGTGAAGCATATTTGCTGA
    CAGCTGCTGCTATGGCTTCAGCTGATGCCGCTGACGGCACTACTGACGGCATCATTGATTTGGACAATGTCAATGAGCAAACCAACTCTTATCAGTTTGTTGGCACCCAAGCA
    GATGACCTGGCCGGCATTGATATCTCAGCTGCTGGTGATGTTGATGGCGATGGCAAAAATGACTTCTTGATCGGTGCTCGGGCAGCAGATGGTGGCGGCGCTGGCTCGGGTGA
    GGCCTATCTGTTGACTGCAGCAGCACTTGCTTCAGCTGATGCAGCTGATGGCACCACTGATGGGATTATCGATCTAGATAATGTCAATGAGCAGACTAACTCTTATCAGTTCG
    TTGGTACGGAAGTTGGCGATGATGCGGGAATTAGCGTGTCATTTGTCGGTGATGTTGACAATGATGGTAAGGACGATCTGTTGATTGGTGCACGTAATGCTGACGGCGGTGGC
    TCCAACTCTGGTGAAGCCTATCTAATGTCTATTGCTTCACTGGCGACTGCTGATGCAGCTGATGGCACCATTGATGGTGTTATCGATTTGGAT
    d) Integration of transposons in Pseudovibrio denitrificans
    Here, we tried to proved that there was actually a random integration of the transposon in the strain's genome. To achieve this, we performed a reverse PCR.
    paragraphe romain sur l'image

    The transformation of Pseudovibrio denitrificans has been verified by the study of its phenotype and its genotype. We therefore confirm the efficiency of the transposon system in our bacteria. c


    Creation of Transposon Plasmid

    The second part of our work consisted in introducing our construction in this plasmid. A problem we had to face though was the presence of biobrick restriction sites in our plasmid. We decided to modify our Transposon plasmid so it would no longer have those restriction sites.
    Here is the schema of our project.
    Our aim was to merged pSB1C3 and pNK2 into a single plasmid, in order to have a plasmid that would replicate in non-pir cells. Also, our goal was to have a plasmid matching the requirements of the biobrick format. Here we used the pSB1C3 plasmid as the backbone.
    To isolate the two plasmids it would be possible to digeste the merged plasmid with BglII and then extract by gel eletrophoresis our Transposon plasmid, which only contains the core elements needed for the transposition. Between the two transposable elements iS10 we will eventually integrate the biobrick prefix and suffix to be able to integrate any biobrick in a genome.

    As a first step, we have to amplify and assemble each element by the Golden Gate assembly method.
    1. Mutation of oRiVR6Kgamma The first issue we encountered was the presence of XbaI restriction sites in the OriVR6Kgamma sequence. Thus, before amplifying it with Golden Gate overhangs, we had to induce mutations in that site of the plasmid using PCR mutagenesis.
      pNK2 plasmid was amplified with primers allowing us to modify the XbaI site : 5' TCTAGA 3' en 5' ACTAGA 3'.
      After amplification of the plasmid, we purified the PCR products obtained and transformed several colonies we got. Many colonies were inoculated in liquid cultures supplemented with kanamycin (50µg/mL) and the pNK2 plasmid was finally extracted. Those plasmids were digested by XbaI in order to verify the mutation of the XbaI site.