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Team:Evry/Biology/Transposons
From 2014.igem.org
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<div align="center">a) Amplification of transposon</div> | <div align="center">a) Amplification of transposon</div> | ||
<br/> The transposon in pNK2 there is kanamycin gene. So we used PCR to amplify this gene. | <br/> The transposon in pNK2 there is kanamycin gene. So we used PCR to amplify this gene. | ||
| - | <br/>There is an amplification of the gene in the cells transformed. | + | <br/>There is an amplification of the gene in the cells transformed. All sequences them amplified come from ADNg and not from plasmid. |
| + | <img src="https://static.igem.org/mediawiki/2014/6/6c/Kan.JPG"/><br> | ||
| + | We obtains this gel for 15 colonies of Pseudovibrio <i>denitrificans</i>. | ||
| + | |||
<br/>It's first verification. | <br/>It's first verification. | ||
<br/> | <br/> | ||
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<br/> The amplification of transformed cells with primer 16S, which amplified the sequence of the ribosome 16S. | <br/> The amplification of transformed cells with primer 16S, which amplified the sequence of the ribosome 16S. | ||
The PCR product is purified and send to sequence. | The PCR product is purified and send to sequence. | ||
| + | <img src="https://2014.igem.org/File:Verif_pseudo.jpg"/><br> | ||
| + | |||
<br/> | <br/> | ||
<div align="center">c) Amplification of specific sequence of Pseudovibrio <i>denitrificans</i></div> | <div align="center">c) Amplification of specific sequence of Pseudovibrio <i>denitrificans</i></div> | ||
Revision as of 22:43, 17 October 2014
Biology - Transposons
Transposon
A transposon is a sequence of DNA able to move by itself within the genome preferentially in genic regions; characterized as mobile genetic element or transposable element. This event occur according differents transposition mechanism. One of them called cut-and-paste mechanism require an enzyme : transposase. Thienzyme is encoded within some transposons. Transposase binds to the end of the transposon, which is consist of inverted repeats and catalyze the movement of transposon for insertion and excision.
Transposas Tn10 / Is10
The complex Tn10/IS10 is involved in the non-replicative cut-and-paste mechanism.; the transposable segment is excised by cleavage at the transposon ends and then re-inserted in a target DNA site.
Tn10 transposase protein is made up of 402 amino-acid, which recognise inverted repeats insertion sequence; Is10-right and Is10-left.
Tn10 is a composite bacterial transposon subject to a strong positive and negative regulation.
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 for the transposase protein Tn10. The two ends of IS-10 have a similar terminal inverted repeat of 23bp, correspond to the transposase-binding site. However some genetic drift between both result in different functionality; Is10-right is fully functional while Is10-left is partially functional.
Our project
We tested some plasmids, and some methods to transform Pseudovibrio denitrificans but unsuccessfully. So the integration in the genome was tested. For it the researcher Brian Jester gave us the plasmide pNK2 as well as cells DH5α pir.In fact the plasmid pNK2 contains a particular origine of replication OriVR6Kgamma. The ori VR6K gamma is controlled by pi protein, which is encoded by pir gene and binds to site and allowing the replication.Hence, the oriVR6K gamma can only be replicated in strain with pi protein. This ori was already used in iGEM competition in 2009 by a french team. The gamma origin is adjacent to pi protein binding site and other site bound by proteins from the host bacterial cell, which includes in its own reproduction.
Insertion of transposon
- Transformation Pseudovibrio denitrificans with pNK2
- Phenotypic verification
- Genotypic verification
The plasmid pNK2 is tested in Pseudovibrio denitrificans. For this the cells is transformed by electroporation. The selection of cells is made in medium marine broth 1X with kanamycine 50µg/mL. In fact Pseudovibrio denitrificans can survive with kanamycine 25µg/mL. link name
In this image we can see that only the transformed cells can survive at this concentration of antibiotic. This is a phenotypic verification that there is integration of transposons.
The transposon in pNK2 there is kanamycin gene. So we used PCR to amplify this gene.
There is an amplification of the gene in the cells transformed. All sequences them amplified come from ADNg and not from plasmid.
We obtains this gel for 15 colonies of Pseudovibrio denitrificans.
It's first verification.
The amplification of transformed cells with primer 16S, which amplified the sequence of the ribosome 16S. The PCR product is purified and send to sequence.
