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IGEM Evry 2014

Biology - Genome Assembly

De novo Genome assembly

Antibiotic resistance

Concerning the antibiotics, we look at specific antibiotics such as kanamycin, erythromycin, tetracycline, ampicillin, and chloramphenicol and other type of resistance gene. For the kanamycin and erythromycin, we found not relevant annotation. However, for the other ones, tetracycline, ampicillin, and chloramphenicol, we found specific annotations.
Regarding the tetracycline, we found that it possessed one Tetracycline repressor protein class H , two Tetracycline resistance protein, class C and nine Bacterial regulatory proteins, tetR family annotated genes. This result are coincident with our experiment where we found that Pseudovibrio is resistant to tetracycline.
About the ampicillin, one Metallo-beta-lactamase superfamily protein, Beta-lactamase, Beta-lactamase type II precursor, two Beta-lactamase precursor, Beta-lactamase hydrolase-like protein HTH-type transcriptional activator AmpR and five Putative beta-lactamase HcpC precursor were predicted by the Prokka software. Also this result is coincident with our experiment.
For the Chloramphenicol antibioctics, we predicted that the chloramphenicol phosphotransferase-like protein, Chloramphenicol 3-O phosphotransferase and Chloramphenicol acetyltransferase are present in our strain. The last one is used as a reporter gene in molecular biology. But in contrary to other antibiotics, the test does not reveal, that our strain is resistant to the Chloramphenicol.
Finally we look at other antibiotic resistance and we see that other antibiotics such as the Bleomycin are predicted. Bleomycin is used as a chemotherapy agents for the Hodgkin's lymphoma. Also it possessed the Multidrug resistance protein, MdtA, MdtA precursor, MdtB, MdtK, MdtN, MdtH, MdtG, which are known to play an important role in antibiotic resistance.

Restriction enzyme

During the transformation tests of our strain, we supposed that Pseudovibrio Denitrificans, was able to degrade DNA. Indeed all our electroporation attempts failed. They could not keep the plasmid inside them. One relevant research led us to the discovery of a Type I Restriction enzyme EcoKI. This site specific DNA methylase is not very common but can be found in a wide range of Gammaproteobacteria and so potentially into PseudoVibrio Denitrificans. We blasted the sequence of EcoRI gene with our bacteria genome, and we found that Pseudovibro have the gene. EcoKI recognises the sequence 5’ AAC(N)6GTGC-3’ and acts depending on the methylation state of the DNA substrate. It can be a methyltransferase or an endonuclease. Psb1c3, first plasmid that we tried to transform ( E.Coli Ori) does not have the target sequence of EcoRI. Otherwise, in one plasmid sent by Dr Thomas Drepper from Heinrich-Heine-Universität Düsseldorf, Institute of Molecular Enzyme Technology, Group of Bacterial Photobiotechnology, which was from a bacterium very closed with Pseudovibro and which we tried as well to transform, there is the target sequence of the enzyme. At this moment, Pseudovibro was not sequenced, so we could not find the localisation of the EcoKI gene. In order to integrate our constructions just in time, we opted for a transposon strategy.

Figure 1: Sequence of the pRhokHi vector and the hightlight of the EcoKI target sequence


Concerninrg the nitrate and nitrite a potential gene transporter NarK Nitrate/and Nitrite was predicte. Also we discovered the protein NapE was predict in the genome of Pseudovibrio Denitrificans. This enzyme is known to reduce nitrate to nitrite. Other some other genes such as Respiratory nitrate reductase 1 alpha and game chaine and other which are involve in the nitrification process. It possessed also Required for formate-dependent nitrite reduction. Not required for the biosynthesis of any of the c-type cytochromes nor for the secretion of the periplasmic cytochromes. They are involve in the reduction of nitrite to amoniac





Figure2 : Anaerobic pathway degradation for phenol