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Citrobacter freundii Characterisation:
Genome sequencing
One major advantage of E. coli over C. freundii is the fact that there is a lot of sequence information available on the former, while no whole genome sequences exist for the latter. In order to get the ball rolling, the genomes of two C. freundii strains (the type strain, ATCC 8090 and another strain our lab had, called SBS 197) were sequenced in Newcastle by Drs Wendy Smith and Anil Wipat with IonTorrent Sequencing.
To the best of our knowledge, no other iGEM projects have yielded genome sequencing data before, so this is a first both for our team, Europe and iGEM in general.
While a complete assembly of the sequence reads could not be done, these sequences constitute the first step towards unraveling the genome of our proposed chassis organism and even in this form they can provide valuable information.
Since our project was concerned with making synthetic biology safer, we wanted to see whether Citrobacter freundii contains any antibiotic resistance genes in its genome. With the help of the sequencing data (Figure 1), we have found that both strains have got several beta-lactamases encoded within their genomes, which normally confer these bacteria resistance to antibiotics with beta-lactam rings, such as ampicillin.
Figure 1 – A snapshot of the sequencing reads that covered one of the beta-lactamase regions in Citrobacter freundii, showing good sequence coverage.
This is a puzzling find, as neither strain shows any resistance to carbenicillin, a synthetic ampicillin analogue. Nonetheless, a lot of gram negative bacteria carry beta-lactamases within their genomes, so even if Citrobacter freundii were released into the environment, it should not lead to an increase in the spread of antibiotic resistance.
The raw sequencing files (zip files) and the contigs that have been assembled de novo (.fa format) can be accessed at the public Dropbox folder
Sequencing Files along with pdf files of the assembly reports. In addition, the automated annotation spread sheets (done by RAST) for both strains can also be accessed from this location.
