Latest revision as of 03:55, 30 October 2008

Organism

Most of our work this summer is founded upon the diverse metabolism of the bacterium Shewanella oneidensis MR-1. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish S. oneidensis as a interesting chasis for synthetic biology.

So, who is this "Shewie"?

This summer we worked with Shewanella oneidensis MR-1, a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These characteristics of S. oneidensis MR-1 make it an important organism for toxin-reduction based bioremediation and biotechnology applications.

Molecular Biology with Shewanella oneidensis

The Shewanella oneidensis MR-1 genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in S. oneidensis MR-1, it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html pCDF-Duet vector] from Novagen work in S. oneidensis. However, they are not pir+, so the R6K pir+ dependent origin does not work for them. S. oneidensis MR-1 grows at 30 ºC, can be electroporated (see protocol in our Notebook) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini). Together, these characteristics make S. oneidensis MR-1 a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.

S. oneidensis MR-1 colonies from a transformation

Online resources for working with S. oneidensis

-[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]

-[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]

-[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| In silico PCR of S. oneidensis genome]

Chassis and the Registry

To facilitate easy manipulation in different organisms, it may be advantageous for the Registry to standardize a chassis specification sheet. Below, we provide a quick summary of S. oneidensis MR-1 following what we think may be a suitable documentation format. Since iGEM teams frequently work with species other than E. coli, if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms. A PDF version with links is available.

@@ Line 61: / Line 61: @@
 ==So, who is this "Shewie"?==
-This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997).  Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV).  This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007).  When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current.  These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002).  These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.
+This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997).  Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV).  This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007).  When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current.  These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002).  These characteristics of ''S. oneidensis MR-1'' make it an important organism for toxin-reduction based bioremediation and biotechnology applications.
 ==Molecular Biology with <i>Shewanella oneidensis</i>==
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 {|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" width="100%"
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-The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism.  It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002).  When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997).  We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html| pCDF-Duet vector] from Novagen work in ''S. oneidensis''.  However, they are not pir+, so the R6K pir+ dependent origin does not work for them. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates.  It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini).  Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.
+The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism.  It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002).  When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997).  We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html pCDF-Duet vector] from Novagen work in ''S. oneidensis''.  However, they are not pir+, so the R6K pir+ dependent origin does not work for them. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates.  It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini).  Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.
 |<div style="text-indent:0pt;color:black">[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]</div>
 |-

Team:Harvard/Shewie

From 2008.igem.org