Cloning

We performed all the cloning in E.coli, and did experiments on Asaia transformation, growth and resistance to antibiotics in parallel.

As E. coli origin does not work in Asaia, we added an origin that was compatible with this bacteria (which was recovered from a GFP-plasmid transformed into the Asaia we received from Italy). On the opposite, Asaia's origin does work in E. coli, which is very useful for our sets of experiments.

BioBricks

All our parts are in the pSB1C3 backbone vector (abbreviate C3). When you click on the part's name, you are redirected to the partsregistry corresponding page.

First the legend:

• Ori = Origin of replication for Asaia and E. coli
• S = Strong Promoter
• W = Weak Promoter
• Immu = immunotoxin
• P25 = Pfs25 protein
• P28 = Pfs28 protein
• Kan = Resistance to Kanamycin
• Tet = Resistance to Tetracycline

We made basic parts to build the complexest one.

[http://partsregistry.org/Part:BBa_K320000 *C3 + Ori] We made this plasmid because we needed an origin of replication that works in Asaia. After experiences we concluded that it also work with E. coli.

[http://partsregistry.org/Part:BBa_K320001 *C3 + Immu] The immunotoxin is the main part of the project, we obtained it by synthetization. For a better understanding, go there.

[http://partsregistry.org/Part:BBa_K320002 *C3 + S] We made a new strong promoter permitting use to test the expression of the immunotoxine. We called it strong because we used the constitutive sequence which should give the biggets level of expression.

[http://partsregistry.org/Part:BBa_K320010 *C3 + W] This promoter has been made to have a second level of expression for our proteins, which is lower than the strong one.

We made the following BioBricks from the aboves :

[http://partsregistry.org/Part:BBa_K320003 *C3 + Ori + Tet]

[http://partsregistry.org/Part:BBa_K320004 *C3 + Ori + Kan]

[http://partsregistry.org/Part:BBa_K320005 *C3 + S + CFP]

[http://partsregistry.org/Part:BBa_K320006 *C3 + S + Immu]

[http://partsregistry.org/Part:BBa_K320007 *C3 + S + P25]

[http://partsregistry.org/Part:BBa_K320008 *C3 + S + Immu + ori + Kan]

[http://partsregistry.org/Part:BBa_K320009 *C3 + S + P28 + Ori + Tet]

[http://partsregistry.org/Part:BBa_K320011 *C3 + Ori + Amp]

[http://partsregistry.org/Part:BBa_K320012 *C3 + W + Immu]

[http://partsregistry.org/Part:BBa_K320013 *C3 + W + P25]