Team:Washington/Magnetosomes/GibsonVectors

From 2011.igem.org

Gibson Vector Toolkit

About Gibson Cloning/Assembly

Gibson Cloning/Assembly is a cloning method that allows multiple inserts in one time isothermal reaction for assembling overlapping DNA fragments. During the cloning process, this does not only saves a lot of time and efforts, but also allows more complexity when building the circuit.

What happened last year?

The Gibson Cloning method is definitely not a new terminology to the iGEM community. This cloning method was published in Nature protocols 2009 ( Please see Enzymatic assembly of DNA molecules up to several hundred kilobases, Gibson et al. (2009).) and it has gained a lot of attention from the iGEM community since then.

In 2010, the Cambridge iGEM team proposed the proposed a BioBrick standard for Gibson Assembly (RFC 57) which enables simultaneous assembly of multiple fragments with no scar sequences. We found that this Biobrick Standard was incapable of giving high yields even in two-fragments assembly. The primary problem is the self-complementarity of the two NotI sequences embedded in the BioBrick prefix and suffix which prevents the insert from being incorporated into the vector.

• E=ECoRI, N=NotI, X=XohI, S=SpeI, P=PstI

Therefore, to combat the problem, a “gibson reaction compatible” prefix and suffix were designed based on BglBrick standard to increase the cloning efficiency.

• E=ECoRI,S1=Spacer 1, Bg=Bgl S2=Spacer2, P=PstI

What about this year?

Seeing that this is such a great method to do cloning...we CONTINUE WITH THE INVESTIGATION! AND WE MADE/SUBMITTED MORE VECTORS that are compatible with Gibson cloning in a hope to ease everyone’s pain when doing cloning. (bring the iGEM to a differnt level of cloning)

New vectors... a diagram a table showing the antibiotics, copy number

Next level of iGEM project: more complex circuit.

And we decided to look at magnetosome