Team:Washington/Magnetosomes/GibsonVectors

From 2011.igem.org

Gibson Vector Toolkit

About Gibson Cloning/Assembly

Gibson Cloning/Assembly is a novel synthetic biology tool that allows multiple gene-inserts during a single isothermal reaction that is used for assembling overlapping DNA fragments. This method is gaining popularity as it tends be more efficient, saving a great amount of time during the cloning process. Overall, Gibson cloning allows teams to built more complex circuits with ease.

What happened last year?

The Gibson Cloning method is definitely not a new method to be introduced to the iGEM community. In 2010, the Cambridge iGEM team created the RFC 57 document which outlines a protocol for Gibson Assembly using Standard BioBricks that would allow multiple gene inserts during a single cloning event. However, while creating our Magnetosome Toolkit, we found that this BioBrick standard was incapable of producing high yields of Gibson products for even two-gene assemblies.

The primary problem with this standard pSB vector is the self-complementary of the two NotI sequences embedded in the BioBrick prefix and suffix. These sequences prevent gene inserts from being incorporated effeciently 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 continued with the investigation and made the Gibson Assembly Toolkit!

We call our new vectors plamid Gisbon Assembly (pGA) vectors. And we were able to show that the cloning efficiency of pGA vector is better than the pSB vector.

(Stay tuned for our results)

We submitted 5 pGA vectors of different copy numbers and antibiotic resistances to the Registry. (All of them have pLac GFP)

pGA1A3: high copy plasmid backbone with Amp resistance. pGA1C3: high copy plasmid backbone, with Chloramphenicol resistance. pGA3K3, pGA 4C5 and pGA 4A5 are low copy plasmid backbone, which are good for more gene fragment inserts.

pGA4C5_pLacGFP: a low copy plasmid backbone which has Chloramphenicol resistance

To make the pGA vectors, we first amplified the backbones and the pLac GFP insert respectively. Then we performed Gibson reaction to combine them together to make the pGA vectors.

a table showing the antibiotics, copy number

References

1.