Team:Cambridge/Gibson/Mechanism

Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.

Creating overlapping DNA sequences

Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.

By using two ~40nt oligonucleotides as primers, we can add 20 bp of sequence A to sequence B and 20 bp of sequence B to sequence A. We are then ready to use Gibson Assembly. The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.

Note:

• The end of sequence A with the extension now looks like

.....endA+begB

• The beginning of sequence B looks like

endA+begB.......

• This results in there now being a total overlap of 40bp between the two fragments, enough to perform Gibson assembly.

Gibson Assembly

Gibson Assembly master mix contains 3 enzymes:

• T5 exonuclease
• Phusion polymerase
• Taq ligase

The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA

Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.

We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.

Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.

Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.

And finally we have our finished piece of DNA.