Instruction Manual of this method

We’ll explain how you should use this method using an example of BBa_J04450

At the beginning…

You should choose two non-essential genes from the list. It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).

Attention

You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)

1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers. By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.

Primer 1 : Cm primer (forward)

Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)

Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2

Primer 4 : VR + homology site of Cm resistant gene

2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of Bba_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.

Primer of yahA (from gene list)

Primer of yahC (from gene list)

3. Next, you should make the following insertion fragment, “yahA homology site+Bba_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.

4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.


You should try this method!