Using the Gibson assembly technique to ‘hot’ swap a gene


This is an example of how to exchange one gene for another whilst retaining the desired promoter, ribosomal binding site (rbs) and terminator DNA sequences of the gene to be replaced based on Biobrick parts from the Registry of Standard Biological Parts. In this example we will directly replace the gene for Red Fluorescent Protein (RFP), mCherry with the gene for a super folder Green Fluorescent Protein (GFP), whilst keeping the plasmid backbone and regulatory sequences of the gene to be replaced. The process of designing the necessary oligo primers for later Gibson DNA assembly will be illustrated.

Mining the Registry of Standard Biological Parts for the DNA Part


Go to the Registry of Standard Biological Parts web address http:partsregistry.org/
and use the search box to recall the relevant Biobrick part.

In this example we are searching for the RFP Biobrick with the part number BBa_J69512.

Stacks Image 1969

Having pulled the part page describing the RFP Biobrick we need to download the DNA sequence file in the Genbank format (.gb) using the Tools tab.

Stacks Image 1970

The page downloaded looks like this.

Stacks Image 1971
Stacks Image 1972
Stacks Image 1973
Clicking on the ‘download as .gb file’ gives you the option to open the file in a DNA editor program, such as the free ‘A Plasmid Editor’ (Ape) or save the file. Ape is a simple, excellent tool for handling DNA sequences and building graphical maps. It is cross-platform, and is based on the use of Genbank annotations for archiving and exchange of DNA features. The softyware can be download from (www.biology.utah.edu/jorgensen/wayned/ape).
Stacks Image 1974
The tool for the conversion of a Biobrick to a Genbank format file was contributed by University of Cambridge iGEM2010 team and can be accessed at (http://2010.igem.org/Team:Cambridge/Tools/GenBank) using the Biobrick part number.
Stacks Image 1975
Working with the Ape DNA Editor to Create a Plasmid (pSB3K3) with RFP (mCherry)


The advantage of the conversion from the Biobrick to Genbank file format is that the annotation of the DNA is retained in Ape. All recognized features (promoters, ribosomal binding sites and sequences of interest are highlighted in colours, which can be edited). These features will help in the design of primers for Gibson assembly.

Step 2. Using the same procedure outlined in step 1 create an Ape file for the plasmid pSB3K3 which has the part number (http://partsregistry.org/Part:pSB3K3). Cut & paste the DNA sequence from the RFP (mCherry) BBa_J69512 into the backbone plasmid vector pSB3K3 and use this new sequence as a template to design primers for PCR amplification of the vector sequence including promoter, RBS and terminators flanking BBa_J06504 RFP (mCherry), this part is the gene open reading frame (ORF).

Stacks Image 1976

Step 3. Using the same procedure outlined in steps 1 and 2 create an Ape file for the super folder GFP Biobrick BBa_I746916 (http://partsregistry.org/Part:BBa_I746916) then using the Ape file pSB3K3 RFP (mCherry) BBa_J69512 replace the mCherry ORF with the super folder GFP. This will be the in silico template file for designing the oligo primers for Gibson assembly

Stacks Image 1977
To summarize we want to replace the mCherry Biobrick (BBa_J06504) (which is a sub-part of the larger Biobrick BBa_J69512 cloned in to pSB3K3) with the super folder GFP Biobrick (BBa_I746919) (which is sourced from the larger Biobrick BBa_I746914 cloned in to pSB1A2).
Stacks Image 1978