Team:Edinburgh/Project

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Genomic BRIDGEs

Plasmid transformation protocols are the backbone of modern day synthetic biology, allowing for bacteria such as E. coli to take up foreign genetic material and express it as part of their cellular mechanisms. However, since only a relatively low number of cells are actually transformed in the process, selection markers are necessary to identify the cells that have acquired the plasmid; this usually takes the form of an antibiotic resistance gene built into the plasmid, which has the undesired effect of giving the transformed cells resistance to commonly-used antibiotics.

What if synthetic biologists were able to utilise an efficient two-step recombination method for markerless gene insertion and deletion? In 2008, Wei Sun, Shifeng Wang, and Roy Curtiss III of Arizona State University published such a protocol, based on the lambda red recombinase system (a simple method for disrupting chromosomal genes in bacteria such as E. coli using PCR products). The 2010 University of Edinburgh iGEM team has adapted their method to take advantage of the reusability of BioBricks, such that synthetic biologists can target critical areas of the E. coli genome with even greater efficiency.

BRIDGE stands for BioBrick Recombination In Direct Genomic Editing. It is an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product.

Our Project

Our BRIDGE construct contains two selection markers in order to successfully complete the protocol (described in greater detail here): cat, which confers chloramphenicol resistance, and sacB, which is toxic when the host is grown on sucrose. Both the construct and the desired gene are inserted by homologous recombination using the lambda red recombinase system. For this we require up- and down-stream sequences of the genes that we wish to replace.

To prove the principle of BRIDGE we removed a non-essential, constitutively expressed gene from the E. coli genome and replaced it with a well-known marker, GFP. This could have been extended further: we also had several genes from a past project idea which we could delete to increase fatty acid synthesis, and other genes we could introduce which would result in the production of long chain alkenes from the excess fatty acids. This is not useful for our current project but it would be a nice way to demonstrate the effectiveness of BRIDGE.

Eventually, we hoped that BRIDGE could be used to introduce whole light producer-sensor constructs, to demonstrate its ability for utilisation in further work using BioBricks.

Table of Contents

• The protocol proper, explaining the technical details of BRIDGE.
• The BioBricks we submitted as part of developing the BRIDGE protocol.
• A summary of what we achieved developing the BRIDGE protocol.
• Our vision of the future of the BRIDGE protocol, and where we would like to go next.
• References used throughout the section.