"
Team:Edinburgh/Project/Protocol
From 2010.igem.org
| Line 135: | Line 135: | ||
<br> | <br> | ||
<center><p><img src="https://static.igem.org/mediawiki/2010/5/5c/Ed10-OriginalBridge.JPG" width="800" height="441" border="0" /></p><br> | <center><p><img src="https://static.igem.org/mediawiki/2010/5/5c/Ed10-OriginalBridge.JPG" width="800" height="441" border="0" /></p><br> | ||
| + | <p><b>Figure 1:</b> The strategy for markerless deletion of a chromosomal gene by two-step recombination. (A) A DNA fragment carrying the <i>cat-sacB</i> genes, flanked by two regions homologous to the DNA sequences bordering the target site, is integrated into the chromosome. (B) A DNA fragment carrying the desired deletion or insertion, again flanked by two long regions homologous to the DNA sequences bordering the target sites, replaces the <i>cat-sacB</i> genes through homologous recombination.</p> | ||
<p>Image: Appl Environ Microbiol. 2008 July; 74(13): 4241–4245 (Fig. 1)</p><br></center> | <p>Image: Appl Environ Microbiol. 2008 July; 74(13): 4241–4245 (Fig. 1)</p><br></center> | ||
<br> | <br> | ||
Revision as of 14:52, 14 October 2010
BRIDGE: The concept
BRIDGE stands for BioBrick Recombineering 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.
Figure 1: The strategy for markerless deletion of a chromosomal gene by two-step recombination. (A) A DNA fragment carrying the cat-sacB genes, flanked by two regions homologous to the DNA sequences bordering the target site, is integrated into the chromosome. (B) A DNA fragment carrying the desired deletion or insertion, again flanked by two long regions homologous to the DNA sequences bordering the target sites, replaces the cat-sacB genes through homologous recombination.
Image: Appl Environ Microbiol. 2008 July; 74(13): 4241–4245 (Fig. 1)
The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene, the other sacB, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic.
The second step involves swapping the construct for another piece of DNA (e.g. a BioBrick construct). After this we can select for those with the new gene by growing the cells on sucrose.
BRIDGE: The advantages
BRIDGE has a significant advantage over the current method of BioBrick insertion. For one, it is vector independent - whole PCR constructs can be inserted directly into the genome in two steps in under a week, compared to the lengthy process of vector digestion and ligation required with normal BioBricks.
The other major advantage is that it will not leave a lasting marker in the genome. With most BioBricks we have to leave a marker (antibiotic resistance, GFP, etc) in our constructs so that we can guarantee their presence. This becomes an issue, a) when you want to use the organism in an industrial or environmental capacity, and b) when you want to insert multiple constructs (there is only a limited number of markers out there). With this system, the markers are removed every time you insert a new gene, so they can be used again and again indefinitely. You could essentially replace the entire genome with new genes.
BRIDGE: The protocol
The idea here is that I will write up the method that gave us the best results in the shortest time. The protocol that is up here by the wiki freeze may not be the optimum, but will be based on our success so far. To see the progression of this protocol, visit the "BRIDGE" page in the lab notes drop down menu.
References
Sun, W., Wang, S. & Curtiss, R. (2008). Highly Efficient Method for Introducing Successive Multiple Scarless Gene Deletions and Markerless Gene Insertions into the Yersinia pestis Chromosome Appl Environ Microbiol. 2008 July; 74(13): 4241–4245.
