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Team:Edinburgh/Project/Protocol
From 2010.igem.org
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.
Introduction
The bulk of this protocol has been developed from the Gene Bridges "Quick and Easy BAC Modification Kit by Red/ET Recombination" protocol, version 2.4 from 2005. We have had to edit in places as we are not using vectors and have two strains of E.coli.
I will give you a brief overview of how to make your constructs by traditional biobricking methods and will then go into detail about how to transform and select for you recombinants.
The two E.coli strains we used were JM109 and DH5alpha. When transformed with the Red/ET these are tetracycline resistant, but you be aware that the two strains require different concentrations of tetracycline in liquid cultures as JM109 has greater antibiotic resistance than DH5alpha. These concentrations are given in the protocol but remember that if you alter the volume of you liquid cultures you should also alter the volume of tet15 added.
You should also bear in mind that the Red/ET plasmid will not replicate at 37C so will be lost from cells grown at 37C for more than an hour. For this reason we grew our transformants at both 30C and 37C to retain the plasmid and to gain decent growth for characterisation and determination of results.
For this experiment you will need negative controls for your transformations to confirm that growth is definitely due to the presence of the chloramphenicol resistance gene. We grew 1 liquid culture for each strain, prepared 2 transformations for each (one with and one without DNA), grew each transformant at both 30C and 37C and then plated each of these onto cml40 at both a high and a low concentration of cells. By the end of the first step you should have 16 plates: JM109/DH5alpha +/-DNA at 30/37C and concentrated/not concentrated.
Sections:
- Creating homology between target and constructs
- Preparing cells with Red/ET
- Preparing cells for electroporation
- Electroporation and recovery steps
- Selecting for recombinants
- Stating again for the second step
Materials
- A good stock of sterile LB
- A large supply of agar plates with cml40
- Agar plates with tet15
- Tetracycline (15mg/ml)
- Chloramphenicol (40mg/ml)
- L-arabinose
- Access to electroporation technology
- Sterile electroporation cuvettes
- Sterile water
- Eppendorfs
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.
