Difference between revisions of "Part:BBa K1019008"

Revision as of 22:44, 27 September 2013

pEBS Expression Plasmid

We made use of [http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid Clontech’s In-Fusion HD Plus cloning kit] to develop a highly modular, inducible vector suitable for the expression of any gene and compatible with biobrick assembly standards. If choosing to exploit the ease-of-use of the In-Fusion protocol this plasmid provides a pre existing RBS, start, and stop codons for straightforward expression of your gene of interest. These are flanked by the biobrick prefix and suffix to enable standard ligation assembly if you wish to employ a unique RBS. Also included on this high-copy number plasmid are an ampicillin resistance marker and inducible expression of the inserted gene via the lac promoter.

The In-Fusion kit makes use of homologous recombination to attach fragments of DNA with homology regions at their ends in an easy one-step reaction, thus alleviating the tedium associated with traditional ligation-based cloning. A unique BseRI site in our plasmid allows linearization between the integrated start and stop codons, thus allowing any coding region amplified with the appropriate homology regions to be seamlessly cloned into the plasmid and expressed with ease.

This new plasmid, pEBS (“E. coli biofilm system”) originated from the commercially-available pMAL vector.

In-Fusion Protocol

This protocol makes use of a pre existing RBS, start, and stop codons and only requires insertion of whatever gene you wish to express. The vector is prepared for in-fusion by digesting with BseRI, this will produce a linearized plasmid of 5494bp. The gene to be expressed is amplified using primers complimentary by 15 bp's of homology to the ends of the linearized vector:

                        <---homology--> 
5'-aggaggtaaaaaatg-GENE-TAATAAtactagtag-3'
   <--homology--->

This will allow the In-Fusion reaction seamlessly clone in your gene based on the following protocol:

1. Linearize pEBS with BseRI
2. Add 2 µL total DNA (3:1 volumes insert:linearized vector) to 0.5 µL 5X In-Fusion HD Enzyme premix in a 1.5mL microcentrifuge tube; place at 55C for 30 minutes
3. Place SOC outgrowth media at room temperature and put Stellar competent cells to thaw on ice during this 30 minutes
4. After the 30 minute incubation period add 50 µL Stellar competent cells to the reaction tube and incubate on ice for 20 minutes
5. 40 seconds heat shock at 42C; transfer immediately to ice for 2 minutes
6. Add 200 µL SOC media to the tube. If using pEBS it has ampicillin resistance and thus can be plated on an LB agar with ampicillin immediately. Plate all 262.5 µL and incubate overnight at 37C

NB: Each tube of Stellar competent cells contains 120 µL of liquid so it is best to do two in-fusion reactions simultaneously so as not to waste expensive cells. You can use 60 µL in each reaction or do 50 µL and plate the remainder on and LB agar+AMP plate as a negative control.

Ligation based cloning

As the homology regions described above are flanked by the biobrick prefix and suffix you also have the option of digesting the vector at these sites and cloning in appropriately-digested genes via ligation. This method, however, does not conserve the RBS included in the original vector so you will be required to include your own.

Sequence and Features