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Revision as of 03:30, 27 September 2012

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Cloning and Transformation

Because we didn’t receive the pSB1C3 plasmid (tube received in the kit containing plasmid was dry and we couldn’t get any mass), we took the BioBrick BBa_J04450 (Plate 1, position A3) from the kit and transformed CaCl2-competent cells (E. coli strain DH5α) following a heat-shock protocol, and cells were plated on LB-agar containing Chloranphenicol.

Over-night red colonies (the BioBrick contains a device that expresses RFP, that’s the reason we used this part!) were used for liquid cultures (LB + Chloranphenicol). Minipreps of pSB1C3 were done using the AxyPrep Plasmid Miniprep Kit (Axygen).

The obtained plasmid was cut using EcoRI and PstI restriction enzymes (both from Invitrogen) at 37 °C for 1 hour. We performed a preparative 1%-agarose gel with the product of the reaction, and the pSB1C3 backbone was purified from its respective band (in relation with 1 Kb-DNA ladder marker) through a commercial kit. After a lot of time spent in cloning and troubleshooting, we accomplished the ligation of devices into pSB1C3 backbone following the next protocol:

1. Dry gBlocks (200 ng) were resuspended in 20 μl of bi-distilled water.
2. 5 μl were used to digest with restriction enzymes EcoRI and PstI (both from Invitrogen). Enzymes were inactivated at 65 °C.
3. 25 ng of digested gBlocks (devices 1 to 4) and 30 ng of backbone were ligated using T4 ligase (Invitrogen), at room temperature for 12 hours.
4. 5 μl of ligation product were used to transform TOP10 CaCl2-competent cells. We followed a conventional heat-shock protocol, with the next exception: cells were recovered, after heat-shock, with fresh LB medium for 2 hours.
5. Transformated cells were plated on LB-agar containing Chloranphenicol, and incubated over night.
6. White colonies (containing pSB1C3 + insert) were used for liquid cultures (LB + Chloranphenicol). Plasmid minipreps were done using an Alkaline Lysis Method.

Plasmids containing, or not, the desired inserts were evaluated by restriction (EcoRI + PstI) and 1%-agarose gel using a 100 bp ladder marker. We selected those plasmid wich had the expected size insert.

Even though we didn’t have enough time to do the sub-cloning of the devices into yeast-expression plasmid and characterize their functioning, currently we are proceeding to obtain these plasmids and conduct co-culture experiments in transformed yeast strains and we will have the results soon.