Team:Exeter/lab book/novpol/wk3

Operon Construction  |  Glycobase

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**Monday 23/07/12**

Preparation of the IDTE Buffer

To re-suspend our two genes that arrived on the 19th July, WbnJ and WfcA were first centrifuged for 10 seconds to ensure the DNA was at the bottom of the tube. IDTE buffer was made by adding Tris (11.31g) to MilliQ H2O (40mL) and swirled gently. A stir bar was added and mixed thoroughly for 1 minute. 1M NaOH was added drop-wise until all Tris dissolved and the pH reached approximately 9. The total solution was topped up to 50mL using MilliQ H2O to make a 10mM Tris solution, and autoclaved for 20 minutes. The IDTE buffer was completed by adding EDTA (1.21g) to MilliQ H2O (60mL) and swirled gently. A stir bar was added and mixed thoroughly for 1 minute. 1M HCl was added drop-wise until the pH dropped to 7.5. The total solution was topped up to 100mL using MilliQ H2O to make a 0.1mM EDTA solution, and then autoclaved for 20 minutes.

**Tuesday 24/07/12**

IDT Re-suspension, 3A assembly for the Promoter-RBS and Transformation

The two synthesised DNA genes were re-suspended using IDTE buffer (40µL of 10mM Tris, 0.1mM EDTA), held between pH 7.5-8.0, into two fresh Eppendorf tubes, to reach an approximate concentration of 50ng/µL stock solution. Both tubes were vortexed for 20 seconds. Both tubes were left to incubate at room temperature for 30 minutes and were then centrifuged for 1 minute. To dilute WbnJ and WfcA genes for the transformation procedure, both respective stock solutions (2µL) were added to MilliQ H2O (999µL) to reach an approximate concentration of 0.1ng/µL. Tubes were centrifuged briefly to spin down any liquid. To BioBrick the two promoters (pBAD weak and pBAD strong) onto the RBS part, three antibiotic assembly (3A Assembly) was used. The promoters and RBS were added to a digested ampicillin plasmid.

Assembled plasmids were then transformed into Top10 E.coli competent cells (Invitrogen) onto ampicillin containing antibiotic plates. Remaining hydrated DNA stock solution from the IDT re-suspension was stored at -20°C.

**Wednesday 25/07/12**

Transferred cultures from plates to liquid medium containing ampicillin. Ampicillin, kanamycin, tetracycline and chloramphenicol plates were prepared and stored in the fridge.

**Thursday 26/07/12**

Performed a Mini prep of the promoter-RBS BioBricks and added cultures to liquid medium.

To verify BioBrick parts were cloned successfully, gel electrophoresis was used. Agarose was made (1x 50mL TAE buffer, 0.5g agarose and then microwaved until melted). Ethidium bromide (EtBr, 1µL) was added to the agarose gel, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted. As the agarose gel was setting, plasmid WbnJ and WfcA DNA was digested: 1µL of EcoR1-HF, 1µL of PstI, 5µL of 10x NEBuffer2, 0.5µL 100x BSA, 500ng plasmid DNA and enough MilliQ H2O to make a total volume of 50µL. Both Eppendorfs containing 50µL total solution was put in an incubator at 37°C and left overnight.

Glycosyltransferase WbbC could not be synthesised without a point-mutation present, WbbC was amplified from the laboratory strain BL21(DE3). To do this, ampicillin was defrosted (as this would select BL21(DE3) only). BL21(DE3) was moved from the -80°C freezer and inoculated in a single Falcon tube containing 5mL LB broth with 5µL ampicillin to make an 1000-fold dilution via dropping the pipette tip. The inoculated LB broth containing the pipette tip was put in a shaking incubator at 37°C at 250rpm and left overnight.

**Friday 27/07/12**

Electrophoresis of WbnJ and WfcA to verify they had been cloned successfully.