Team:Exeter/lab book/novpol/wk1

Operon Construction  |  Glycobase

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Tuesday 10th July - Today we had our laboratory induction, we were shown where all the equipment was and given a demonstration.

LB agar and Broth were then prepared.

Tryptone (10mg) was added to a 1L beaker containing a stir bar. MilliQ water (≈100mL) was then added to the 1L beaker and stirred using a magnetic stirrer. Sodium Chloride (10mg) was added to the stirring solution. Yeast extract (5g) was then added and finally agar (15g) was added to the stirring solution. The remaining MilliQ water (≈900mL) was transferred to the stirring solution and left to mix for five minutes. The LB agar mixture was then transferred to a 1L Duran bottle and then autoclaved.

Wednesday 11th July - Today we performed the Biobrick extraction and Transformation.

DNA was re-suspended in 10µL of MilliQ water into four selected wells of the Biobrick plates. These were: pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000) and a double terminator (BBa-_B0014). These were left for five minutes and then transferred to labelled Eppendorf tubes. The tubes were centrifuged briefly to spin down any liquid. Each BioBrick DNA part (1µL) was pipetted gently into 100µL of Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously. Three Eppendorf tubes were set up: Tube 1 for 50µL E.coli + 1µL double terminator, Tube 2 for 25 µL E.coli + 1µL pBAD/AraC weak, Tube 3 for 25µL E.coli + 1µL pBAD/AraC strong. Competent cells were then placed on ice for 30 minutes and then heat-shocked for exactly 30 seconds in a 42°C water bath, making sure not to mix or shake. Afterwards, they were quickly placed on ice for 2 minutes. Pre-warmed SOC medium (250µL for Tube 1 and 125µL for Tubes 2 and 3) was added and then each Eppendorf tube was secured in a shaking incubator and incubated at 37°C for 1 hour at 220rpm.

LB agar plates were made, whilst incubating, containing ampicillin or kanamycin for the double terminator. Therefore 400µL ampicillin was added to 400mL of LB agar, and 50µL kanamycin was added to 50mL of LB agar to create a 1000-fold dilution of the antibiotic. After the transformed E.coli cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto their respective LB agar plates containing the correct antibiotic. The remaining transformation mix was stored at 4°C and the eight inoculated LB agar plates were inverted and placed in an incubator at 37°C overnight.

Thursday 12th July - The cultures we plated out were transferred to liquid medium.

The selection antibiotic, ampicillin, was defrosted. Ampicillin (5µL) was added to each bottle to make a 1000-fold dilution (since 5mL LB broth used). Three isolated colonies from the 100µL spread plate that contained our cloned BioBrick parts were picked and inoculated in three separate bottles containing 5mL LB broth. Each bottle was inverted and then placed in a 37°C incubator and left overnight.

Friday 13th July - Genes were mini-prepped and then separated on gel electrophoresis.

Overnight cultures were transferred into new Falcon tubes and centrifuged at 3900rcf for 2 minutes at 21°C. The supernatant was discarded (being careful not to disturb the pellet). The pellets were re-suspended in 250µL of Re-suspension Buffer by pipetting the solution up and down. Added 250µL of lysis solution and then immediately followed with 350µL of neutralisation buffer. Each Falcon tube was then centrifuged for 5 minutes at 16100rcf at 21°C. Withdrew 850µL of the supernatant and transferred to a geneJET Miniprep column. The column was then centrifuged for 1 minute at 16100rcf at 21°C. The flow-through was discarded (as this contained the sugars, metabolites etc.) and then 500µL of Wash solution was added to the geneJET Miniprep column. This was centrifuged again for 1 minute. Any flow-through was discarded and washed again with extra Wash solution (500µL). This was centrifuged again for 1 minute. The flow-through was emptied and centrifuged again for 1 minute at 21°C with an empty column. The supernatant obtained was transferred to clean, labelled Eppendorfs. 50µL of MilliQ water was added to each Eppendorf and left for a couple of minutes. The concentration of plasmid DNA obtained in each Eppendorf was measured at the Nanodropping machine (in ng/µL, see: Results).

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 the agarose had dissolved. Ethidium bromide (EtBr, 1µL) was added to the agarose gel in the fume cupboard, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted.

The BioBrick parts were removed from the circular plasmids by transferring each cloned plasmid containing different BioBrick parts to different Eppendorf’s containing the Master Mix. This consisted of: 500ng/µL DNA of interest, 1µL EcoR1-HF, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and enough MilliQ water to make a 50µL total volume. The resulting Master Mix was multiplied by four to compensate for determining the sizes of the four BioBrick parts selected. The DNA-Master Mix solution was left to incubate for 10 minutes at 37°C for the plasmids to be digested.

Loading buffer (4µL) was added to each DNA sample. DNA of each sample (25µL) was added to different wells, including the DNA hyperladder (10µL). Gel electrophoresis was then run for approximately 40 minutes at 150V.