Team:Exeter/lab book/novpol/wk2

Operon Construction  |  Glycobase

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Monday - Transformation of E coli performed for the ribosome binding site and TetR promoter biobrick.

DNA was re-suspended in 10µL of MilliQ water by pipetting up and down on the well corresponding to the RBS BioBrick (BBa_B0034). The wells containing the biobrick were left for five minutes and then transferred to a labelled Eppendorf tube and spun down briefly in a centrifuge. The suspended RBS DNA (1µL) was pipetted gently into 25µL of E.coli competent cells (Invitrogen), making sure not to mix too rigorously. Competent cells were then incubated 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) was added and then the Eppendorf tube containing the transformed E.coli was secured in a shaking incubator and incubated at 36.8°C for 1 hour at 220rpm.

Whilst incubating, two LB agar plates were made; one would be used for spreading 20µL of transformed E.coli competent cells and the other for 100µL. Therefore 500µL ampicillin was added to 500mL of LB agar to create a 1000-fold dilution.

After the transformed E.coli competent cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto LB(amp) agar plates. Remaining transformation mix was stored at 4°C and the two inoculated LB agar plates were inverted and placed in an incubator at 37°C overnight.

Tuesday - Cultures containing the RBS added to liquid medium.

Ampicillin, the selection antibiotic, was defrosted. Three isolated colonies from the 100µL spread plate were picked and inoculated in three separate bottles containing 5mL LB broth. 5µL ampicillin was added to each bottle to make a 1000-fold dilution. Each bottle was inverted a couple of times, and then put in a 37°C incubator and left overnight.

Wednesday - RBS mini-prep attempt seemed to not pellet so began transformation procedure again.

Overnight cultures were transferred into new Falcon tubes and centrifuged at 3901rcf for 10 minutes at 4°C. It was noticeable that there was no cloudy solution after transferring to new Falcon tubes. Afterwards, when the supernatant was discarded, there was not enough pellet to continue with the mini-prep procedure, and therefore the process of incubating the transformed E.coli overnight in a 37°C incubator was repeated but picking four colonies from the 100µL spread plate instead of three.

Began the transformation procedure (see Monday 16th July) again in case incubation failed to work.

Thursday - Mini-prep and gel electrophoresis of the TetR promoter and RBS.

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.