Team:Exeter/lab book/novpol/wk3

Operon Construction  |  Glycobase

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Monday 23rd July - 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 water (40mL) and swirled gently. A stir bar was added and mixed thoroughly for 1 minute. Sodium hydroxide (1M) was added drop-wise until all Tris dissolved and the pH reached approximately 9. The total solution was topped up to 50mL using MilliQ water to make a 10mM Tris solution, and autoclaved for 20 minutes. Added EDTA (1.21g) to MilliQ water (60mL) and swirled gently and this was added to make THE IDTE buffer. 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 water to make a 0.1mM EDTA solution, and then autoclaved for 20 minutes.

Tuesday 24th July - 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), pH between 7.5-8.0, into two Eppendorf tubes, to reach an approximate concentration of 50ng/µL stock solution. Both tubes were centrifuged for 20 seconds and left to incubate at room temperature for 30 minutes. Both tubes 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 water (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. A master-mix was made up for each promoter, the RBS part and linear plasmid backbone (pSB1C3) in separate Eppendorf tubes. Because of the small concentration of pSB1C3, up to 250ng of DNA was made (hence all the master-mix volumes were halved). Therefore:

Each Eppendorf tube was incubated at 37°C for 10 minutes and then at 80°C for 20 minutes immediately. Afterwards, 2µL of each restriction digested DNA in each of the four tubes were transferred to two Eppendorf tubes. Each tube contained 2µL of RBS, 2µL pSB1C3, 2µL 10x T4 DNA ligase buffer, 1µL T4 DNA ligase and 11µL MilliQ water. However, 2µL of pBAD weak was pipetted into one tube and 2µL of pBAD strong was pipetted into the second tube. Both tubes were left to incubate for 10 minutes at room temperature, and then placed in an incubator at 80°C for 20 minutes. 2µL of WbnJ and WfcA, and 1µL from each Eppendorf tube containing pBAD weak and pBAD strong (ligated to the RBS part) were pipetted gently into separate 25µL E.coli competent cells (Invitrogen) tubes, making sure not to mix too rigorously. Competent cells were then incubated on ice for 30 minutes. They were 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 to each of the four Eppendorf tubes and then each tube was secured in a shaking incubator and incubated at 37°C for 1 hour at 220rpm. Whilst incubating, four LB agar plates were made-up. Half would be used for spreading 20µL of transformed E.coli competent cells and the other half for 100µL of transformed E.coli competent cells, so that we would be able to pick out enough isolated colonies. 300µL ampicillin was added to 300mL of LB agar to create a 1000-fold dilution. Furthermore, to select for the pSB1C3 plasmid containing ligated promoter-RBS, two LB agar plates (for spreading 20 and 100µL transformed cells) with an 1000-fold dilution of chloramphenicol was made-up by adding 150µL chloramphenicol to 150mL of LB agar. After the transformed E.coli competent cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto their respective LB agar plates. Remaining transformation mix was stored at 4°C and the six inoculated LB agar plates were inverted and placed in an incubator at 37°C overnight. Remaining hydrated DNA stock solution from the IDT re-suspension was stored at -20°C.

Wednesday 25th July - Transferred cultures from plates to liquid medium. Also prepared lots of ampicillin, kanamycin, tetracycline and chloramphenicol plates and stored in the fridge.

When all the spread plates were taken out of the 37°C incubator, colonies only appeared on the ampicillin spread plates containing 20µL and 100µL WbnJ and WfcA. Therefore only colonies carrying recombinant WbnJ/WfcA plasmids were added to LB broth. Ampicillin, the selection antibiotic, was defrosted. Three isolated colonies from the 100µL spread plate that contained WbnJ plasmids and WfcA plasmids were picked and inoculated in two separate bottles containing 5mL LB broth (see: Preparation of LB Agar and Broth) via dropping the pipette tip. 5µL ampicillin was added to each bottle to make a 1000-fold dilution (since 5mL LB broth used). Each bottle was inverted a couple of times, and then put in a 37°C incubator and left overnight.

Thursday 26th July - Mini-prep and added cultures to liquid medium.

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). Because 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 (see: Preparation of LB Agar and Broth) 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 27th July - electrophoresis of genes WbnJ and WfcA to prove it had worked.

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 Eppendorfs 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.