Team:Exeter/lab book/1gp/wk5

Operon Construction  |  Glycobase

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•To BioBrick WbnJ and WbbC(dodgy) onto the terminator, three antibiotic assembly (3A assembly) was used.

• A master-mix was made up for WbnJ, WbbC(dodgy), the terminator part (x2 as two genes) and linear plasmid backbone (pSB1A3) in four, separate 1.5mL Eppendorf tubes. Therefore:

• Tube 1 (pSB1A3 master-mix) contained 0.5µL EcoRI-HF, 0.5µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and 18.0µL MilliQ H2O;

• Tube 2 (WbnJ) contained 1µL EcoR1-HF, 1µL SpeI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 1.49µL WbnJ plasmid DNA (starting concentration = 335.6ng/µL) and 41.00µL MilliQ H2O;

• Tube 3 (WbbC(dodgy)) contained 1µL EcoRI-HF, 1µL SpeI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 2.75µL WbbC(dodgy) plasmid DNA (starting concentration = 181.8ng/µL) and 39.75µL MilliQ H2O;

• Tube 4 and 5 (Terminator) contained 1µL XbaI, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 10.37µL terminator DNA (starting concentration = 48.2ng/µL) and 32.13µL MilliQ H2O.

• 4µL of tube 1 contents was transferred to a new 1.5mL Eppendorf tube containing 4µL pSB1A3 linearized plasmid backbone with a total volume of 8µL and this was restriction digested (NOT tube 1).

• Each Eppendorf tube was incubated at 37°C for 30 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, fresh Eppendorf tubes. Each tube contained 2µL of terminator, 2µL pSB1A3, 1µL 10x T4 DNA ligase buffer, 0.5µL T4 DNA ligase and 2.5µL MilliQ H2O. However, one tube contained 2µL of restriction digested WbnJ and the other contained 2µL of restriction digested WbbC(dodgy).

• Both tubes were left to incubate for 30 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 Top10 E.coli competent cells (Invitrogen) tubes, making sure not to mix too rigorously.

• In the meantime, the synthesised genes WbnK and WclY 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, left to incubate at room temperature for 30 minutes, and then centrifuged for 1 minute.

• To dilute WbnK and WclY for the transformation procedure, 2µL of re-suspended WbnK and WclY were added to MilliQ H2O (999µL) to reach an approximate concentration of 0.1ng/µL for both synthesised genes. Each tube was centrifuged briefly to spin down any liquid.

• After this, 2µL of diluted WbnK and WclY were pipetted gently into 25µL Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously. Furthermore, 2µL of ligated WbnJ-terminator-pSB1A3 and ligated WbbC(dodgy)-terminator-pSB1A3 were pipetted gently into 25µL Top10 E.coli competent cells, 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 225rpm.

• Whilst incubating, eight LB agar plates were made-up. All the spread plates for spreading 20µL of transformed E.coli competent cells and 100µL of transformed E.coli competent cells contained ampicillin, the selection antibiotic. 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 their respective labelled LB agar plates.

• 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.

• Remaining hydrated DNA stock solution from the IDT re-suspension was stored at -20°C.

Tuesday 7th July (15.00) – Adding Cultures to Liquid Medium

• When all the spread plates were taken out of the 37°C incubator, colonies appeared on only ampicillin spread plates with WbnK and WclY.

• Ampicillin, the selection antibiotic, was defrosted.

• One isolated colony from the 100µL spread plate that contained WbnK and WclY were picked and inoculated in a single bottle containing 5mL LB broth (see: Preparation of LB Agar and Broth) via dropping the pipette tip.

• 5µL ampicillin was added to the single 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.

Wednesday 8th August (9.00) – Mini-Prepping, 3A assembly and Transformation

•Overnight cultures of WbnK and WclY were transferred into two Falcon tubes and centrifuged at 3900rcf for 2 minutes at 21°C.

•To BioBrick WbnK, WclY and WbbC(dodgy) onto the terminator, three antibiotic assembly (3A assembly) was used.

• A master-mix was made up for WbnK, WclY and WbbC(dodgy), the terminator part and linear plasmid backbone (pSB1C3) in five, separate 1.5mL Eppendorf tubes. Therefore:

• Tube 1 (pSB1C3 master-mix) contained 0.5µL EcoRI-HF, 0.5µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and 18.0µL MilliQ H2O;

• Tube 2 (WbnK) contained 1µL EcoR1-HF, 1µL SpeI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 2.42µL WbnK plasmid DNA (starting concentration = 206.7ng/µL) and 40.1µL MilliQ H2O;

• Tube 3 (WclY) contained 1µL EcoRI-HF, 1µL SpeI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 1.61µL WclY plasmid DNA (starting concentration = 309.6ng/µL) and 40.9µL MilliQ H2O;

• Tube 4 (WbbC(dodgy)) contained 1µL EcoRI-HF, 1µL SpeI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 4.99µL WbbC(dodgy) plasmid DNA (starting concentration = 100.2ng/µL) and 37.5µL MilliQ H2O.

• Tube 5 (Terminator) contained 1µL XbaI, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA, 11.6µL terminator DNA (starting concentration = 43.1ng/µL) and 30.9µL MilliQ H2O.

• 4µL of tube 1 contents was transferred to three 1.5mL Eppendorf tubes containing 4µL pSB1C3 linearized plasmid backbone with a total volume of 8µL and this was restriction digested (NOT tube 1).

• Each Eppendorf tube was incubated at 37°C for 30 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, fresh Eppendorf tubes. Each tube contained 2µL of terminator, 2µL pSB1C3, 1µL 10x T4 DNA ligase buffer, 0.5µL T4 DNA ligase and 2.5µL MilliQ H2O. However, one tube contained 2µL of restriction digested WbnK, another 2µL of restriction digested WclY and the other 2µL of restriction digested WbbC(dodgy.

• These three tubes were left to incubate for 30 minutes at room temperature, and then placed in an incubator at 80°C for 20 minutes.

• 2µL of diluted WbnJ and WfcA from IDT re-suspension, as well as 2µL of ligated WbnK-terminator-pSB1C3, ligated WclY-terminator-pSB1C3 and ligated WbbC(dodgy)-terminator-pSB1C3 were pipetted gently into differnet 25µL Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously. Furthermore, 2µL of ligated WbnJ-pSB1C3 (for sending off to the parts registry) was pipetted gently into 25µL Top10 E.coli competent cells, making sure not to mix too rigorously.

Thursday 9th August (15.00) – Adding Cultures to Liquid Medium

Friday 10th August (9.00) – Mini-Prepping and Gel Electrophoresis

• Overnight cultures were transferred into new, single Falcon tubes and centrifuged at 3900rcf for 2 minutes at 21°C. The remaining Falcon tubes containing dropped pipette tips were stored at -2°C for streak plating to make glycerol stocks.

• The supernatant of the centrifuged Falcon tubes were discarded (being careful not to disturb the pellet).

• The pellets were re-supended in Resuspension Buffer (250µL) by pipetting the solution up and down.

• Lysis solution (250µL) was added and immediately Neutralisation Buffer (350µL).

• Each Falcon tube was then centrifuged for 5 minutes at 16100rcf at 21°C.

• 850µL of the supernatant was withdrawn (being careful not to disturb the cellular debris) and transferred to a geneJET Miniprep column.

• The geneJET Miniprep 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 at 16100rcf at 21°C.

• Any flow-through was discarded and washed again with extra Wash solution.

• This was centrifuged again for 1 minute at 16100rcf at 21°C.

• 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 Eppendorf’s.

• MilliQ H2O (50µL) was added to each Eppendorf and left for a couple of minutes.

• The concentration of WbnJ, WfcA, WbnK, WclY, WbbC(dodgy), ligated WbnJ-pSB1C3 (for Parts Registry), ligated WbnK-terminator-pSB1C3, ligated WclY-terminator-pSB1C3 and ligated WbbC(dodgy)-terminator-pSB1C3 DNA obtained in each Eppendorf was measured at the Nanodropping machine.

• To verify all these constructs had been 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, all DNA constructs previously mentioned were added to different Eppendorf tubes, each containing: 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. The latter two reagents were measured as follows for each plasmid DNA:

• Tube 1 (WbnJ) contained 2.02µL WbnJ plasmid DNA (starting concentration = 247.2ng/µL) and 40.5µL MilliQ H2O;

• Tube 2 (WfcA) contained 1.67µL WfcA plasmid DNA (starting concentration = 299.8ng/µL) and 40.8µL MilliQ H2O;

• Tube 3 (WbnJ-pSB1C3) contained 7.70µL WbJ-pSB1C3 plasmid DNA (starting concentration = 64.9ng/µL) and 34.8µL MilliQ H2O;

• Tube 4 (WbnK) contained 2.42µL WbnK plasmid DNA (starting concentration = 206.7ng/µL) and 40.1µL MilliQ H2O;

• Tube 5 (WclY) contained 1.61µL WclY plasmid DNA (starting concentration = 309.6ng/µL) and 40.9µL MilliQ H2O;

• Tube 6 (WbbC(dodgy)) contained 4.99µL WbbC(dodgy) plasmid DNA (starting concentration = 100.2ng/µL) and 37.5µL MilliQ H2O;

• Tube 7 (ligated WbnK-terminator-pSB1C3) contained 10.8µL ligated WbnK-terminator-pSB1C3 plasmid DNA (starting concentration = 46.4ng/µL) and 39.2µL MilliQ H2O;

• Tube 8 (ligated WclY-terminator-pSB1C3) contained 7.63µL ligated WclY-terminator-pSB1C3 plasmid DNA (starting concentration = 65.5ng/µL) and 34.9µL MilliQ H2O;

• Tube 9 (ligated WbbC(dodgy)-terminator-pSB1C3) contained 7.91µL ligated WbbC(dodgy)-terminator-pSB1C3 plasmid DNA (starting concentration = 63.2ng/µL) and 34.6µL MilliQ H2O.

• Each Eppendorf tube was incubated at 37°C for 1 hour and then at 80°C for 20 minutes immediately.

• Once the agarose gel was left to set for around 15 minutes, the well comb was lifted out.

• 10µL of the nine restriction digested DNA plasmid products were added to 2µL loading buffer to make a 5x dilution.

• DNA hyperladder and restriction digested DNA samples were loaded in the following order: Well 1 - DNA hyperladder, Well 2 - WbnJ, Well 3 - WbnJ-pSB1C3, Well 4 - WfcA, Well 5 - WbnK, Well 6 - WbnK-terminator-pSB1C3, Well 7 - WclY, Well 8 - WclY-terminator-pSB1C3, Well 9 - WbbC(dodgy), Well 10 - WbbC(dodgy)-terminator-pSB1C3, Well 11 - DNA hyperladder. TAE buffer was then added to the gel electrophoresis plate to submerge the agarose gel and therefore the samples.

• Gel electrophoresis was then conducted at 150V for 40 minutes.

• After UV illumination, restriction digested WbnJ, WfcA, WbnK, WclY, WbbC(dodgy) and WclY-terminator-pSB1C3 products were observed. However, WbnJ-pSB1C3 (for Parts Registry), WbnK-terminator-pSB1C3 and WbbC(dodgy)-terminator-pSB1C3 were not seen.