Team:Exeter/lab book/1gp/wk4

Operon Construction  |  Glycobase

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• In duplicate, 1.5mL of overnight BL21(DE3) broth culture was transferred to a fresh, labelled 50mL Falcon tube and pelleted by centrifuging for 2 minutes at 13’000 x g. The supernatant culture medium was removed completely and discarded.

• The pellet was re-suspended thoroughly in 180µL lysis solution T and 20µL RNase A solution was added. The total solution was mixed and incubated for 2 minutes at 22°C.

• 20µL Proteinase K was added to the solution and then mixed. This was then incubated for 30 minutes at 55°C.

• 200µL lysis solution C was added and the solution was vortexed thoroughly for about 15 seconds and then incubated again at 55°C for 10 minutes.

• Whilst this was incubating, 500µL column preparation solution was added to a pre-assembled GenElute miniprep binding column that was seated in a 2mL collection tube. This was centrifuged at 12’000 x g for 1 minute. Afterwards, the eluate was discarded.

• When incubation of extracted genomic DNA of BL21(DE3) was complete, 200µL ethanol (100%) was added to the lysate and mixed thoroughly by vortexing for 5-10 seconds.

• All the lysate-ethanol solution was transferred to the GenElute miniprep binding column, being careful to not pipette too hard as to shear the genomic DNA. The binding column with the lysate-ethanol solution was centrifuged at 6’500 x g for 1 minute. After this, the collection tube was discarded that contained the eluate and the binding column was placed in a new 2mL collection tube.

• 500µL wash solution 1 was added to the column and centrifuged for 1 minute at 6’500 x g. The 2mL collection tube after centrifugation was discarded and the binding column was added to a new 2mL collection tube. 500µL wash solution containing ethanol was transferred to the binding column and centrifuged for 3 minutes at 13’000 x g to dry the column. The column was centrifuged for another minute so all ethanol was removed from the binding column. The collection tube containing eluate (including ethanol) was discarded and the binding column was placed in a new 2mL collection tube.

• 200µL elution solution was pipetted directly onto the center of the binding column and left to incubate at 22°C for 5 minutes. After this, the column was centrifuged for 1 minute at 6500 x g to elute the genomic DNA. This was repeated again to elute as much DNA as possible using the same 2mL collection tube.

• The 2mL collection tube containing genomic DNA was stored on ice (2-8°C), and 1.5µL was used to measure DNA concentration on the Nanodropping Machine.

• To PCR amplify WbbC from the genomic DNA, a two master-mixes were made up containing: 10µL 5ng/µL genomic DNA (starting concentration = 7.8ng/µL for the first PCR tube and 6.6ng/µL for the second PCR tube), 1µL of dNTP’s, 1µL WbbC forward primer (at 100pmol/µL), 1µL WbbC reverse primer (at 100pmol/µL), 10µL 5x PCR buffer, 1µL Phusion polymerase and 26µL MilliQ H2O to make a total volume of 50µL.

• Once this master-mix was made up, the PCR tube containing these reagents was put in a PCR machine that was programmed for two-step PCR (using Phusion). Because of the high TM of the forward and reverse primers for WbbC (roughly 75°C), two-step PCR using extension at 72°C was possible. The machine was programmed as: 98°C for 30 seconds, 98°C for 10 seconds and then 72°C for 2 seconds (this was repeated for 30 cycles), and then finally 72°C for 5 minutes.

• As WbbC was being PCR amplified, an agarose gel for gel electrophoresis was made to verify whether WbbC was amplified. This involved adding 50mL 1x TAE buffer to 0.5g agarose and then microwaving until completely dissolved.

• 1µL ethidium bromide (EtBr) was added to the hot dissolved agarose gel and mixed thoroughly. The entire solution was poured into a gel electrophoresis plate with a well comb inserted. Once left to set for around 15 minutes, the well comb was lifted out.

• When PCR amplification of WbbC was finished, 5µL PCR mix was added to 1µL loading buffer to make a 5x dilution. 5µL of DNA hyperladder was also added to another tube containing 1µL loading buffer (again, to make a 5x dilution).

• Both DNA hyperladder and PCR mix were added to different wells and then TAE buffer was added to the gel electrophoresis plate to submerge the agarose gel and therefore the two samples.

• Gel electrophoresis was then conducted at 200V for 15 minutes.

• After UV illumination, no WbbC products in lanes 2 and 3 were observed. It was discovered that the reverse primer was incorrect and had actually amplified a shorter section of WbbC, rather than the whole gene. Thus the correct reverse primer was ordered.

• Meanwhile, transformed competent E.coli containing recombinant pBAD/AraC promoter plasmids were picked from the 100µL spread plate and inoculated in LB broth (See: Week 30th July-03rd August 2012, Operon Construction: Mary Beton).

Tuesday 31st July (15.00) – IDT Re-suspension and Transformation

• The synthesised gene WbbC (with a point-mutation, which we term WbbC(dodgy)) was re-suspended using IDTE buffer (40µL of 10mM Tris, 0.1mM EDTA), held between pH 7.5-8.0, into a fresh Eppendorf tubes, to reach an approximate concentration of 50ng/µL stock solution.

• The tube was vortexed for 20 seconds, left to incubate at room temperature for 30 minutes, and then centrifuged for 1 minute.

• To dilute WbbC for the transformation procedure, 2µL of re-suspended WbbC(dodgy) was added to MilliQ H2O (999µL) to reach an approximate concentration of 0.1ng/µL in a fresh, labelled Eppendorf. The tube was centrifuged briefly to spin down any liquid.

• After this, 2µL of diluted WbbC was pipetted gently into 50µL Top10 E.coli competent cells (Invitrogen), 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 and then the Eppendorf tube containing recombinant WbbC(dodgy) plasmids 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-up (25µL LB agar in each). One would be used for spreading 20µL of transformed E.coli competent cells and the other for 100µL of transformed E.coli competent cells, so that we would be able to pick out enough isolated colonies. 50µL ampicillin was added to 50mL 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 respective ampicillin-containing LB 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.

Wednesday 1st August (15.00) – Adding Cultures to Liquid Medium

• When all the spread plates were taken out of the 37°C incubator, colonies appeared on the ampicillin spread plates containing 20µL and 100µL Wbbc(dodgy). Therefore only colonies carrying recombinant Wbbc(dodgy) plasmids were added to LB broth.

• Ampicillin, the selection antibiotic, was defrosted.

• Three isolated colonies from the 100µL spread plate that contained Wbbc(dodgy) plasmids 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.

Thursday 2nd August (9.00) – Mini-prepping, Gel Electrophoresis and Preparation for new BioBrick Shipping

• Overnight cultures were transferred into two Falcon tubes (as done in duplicate) 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-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 plasmid WbbC(dodgy) DNA obtained in each Eppendorf was measured at the Nanodropping machine.

• To verify WbbC(dodgy) 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 WbbC(dodgy) DNA was added to two different Eppendorf tubes, each containing: 1µL of EcoR1-HF, 1µL of PstI, 5µL of 10x NEBuffer2, 0.5µL 100x BSA, 500ng plasmid DNA (?.??µL for WbbC(dodgy) as starting concentration = ???.?ng/µL) and enough MilliQ H2O to make a total volume of 50µL (hence for WbbC(dodgy) restriction digest, ??.?µL MilliQ H2O was added).

• To prepare sending off WbnJ, WfcA and WbbC(dodgy) as BioBrick parts for the parts registry, a master-mix was made up for each glycosyltransferase and linearized plasmid backbones (pSB1C3, pSB1T3 and pSB1A3) in separate Eppendorf tubes(hence 7 tubes required). Therefore:

• Tube 1 (WbnJ) contained 1.0µL EcoR1-HF, 1.0µL PstI, 5.0µL 10x NEBuffer2, 0.5µL 100x BSA, ?.??µL WbnJ plasmid DNA (starting concentration = ???.?ng/µL) and ??.?µL MilliQ H2O;

• Tube 2 (WfcA) contained 1.0µL EcoR1-HF, 1.0µL PstI, 5.0µL 10x NEBuffer2, 0.5µL 100x BSA, ?.??µL WfcA plasmid DNA (starting concentration = ???.?ng/µL) and ??.??µL MilliQ H2O;

• Tube 3 (WbbC(dodgy)) contained 1.0µL EcoR1-HF, 1.0µL PstI, 5.0µL 10x NEBuffer2, 0.5µL 100x BSA, ?.??µL WbbC(dodgy) plasmid DNA (starting concentration = ??.?ng/µL) and ??.??µL MilliQ H2O;

• Tube 4 (master-mix for linearized plasmid backbones) contained 0.5µL EcoR1-HF, 0.5µL PstI, 5.0µL 10x NEBuffer2, 0.5µL 100x BSA, and 18.00µL MilliQ H2O.

• 4µL of tube 4 contents was added to three separate 1.5mL Eppendorf tubes containing: 4µL pSB1C3, 4µL pSB1T3 and 4µL pSB1A3.

• 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 glycosyltransferase DNA in each of the three tubes were transferred to respective restriction digested linearized plasmid DNA (hence 2µL restriction digested WbnJ was added to 2µL restriction digested ??? linearized plasmid DNA, 2µL WfcA was added to 2µL restriction digested pSB1A3 DNA and 2µL restriction digested WbbC(dodgy) was added to 2µL restriction digested ??? linearized plasmid DNA in three separate Eppendorf tubes). Each of these three ligation tubes also contained: 1µL T4 DNA ligase buffer, 0.5µL T4 DNA ligase and 4.5µL MilliQ H2O.

• 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.

• These three tubes containing ligated glycosyltransferase-linearized plasmid DNA were stored at -5°C.