Month: November 2014

Cast your minds back to my post concerning bacterial transformation (or just scroll down, whatever). One thing that I forgot to mention was how I extracted pAG4 from the XL-1 blue bacterial cells once it had been replicated. That’s where this post comes in.

Minipreps were performed on four separate 5ml overnight cultures of pAG4 containing XL-1 blues in LB broth. This procedure requires a number of different reagent additions to ultimately extract pure pAG4 from the cells without damaging the plasmid itself. To do this, the cells need to be separated from the culture medium by means of centrifugation, lysed, and cellular debris needs to be centrifuged out of suspension. Leaving only cellular content, including the plasmid.

The processed samples are then run through Qiagen miniprep tubes. Miniprep tubes contain a filter with an affinity for DNA, which allows for all non-target cellular contents to pass through the tube with the aid of wash buffers. The ‘caught’ DNA is then flushed through the tube and collected. Simple.

The 5ml culture, reagents and miniprep tubes can be seen in the above image.

Following snap-freeze cycles from the last protocol, this next one focuses on the purification of my protein sample. I did this using immobilised metal ion affinity chromatography – as previously stated, the protein contains a His-tag, hence the means of its purification. A 1ml HiTrap purification column was used.

This protocol consisted of a number of steps:

1. Wash of the column with ultra pure water
2. Equilibration of the column using the same protein buffer that the protein sample was resuspended in
3. Addition of protein sample in protein buffer
4. Elution of the column with low concentration imidazole buffer
5. Elution of the column with high concentration imidazole buffer
6. Wash of the column with ultra pure water and 20% ethanol

The first image above shows the column after the addition of the protein sample (again, green because it contains GFP). Notice how it forms a noticeably green band at the top of the tube, this is the region in which the protein is most highly concentrated. The second image shows the column after the first elution with low concentration imidazole buffer. For reference, imidazole is employed to elute the column because it competitively binds to the same site that Histidine attaches to. This enables the protein to move down the column once it has detached and not re-attach. The third image shows the product of elution with high concentration imidazole – 10 column volumes were run through the column and collected in separate 1 column volume aliquots, labelled here as 1-10). You can clearly see that the highest concentration was eluted in the second column volume of buffer.

Next week: SDS-PAGE to analyse the raw sample vs my newly purified sample.

The 200ml cultures from the last protocol were aliquoted into 8, 50ml centrifuge tubes and spun down at 3000g to separate the cells containing my protein of interest from the media in which they were cultured- the pellet consisted of a layer of bright green (my protein) and a layer of light brown (cellular debris). The supernatant was then removed and the pellet frozen down in a -80*c freezer. Later, the pellet was resuspended in pH and salt specific Tris protein buffer with added DNAase.

This marks the part of the protocol that requires protein extraction from the cells by lysis – the method of choice was several freeze-thaw cycles in liquid nitrogen (6 to be exact). Multiple drastic change in temperature within a short space of time causes a reducing in cellular integrity and cause the cells to lyse, releasing their contents including my protein of interest: His-GFP-NT. At this stage, the sample was spun down at 3000g to pellet out cellular debris.

    

The first two photos above show the sample during each stage of the freeze-thaw cycles. The latter shows the cellular debris pellet after centrifugation, the green supernatant contains my target protein.

The supernatant was filtered and frozen down, ready for the next part of the protocol – protein purification.

 

The first stage of my project required bacterial transformation for the production of two plasmids: pAG3 and pAG4 – aptly named after my project supervisor. The first is a Histidine-GFP-neurotensin containing plasmid and the latter lacks the neurotensin component as a result of mutagenesis. I used two strains for E.coli for this stage of the project – XL-1 blue and BL21 (DEM).

Site-directed mutagenesis to the pAG3 plasmid was conducted with an optimised PCR running programme and specially designed primers. The mutagenesis product was then digested by dpn1 to ensure that only the plasmid of interest was later transformed into XL-1 blue. XL-1 blue does not contain the T7 promoter region, meaning that this strain in particular is not able to produce protein from this plasmid, simply replicate the plasmid. The image below shows the product of this transformation. You can see the difference between the number of observable colonies between the transformation of pAG4 and pAG3 (scroll down for the other image)- this is typical for plasmids transformed as a product of mutagenesis.

As an experiment running alongside the mutagenesis of pAG3 to pAG4, I induced protein expression from the pAG3 plasmid in a series of steps:

1. Transformation of pAG3 into BL21 E.coli cells (contains the T7 promoter – thus, produces protein)
2. 5ml overnight culture of transformed cells from the previous step
3. Expansion of culture in 200ml media
4. Induction of protein expression with IPTG – incubated overnight

          

The first image above shows the colonies from transformation of pAG3 into BL21(DEM) (note how many more colonies are present compared to transformations of pAG4 into XL-1 blue) – this has a slight green colour due to the expression of the protein, which contains green fluorescent protein (GFP). The same applies to the second image of the overnight culture that had been IPTG induced the previous day – it appears rather green compared to the media it is grown in.

Next week: I will be purifying this protein with immobilised metal affinity chromatography and sending off pAG4 for sequencing.